**Meet the editor**

Dr. Ivan Diaz Padilla received his Doctor of Medicine (M.D.) in 2000 from Autonoma University, in Madrid, Spain. He completed his medical oncology training in 2005 at University Hospital La Princesa, Madrid. After a few years serving as staff general medical oncologist in Madrid, Dr. Diaz-Padilla completed a two-year clinical research fellowship in Drug Development and Gyne-

cologic Oncology at the Princess Margaret Hospital in Toronto, Canada. Dr. Diaz-Padilla's main research interest is directed towards an efficient implementation of new molecularly targeted agents into the gynecological cancers. He currently serves as a Consultant Medical Oncologist at the Centro Integral Oncologico Clara Campal in Madrid, Spain. His clinical activity is focused on gynecological cancer and early-phase clinical trials.

Contents

**Preface IX**

**Receive Care? 3**

**Survivorship 27** Vânia Gonçalves

**Section 2 Pathology 75**

Laurie Elit and Clare Reade

**Section 1 Clinical Epidemiology of Ovarian Cancer 1**

Chapter 1 **In what Setting Should Women with Ovarian Cancer**

Chapter 2 **Quality of Life in Ovarian Cancer Treatment and**

Chapter 3 **Preventive Strategies for Ovarian Cancer 45** L. Cortesi, A. Toss and E. De Matteis

Chapter 4 **Borderline Epithelial Tumors of the Ovary 77**

Gennaro Cormio, Vera Loizzi, Maddalena Falagario, Doriana

**Epithelial Ovarian Cancer, and Survival Following a Mucinous**

Sherri L. Stewart, Jennifer M. Wike, Trevor D. Thompson, Rosemary D. Cress, Amy R. Kahn, Cynthia D. O'Malley and Maria J. Schymura

Scardigno, Donatella Latorre and Luigi E. Selvaggi

Chapter 5 **Implication of Clear Cell and Mucinous Histology 97** Jun Naniwa, Hiroaki Itamochi and Junzo Kigawa

Chapter 6 **Demographic and Clinical Characteristics of Mucinous**

**Epithelial Ovarian Cancer Diagnosis 117**

Chapter 7 **The Genetics of Ovarian Cancer 135** Constantine Gennatas

### Contents

#### **Preface XIII**


Chapter 6 **Demographic and Clinical Characteristics of Mucinous Epithelial Ovarian Cancer, and Survival Following a Mucinous Epithelial Ovarian Cancer Diagnosis 117** Sherri L. Stewart, Jennifer M. Wike, Trevor D. Thompson, Rosemary D. Cress, Amy R. Kahn, Cynthia D. O'Malley and Maria J. Schymura

Chapter 7 **The Genetics of Ovarian Cancer 135** Constantine Gennatas

#### **X** Contents

#### **Section 3 Ovarian Cancer Therapeutics 159**

Chapter 8 **Surgical Treatment of Ovarian Cancer 161** Lucas Minig, M. Guadalupe Patrono, Rafael Alvarez Gallego, Javier Valero de Bernabé and Ivan Diaz-Padilla

Chapter 16 **Ovarian Cancer in vitro Diagnostics: New Approaches to Earlier**

Jorge Tapia, Mercedes de Alvarado, Benjamín Bustos, Vania

Contents **VII**

Malgorzata Banys, Natalia Krawczyk, Andreas D. Hartkopf and

**Detection 349**

Lukoviek and Gregory E. Rice

**Ovarian Cancer 359**

Tanja Fehm

Chapter 17 **Disseminated Tumor Cells and Cancer Stem Cells in**

	- **Section 4 Molecular Pathogenesis and Targeted Therapies 225**

#### Chapter 16 **Ovarian Cancer in vitro Diagnostics: New Approaches to Earlier Detection 349**

Jorge Tapia, Mercedes de Alvarado, Benjamín Bustos, Vania Lukoviek and Gregory E. Rice

#### Chapter 17 **Disseminated Tumor Cells and Cancer Stem Cells in Ovarian Cancer 359**

**Section 3 Ovarian Cancer Therapeutics 159**

Garcia Mata

**VI** Contents

**Ovarian Cancer 205**

Chapter 8 **Surgical Treatment of Ovarian Cancer 161**

Valero de Bernabé and Ivan Diaz-Padilla

Chapter 10 **Molecular Mechanisms of Platinum Resistance in**

**Section 4 Molecular Pathogenesis and Targeted Therapies 225**

Chapter 11 **Biological Significance of Apoptosis in Ovarian Cancer: TRAIL**

Chapter 12 **Protein Kinase G-Iα Hyperactivation and VASP Phosphorylation**

Chapter 13 **The PI3K/Akt/mTOR Pathway in Ovarian Cancer: Biological Rationale and Therapeutic Opportunities 275**

Jeanine Staples and Annekathryn Goodman

Chapter 15 **The Merit of Alternative Messenger RNA Splicing as a New**

Jean-Philippe Brosseau and Sherif Abou-Elela

Nadzeya Goncharenko-Khaider, Denis Lane, Isabelle Matte,

**in Promoting Ovarian Cancer Cell Migration and Platinum**

Alexandra Leary, Edouard Auclin, Patricia Pautier and Catherine

**Mine for the Next Generation Ovarian Cancer Biomarkers 327**

Gonzalo Tapia and Ivan Diaz-Padilla

**Therapeutic Targeting 227**

**Resistance 251**

Lhommé

**Section 5 Biomarkers 325**

Claudine Rancourt and Alain Piché

Janica C. Wong and Ronald R. Fiscus

Chapter 14 **PARP Inhibitors in Ovarian Cancer 303**

Chapter 9 **The Role of Chemotherapy in Recurrent Ovarian Cancer 183**

Lucas Minig, M. Guadalupe Patrono, Rafael Alvarez Gallego, Javier

Miguel Angel Alonso Bermejo, Ana Fernandez Montes, Eva Perez Lopez, Miguel Angel Nuñez Viejo, Jesus Garcia Gomez and Jesus

Malgorzata Banys, Natalia Krawczyk, Andreas D. Hartkopf and Tanja Fehm

Preface

Ovarian cancer can no longer be considered one disease entity, but a heterogeneous group of diseases. Our understanding of its clinical and molecular complexity is improving nota‐ bly over the last decade. This is of utmost importance when it comes to determine the most adequate treatment strategy for each individual patient. Despite not being the most frequent tumor, ovarian cancer has the highest mortality rate amongst gynecological cancers. The ab‐ sence of specific symptoms and the lack of a universally and validated screening strategy leads to a delayed diagnosis, when the tumor has spread beyond the ovaries. Despite most patients are diagnosed at advanced stages, aggressive cytoreductive surgery and combina‐ tion chemotherapy is recommended. This therapeutic approach may control the disease for some time, but a great majority of patients experience relapses within the first two years of primary treatment. Relapsed ovarian cancer is no longer a curable disease and chemothera‐ py is the mainstay of treatment in that scenario. Ovarian cancer has been traditionally con‐ sidered a chemosensitive tumor. However, the development of resistance to cytotoxics is a major problem. New molecularly targeted agents are actively being investigated in an at‐

Stemmed from the complexity of ovarian cancer, high-scale medical specialization is there‐ fore needed. Ideally, ovarian cancer patients should be treated in tertiary institutions, where higher volumes of complex cytoreductive procedures are performed. It is likely that a strong link with a clinical research facility may also derive in benefit for patients, having immediate access to the latest treatment opportunities within clinical trials. Considerable efforts are al‐ so underway in early diagnosis and identification of new biomarkers that may help in pre‐

The present book encompasses most of the key aspects pertaining the current of diagnosis and treatment of ovarian cancer. It is intended to cover topics from clinical epidemiology to the latest advances in biomarker development and new drugs. The present publication has counted with the valuable contribution of renowned international experts in the field of ovarian cancer, to whom I want to express my most sincere gratitude. We hope that our target readership (general gynecologists, medical oncologists, gynecologic oncologists) will

> **Ivan Diaz-Padilla, M.D.** Consultant Medical Oncologist Head, Gynecologic Cancer Program Centro Integral Oncologico Clara Campal Hospital Universitario HM Sanchinarro

find this book as a useful and valuable reference in their daily practice.

tempt to improve the outcome of this patient population.

dicting response to treatment.

### Preface

Ovarian cancer can no longer be considered one disease entity, but a heterogeneous group of diseases. Our understanding of its clinical and molecular complexity is improving nota‐ bly over the last decade. This is of utmost importance when it comes to determine the most adequate treatment strategy for each individual patient. Despite not being the most frequent tumor, ovarian cancer has the highest mortality rate amongst gynecological cancers. The ab‐ sence of specific symptoms and the lack of a universally and validated screening strategy leads to a delayed diagnosis, when the tumor has spread beyond the ovaries. Despite most patients are diagnosed at advanced stages, aggressive cytoreductive surgery and combina‐ tion chemotherapy is recommended. This therapeutic approach may control the disease for some time, but a great majority of patients experience relapses within the first two years of primary treatment. Relapsed ovarian cancer is no longer a curable disease and chemothera‐ py is the mainstay of treatment in that scenario. Ovarian cancer has been traditionally con‐ sidered a chemosensitive tumor. However, the development of resistance to cytotoxics is a major problem. New molecularly targeted agents are actively being investigated in an at‐ tempt to improve the outcome of this patient population.

Stemmed from the complexity of ovarian cancer, high-scale medical specialization is there‐ fore needed. Ideally, ovarian cancer patients should be treated in tertiary institutions, where higher volumes of complex cytoreductive procedures are performed. It is likely that a strong link with a clinical research facility may also derive in benefit for patients, having immediate access to the latest treatment opportunities within clinical trials. Considerable efforts are al‐ so underway in early diagnosis and identification of new biomarkers that may help in pre‐ dicting response to treatment.

The present book encompasses most of the key aspects pertaining the current of diagnosis and treatment of ovarian cancer. It is intended to cover topics from clinical epidemiology to the latest advances in biomarker development and new drugs. The present publication has counted with the valuable contribution of renowned international experts in the field of ovarian cancer, to whom I want to express my most sincere gratitude. We hope that our target readership (general gynecologists, medical oncologists, gynecologic oncologists) will find this book as a useful and valuable reference in their daily practice.

#### **Ivan Diaz-Padilla, M.D.**

Consultant Medical Oncologist Head, Gynecologic Cancer Program Centro Integral Oncologico Clara Campal Hospital Universitario HM Sanchinarro

**Section 1**

**Clinical Epidemiology of Ovarian Cancer**

**Clinical Epidemiology of Ovarian Cancer**

**Chapter 1**

**In what Setting Should Women with**

**Ovarian Cancer Receive Care?**

Additional information is available at the end of the chapter

In Canada, ovarian cancer affects 2600 women and 1750 women die annually from this dis‐ ease.[1] The case fatality rate for ovarian cancer is quite high at 0.67 because women usually present with wide-spread disease. Symptoms of ovarian cancer are non-specific, and there is no effective screening test which identifies ovarian cancer early, when the cure rate is high‐ est.[2] When patients present with advanced disease, long term survival is elusive and the goals of care focus on increasing duration of survival and improving quality of life by man‐

Ovarian cancer is usually managed with a combination of surgery and chemotherapy. The role of surgery is to make a histologic diagnosis, determine the extent of disease spread (staging) and remove as much disease as possible (debulking). The role of chemotherapy is to reverse the vascular permeability of tumour capillaries, thereby decreasing the presence of ascites and pleural effusions, and to cause cellular apoptosis of tumour cells, resulting in

Evaluation of the patterns of care provided to patients with ovarian cancer in Ontario, Canada demonstrated a variety of specialists are involved in the delivery of surgery in‐ cluding gynaecologists, general surgeons and gynaecologic oncologists.[3] The delivery of chemotherapy can be provided by medical or gynaecologic oncologists. Surgery and/or chemotherapy can be delivered in low, medium or high volume centres in rural or urban settings and by teaching or non-teaching faculty.[3] This paper addresses the question of whether the context in which a woman receives care for her ovarian cancer affects her

and reproduction in any medium, provided the original work is properly cited.

© 2013 Elit and Reade; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Laurie Elit and Clare Reade

http://dx.doi.org/10.5772/52936

**1. Introduction**

aging symptoms of disease.

disease regression.

outcome.

#### **Chapter 1**

### **In what Setting Should Women with Ovarian Cancer Receive Care?**

Laurie Elit and Clare Reade

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52936

#### **1. Introduction**

In Canada, ovarian cancer affects 2600 women and 1750 women die annually from this dis‐ ease.[1] The case fatality rate for ovarian cancer is quite high at 0.67 because women usually present with wide-spread disease. Symptoms of ovarian cancer are non-specific, and there is no effective screening test which identifies ovarian cancer early, when the cure rate is high‐ est.[2] When patients present with advanced disease, long term survival is elusive and the goals of care focus on increasing duration of survival and improving quality of life by man‐ aging symptoms of disease.

Ovarian cancer is usually managed with a combination of surgery and chemotherapy. The role of surgery is to make a histologic diagnosis, determine the extent of disease spread (staging) and remove as much disease as possible (debulking). The role of chemotherapy is to reverse the vascular permeability of tumour capillaries, thereby decreasing the presence of ascites and pleural effusions, and to cause cellular apoptosis of tumour cells, resulting in disease regression.

Evaluation of the patterns of care provided to patients with ovarian cancer in Ontario, Canada demonstrated a variety of specialists are involved in the delivery of surgery in‐ cluding gynaecologists, general surgeons and gynaecologic oncologists.[3] The delivery of chemotherapy can be provided by medical or gynaecologic oncologists. Surgery and/or chemotherapy can be delivered in low, medium or high volume centres in rural or urban settings and by teaching or non-teaching faculty.[3] This paper addresses the question of whether the context in which a woman receives care for her ovarian cancer affects her outcome.

© 2013 Elit and Reade; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **2. Quality of care**

The focus of this chapter falls within the rubric of quality of care. The Institute of Medicine has defined quality of care as "the degree to which health services for individuals and popu‐ lations increase the likelihood of desired health outcomes and are consistent with current professional knowledge".[4] Good quality means providing patients with appropriate serv‐ ices in a technically competent manner, with good communication, shared decision making and cultural sensitivity.[4] Quality assurance can be defined as all those planned and sys‐ tematic actions necessary to provide adequate confidence that a product or service will satis‐ fy given requirements for quality.[5]

To demonstrate the concepts of structure, process and outcomes as ways to measure quality of care in ovarian cancer, we will review population-based studies published over the last 10 years. We have restricted our scope to population-based studies because they provide out‐ comes for the whole population in a region and avoid biases inherent with single institution studies (ie., related to socioeconomic status, race or comorbidities). As well, populationbased studies allow us the opportunity to identify where variations in care may lead to su‐ perior outcomes for the population. If these processes and/or structures are incorporated

In what Setting Should Women with Ovarian Cancer Receive Care?

http://dx.doi.org/10.5772/52936

5

A systematic search of the published English language literature from Jan 1, 2000 to Jun 29, 2012 was undertaken in order to present an unbiased view of the current population-based literature in the field of quality of care. Several key articles were identified[9-11] and MeSH terms from these references were used to create a search strategy for PubMed (Figure 1).

**female[mh]+AND+(th[sh]+OR+su[sh]+OR+rt[sh]+OR+dt[sh]+OR+surgery[tiab]+OR+surgi**

The search yielded 1178 articles of which 172 were identified as potentially relevant by title and abstract. To be included the article had to include population-based data collection re‐ lated to primary management of ovarian cancer. The article needed to report on structure or processes of care in relation to outcomes. Articles were excluded if they were reporting on screening for ovarian cancer, pre-cancerous or benign conditions; if they were focused solely on quality of life, biologic therapies, biomarkers and personalized medicine, survivorship or palliative care. We identified two systematic reviews of quality of care indicators.[12,13] However, in both cases the authors did not restrict their study inclusion to population-based

The 30 population-based studies in this review represent findings from many high-income countries, including Australia (1), Canada (3), USA (12), Austria (2), Finland (2), Germany

**http:/ / www.ncbi.nlm.nih.gov/ entrez/ query.fcgi?CMD=search&term=(genital**

**cal\*[tiab]))+AND+(health planning[mh]+OR+"health care quality, access, and** 

**evaluation"[mh]+OR+"outcome and process assessment (health care)"[mh])+AND+(outcome\*[ti]+OR+population\*[ti]+OR+treatment outcome[majr])+AND+2000:2012[dp]+AND+english[la]&db=PubMed**

reports, therefore these studies are not included in our analysis.

into practice, they may lead to improved health outcomes.

**3. Methods**

**neoplasms,** 

**Figure 1.** PubMed search strategy

**4. Results**

Donabedian originally coined the phrase quality of care.[6,7] He assessed quality of care by looking at the triad of *structures, processes and outcomes*.

**Structure** attributes describe the physical and organizational settings in which care is pro‐ vided and evaluate whether these characteristics are conducive to the kind of care that can be expected to improve health and to be acceptable to patients and the community. Evalua‐ tion of the adequacy of facilities, qualifications of medical staff, availability of equipment, and organizational structure and operations of programs within the institution providing care fall under the category of structure of care. Often-evaluated structural variables include the demographic characteristics, training and experience of care providers and the environ‐ ment in which they work. Other structural variables of interest include access to specific technologies, access to intensive care facilities and nurse-to-patient ratios. The most com‐ monly cited variable used as a surrogate for assessing surgical quality is hospital or physi‐ cian case volume.

**Process** of care describes the care the patient actually receives and evaluates the degree to which interventions provided to patients correspond to what is known or believed to be most effective in improving health. This includes: 1) the patient's activity in seeking care and carrying it out, and 2) the practitioner's activities in making a diagnosis and recom‐ mending and implementing therapy. Whether care is effective can be judged according to the evidence from good studies demonstrating a link between a particular process (ie., de‐ bulking surgery) and better outcomes (ie., prolonged survival). Process indicators are easily measured in a timely fashion and can provide actionable feedback for quality improvement initiatives. Other examples of process variables include guidelines for surgery and use of care pathways. These variables are usually used in the context of quality assessment audits.

**Outcomes** are the actual changes in health and wellbeing obtained by patients and com‐ munities, and the degree to which the care provided is acceptable. In other words, outcome is the effect of care on the health status of patients and populations. This may be improve‐ ment in patient knowledge, behaviour and satisfaction. Endpoints of interest in ovarian can‐ cer could include 30-day peri-operative mortality, overall survival, and quality of life. Overall survival data takes a long time to mature and it reflects a culmination of many proc‐ esses and structures that have contributed to care. There is currently a strong focus on out‐ comes for patients with ovarian cancer, especially in the context of health care payers obtaining high quality care for the health care dollars spent.[8]

To demonstrate the concepts of structure, process and outcomes as ways to measure quality of care in ovarian cancer, we will review population-based studies published over the last 10 years. We have restricted our scope to population-based studies because they provide out‐ comes for the whole population in a region and avoid biases inherent with single institution studies (ie., related to socioeconomic status, race or comorbidities). As well, populationbased studies allow us the opportunity to identify where variations in care may lead to su‐ perior outcomes for the population. If these processes and/or structures are incorporated into practice, they may lead to improved health outcomes.

#### **3. Methods**

**2. Quality of care**

cian case volume.

fy given requirements for quality.[5]

4 Ovarian Cancer - A Clinical and Translational Update

looking at the triad of *structures, processes and outcomes*.

obtaining high quality care for the health care dollars spent.[8]

The focus of this chapter falls within the rubric of quality of care. The Institute of Medicine has defined quality of care as "the degree to which health services for individuals and popu‐ lations increase the likelihood of desired health outcomes and are consistent with current professional knowledge".[4] Good quality means providing patients with appropriate serv‐ ices in a technically competent manner, with good communication, shared decision making and cultural sensitivity.[4] Quality assurance can be defined as all those planned and sys‐ tematic actions necessary to provide adequate confidence that a product or service will satis‐

Donabedian originally coined the phrase quality of care.[6,7] He assessed quality of care by

**Structure** attributes describe the physical and organizational settings in which care is pro‐ vided and evaluate whether these characteristics are conducive to the kind of care that can be expected to improve health and to be acceptable to patients and the community. Evalua‐ tion of the adequacy of facilities, qualifications of medical staff, availability of equipment, and organizational structure and operations of programs within the institution providing care fall under the category of structure of care. Often-evaluated structural variables include the demographic characteristics, training and experience of care providers and the environ‐ ment in which they work. Other structural variables of interest include access to specific technologies, access to intensive care facilities and nurse-to-patient ratios. The most com‐ monly cited variable used as a surrogate for assessing surgical quality is hospital or physi‐

**Process** of care describes the care the patient actually receives and evaluates the degree to which interventions provided to patients correspond to what is known or believed to be most effective in improving health. This includes: 1) the patient's activity in seeking care and carrying it out, and 2) the practitioner's activities in making a diagnosis and recom‐ mending and implementing therapy. Whether care is effective can be judged according to the evidence from good studies demonstrating a link between a particular process (ie., de‐ bulking surgery) and better outcomes (ie., prolonged survival). Process indicators are easily measured in a timely fashion and can provide actionable feedback for quality improvement initiatives. Other examples of process variables include guidelines for surgery and use of care pathways. These variables are usually used in the context of quality assessment audits. **Outcomes** are the actual changes in health and wellbeing obtained by patients and com‐ munities, and the degree to which the care provided is acceptable. In other words, outcome is the effect of care on the health status of patients and populations. This may be improve‐ ment in patient knowledge, behaviour and satisfaction. Endpoints of interest in ovarian can‐ cer could include 30-day peri-operative mortality, overall survival, and quality of life. Overall survival data takes a long time to mature and it reflects a culmination of many proc‐ esses and structures that have contributed to care. There is currently a strong focus on out‐ comes for patients with ovarian cancer, especially in the context of health care payers

A systematic search of the published English language literature from Jan 1, 2000 to Jun 29, 2012 was undertaken in order to present an unbiased view of the current population-based literature in the field of quality of care. Several key articles were identified[9-11] and MeSH terms from these references were used to create a search strategy for PubMed (Figure 1).

**http:/ / www.ncbi.nlm.nih.gov/ entrez/ query.fcgi?CMD=search&term=(genital neoplasms, female[mh]+AND+(th[sh]+OR+su[sh]+OR+rt[sh]+OR+dt[sh]+OR+surgery[tiab]+OR+surgi cal\*[tiab]))+AND+(health planning[mh]+OR+"health care quality, access, and evaluation"[mh]+OR+"outcome and process assessment (health care)"[mh])+AND+(outcome\*[ti]+OR+population\*[ti]+OR+treatment outcome[majr])+AND+2000:2012[dp]+AND+english[la]&db=PubMed**

**Figure 1.** PubMed search strategy

The search yielded 1178 articles of which 172 were identified as potentially relevant by title and abstract. To be included the article had to include population-based data collection re‐ lated to primary management of ovarian cancer. The article needed to report on structure or processes of care in relation to outcomes. Articles were excluded if they were reporting on screening for ovarian cancer, pre-cancerous or benign conditions; if they were focused solely on quality of life, biologic therapies, biomarkers and personalized medicine, survivorship or palliative care. We identified two systematic reviews of quality of care indicators.[12,13] However, in both cases the authors did not restrict their study inclusion to population-based reports, therefore these studies are not included in our analysis.

#### **4. Results**

The 30 population-based studies in this review represent findings from many high-income countries, including Australia (1), Canada (3), USA (12), Austria (2), Finland (2), Germany (1), Netherlands (3), Norway (1), Switzerland (1), UK (3), and Japan (1). Twenty-five unique studies report the impact of structure on outcomes, and 13 studies report the impact of vari‐ ous processes on outcomes. Included are 91,866 patients.

**Study Country Data Source Number of**

Grossi 2002[18] Australia Retrospective database +

Engelen 2006[26] Netherlands Retrospective database +

to find an association between structure and survival

Kumpulainen 2006 and

2009[29,30]

Stockton 2000[14] UK Retrospective database 989 Yes Olaitan 2001[15] UK Prospective cohort 595 n/a Carney 2002[16] USA Retrospective database 734 Yes Elit 2002[17] Canada Retrospective database 3,815 Yes

Kumpulainen 2002[19] Finland Retrospective database 3,851 Yes Cress 2003[20] USA Retrospective database 1,088 n/a Harlan 2003[21] USA Retrospective database 1,167 n/a Ioka 2004[22] Japan Retrospective database 2,450 Yes Diaz-Montez 2005[23] USA Retrospective database 2,417 n/a Bailey 2006[24] UK Prospective cohort 361 No\* Earle 2006[25] USA Retrospective database 3,067 Yes Elit 2006[11] Canada Retrospective database 2,502 No

chart review

chart review

Finland Prospective cohort 275 Yes

n/a: not applicable—these studies used surrogate outcomes, \*the authors of this study reported it was underpowed

Goff 2006 and 2007[27,28] USA Retrospective database 10,432 n/a

Oberaigner 2006[31] Austria Retrospective database 911 Yes Paulsen 2006[32] Norway Prospective registry 198 Yes Schrag 2006[33] USA Retrospective database 2,952 Yes Elit 2008[34] Canada Retrospective database 1,341 No Bristow 2009[35] USA Retrospective database 1,894 Yes Marth 2009[36] Austria Prospective cohort 1,948 Yes Vernooij 2009[37] Netherlands Retrospective cohort 1,077 Yes Mercado 2010[38] USA Retrospective cohort 31,897 Yes Rochon 2011[39] Germany Prospective cohort 476 No

**Table 1.** Studies reporting on structural variables in relation to outcomes for ovarian cancer

**patients**

In what Setting Should Women with Ovarian Cancer Receive Care?

434 No

632 Yes

**Did structure impact**

7

**survival?**

http://dx.doi.org/10.5772/52936

#### **4.1. Outcomes**

The 5-year overall survival rate is the indicator of most interest to clinicians caring for pa‐ tients with ovarian cancer. Other outcomes of interest include quality of life, patient satisfac‐ tion, and cost. However, when 5-year survival rates are so poor, surrogate outcomes, including progression-free survival (PFS), can be used to reflect small changes in outcomes that are important to patients and society. Changes in processes or structures that result in improved surrogate outcomes should eventually be reflected in improved 5-year survival rates. Surrogate outcomes in ovarian cancer include PFS and 30 or 60-day mortality.

#### **4.2. Structure**

In 25 unique population-based studies of quality of care in ovarian cancer, structural varia‐ bles evaluated include a hospital's annual ovarian cancer surgical volume, physician annual ovarian cancer surgical volume, hospital type (university affiliated vs community hospital), and physician type (gynaecologic oncologist, general gynaecologist, or general surgeon). These studies are listed in Table 1.

Studies evaluating hospital volume demonstrate hospitals with higher volumes of ovarian cancer surgery per year are often associated with better long-term survival (Table 2). The improvement in overall survival did not appear to be a reflection of peri-operative deaths, because the 30 and 60-day mortality was not affected by hospital volume in the studies eval‐ uating those outcomes. The long-term survival advantage produced by high-volume hospi‐ tals is due to other differences in structures and processes of care in these institutions.

Studies evaluating physician volume did not demonstrate a uniform improvement in sur‐ vival when high-volume physicians operated on patients with ovarian cancer (Table 3). Findings were inconclusive for both shorter and longer-term survival.

In just over half of the studies identified, hospitals classified as teaching facilities or univer‐ sity hospitals were associated with better short and long-term survival outcomes (Table 4). Usually these specialized facilities provide access to physicians with expertise in complicat‐ ed gynaecologic oncology surgical procedures necessary for appropriate surgical manage‐ ment of ovarian cancer patients.

Studies evaluating physician specialization usually compare outcomes for patients operated by gynaecologic oncologists versus general gynaecologists versus general surgeons. Opera‐ tion by a gynaecologic oncologist was associated in most studies with better outcomes in terms of long-term survival (Table 5). It is likely that general surgeons are more likely to per‐ form emergency surgeries in advanced situations like bowel obstruction. However, the dif‐ ference in outcomes persisted even after adjusting for prognostic factors like the Charlson comorbidity score.


(1), Netherlands (3), Norway (1), Switzerland (1), UK (3), and Japan (1). Twenty-five unique studies report the impact of structure on outcomes, and 13 studies report the impact of vari‐

The 5-year overall survival rate is the indicator of most interest to clinicians caring for pa‐ tients with ovarian cancer. Other outcomes of interest include quality of life, patient satisfac‐ tion, and cost. However, when 5-year survival rates are so poor, surrogate outcomes, including progression-free survival (PFS), can be used to reflect small changes in outcomes that are important to patients and society. Changes in processes or structures that result in improved surrogate outcomes should eventually be reflected in improved 5-year survival

In 25 unique population-based studies of quality of care in ovarian cancer, structural varia‐ bles evaluated include a hospital's annual ovarian cancer surgical volume, physician annual ovarian cancer surgical volume, hospital type (university affiliated vs community hospital), and physician type (gynaecologic oncologist, general gynaecologist, or general surgeon).

Studies evaluating hospital volume demonstrate hospitals with higher volumes of ovarian cancer surgery per year are often associated with better long-term survival (Table 2). The improvement in overall survival did not appear to be a reflection of peri-operative deaths, because the 30 and 60-day mortality was not affected by hospital volume in the studies eval‐ uating those outcomes. The long-term survival advantage produced by high-volume hospi‐ tals is due to other differences in structures and processes of care in these institutions.

Studies evaluating physician volume did not demonstrate a uniform improvement in sur‐ vival when high-volume physicians operated on patients with ovarian cancer (Table 3).

In just over half of the studies identified, hospitals classified as teaching facilities or univer‐ sity hospitals were associated with better short and long-term survival outcomes (Table 4). Usually these specialized facilities provide access to physicians with expertise in complicat‐ ed gynaecologic oncology surgical procedures necessary for appropriate surgical manage‐

Studies evaluating physician specialization usually compare outcomes for patients operated by gynaecologic oncologists versus general gynaecologists versus general surgeons. Opera‐ tion by a gynaecologic oncologist was associated in most studies with better outcomes in terms of long-term survival (Table 5). It is likely that general surgeons are more likely to per‐ form emergency surgeries in advanced situations like bowel obstruction. However, the dif‐ ference in outcomes persisted even after adjusting for prognostic factors like the Charlson

Findings were inconclusive for both shorter and longer-term survival.

rates. Surrogate outcomes in ovarian cancer include PFS and 30 or 60-day mortality.

ous processes on outcomes. Included are 91,866 patients.

6 Ovarian Cancer - A Clinical and Translational Update

**4.1. Outcomes**

**4.2. Structure**

These studies are listed in Table 1.

ment of ovarian cancer patients.

comorbidity score.

n/a: not applicable—these studies used surrogate outcomes, \*the authors of this study reported it was underpowed to find an association between structure and survival

**Table 1.** Studies reporting on structural variables in relation to outcomes for ovarian cancer


Several studies have reported a link between structural variables (hospital volume, physi‐ cian volume, hospital type and physician specialization) and outcomes. Population-based studies published over the past ten years identify more consistent evidence linking in‐ creased hospital volume and increased physician specialization with long-term outcomes than for other structural variables. Surgery by a gynecologic oncologist appears to pro‐ vide superior outcomes in terms of long term survival. These studies pertain to the sur‐ gical management of patients with ovarian cancer. The single study looking at chemotherapy for ovarian cancer patients found no association between oncologist vol‐ ume of chemotherapy and outcomes.[11] Of note, no study demonstrated worse out‐ comes with higher volumes or specialization of hospitals or physicians. Some jurisdictions have used these findings to implement a strategy of centralization of sur‐ gery for ovarian cancer in an effort to improve quality of surgical care and outcomes.

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There are important limitations in this data. Not all studies were able to obtain individu‐ al data to allow adjustment for every important confounding variable which can impact survival. The majority of these studies were retrospective or dependant on accurate dataentry into databases. It is possible some of the advantages observed for type or volume of provider may be due to more diligent data-entry and documentation of patient demo‐ graphics, stage and treatment received. For example, teaching hospitals may have more accurate and detailed documentation of the surgical procedures provided to patients which may lead to an assumption that they provided more complete surgical care when in fact the differences were in documentation only. The use of re-operation as a surro‐ gate outcome is questionable when discussing physician type, since more specialized physicians are typically the ones making the decision to perform a second operation and this decision is more likely to occur if the primary surgery was performed by a less spe‐

Evidence-based guidelines on the surgical care of women with ovarian cancer generally rec‐ ommend hysterectomy, bilateral salpingo-oophorectomy, and omentectomy. In early-stage disease, staging should be performed, including cytology, peritoneal biopsies, and pelvic and para-aortic lymphadenectomy. In late-stage disease, debulking should be performed, including the removal of all macroscopic tumour. This sometimes requires the use of bowel resection, splenectomy, diaphragmatic and peritoneal stripping.[42-44] Adjuvant or neoad‐ juvant chemotherapy with a combination of a platinum and a taxane agent has been the standard of care for epithelial ovarian cancers over the past ten years.[45] Appropriate sur‐ gery and chemotherapy have a demonstrated impact on outcomes for ovarian cancer pa‐

Next we look at whether the processes evaluated in the literature are related to the four

tients and represent processes of care indicating quality.

structural variables reported, and whether these impact on survival.

[40,41]

cialized surgeon.

**4.3. Process**

**Table 2.** Relationship between hospital volume and patient outcomes


**Table 3.** Relationship between physician volume and patient outcomes


**Table 4.** Relationship between hospital type and patient outcomes


**Table 5.** Relationship between physician specialization and patient outcomes

Several studies have reported a link between structural variables (hospital volume, physi‐ cian volume, hospital type and physician specialization) and outcomes. Population-based studies published over the past ten years identify more consistent evidence linking in‐ creased hospital volume and increased physician specialization with long-term outcomes than for other structural variables. Surgery by a gynecologic oncologist appears to pro‐ vide superior outcomes in terms of long term survival. These studies pertain to the sur‐ gical management of patients with ovarian cancer. The single study looking at chemotherapy for ovarian cancer patients found no association between oncologist vol‐ ume of chemotherapy and outcomes.[11] Of note, no study demonstrated worse out‐ comes with higher volumes or specialization of hospitals or physicians. Some jurisdictions have used these findings to implement a strategy of centralization of sur‐ gery for ovarian cancer in an effort to improve quality of surgical care and outcomes. [40,41]

There are important limitations in this data. Not all studies were able to obtain individu‐ al data to allow adjustment for every important confounding variable which can impact survival. The majority of these studies were retrospective or dependant on accurate dataentry into databases. It is possible some of the advantages observed for type or volume of provider may be due to more diligent data-entry and documentation of patient demo‐ graphics, stage and treatment received. For example, teaching hospitals may have more accurate and detailed documentation of the surgical procedures provided to patients which may lead to an assumption that they provided more complete surgical care when in fact the differences were in documentation only. The use of re-operation as a surro‐ gate outcome is questionable when discussing physician type, since more specialized physicians are typically the ones making the decision to perform a second operation and this decision is more likely to occur if the primary surgery was performed by a less spe‐ cialized surgeon.

#### **4.3. Process**

**Outcomes Number of studies finding an**

8 Ovarian Cancer - A Clinical and Translational Update

DFS: disease-free survival

**outcomes**

**Table 2.** Relationship between hospital volume and patient outcomes

**Table 3.** Relationship between physician volume and patient outcomes

**Outcomes Number of studies finding an**

**Table 4.** Relationship between hospital type and patient outcomes

**Outcomes Number of studies finding an**

**Outcome Number of studies finding an**

**association between higher volume and improved**

**association between higher volume and improved outcomes**

**association between specialized hospitals and improved outcomes**

**association between increased physician specialization and improved outcomes**

**Table 5.** Relationship between physician specialization and patient outcomes

Overall survival 6 3 9 30-day mortality 1 1 2

Survival 1 2 3 30-day mortality 1 2 3 60-day mortality 0 1 1

Overall survival 4 3 7 30-day mortality 1 1 2

Overall survival 7 3 10 DFS 1 0 1 30-day mortality 0 2 2 60-day mortality 0 1 1

**Number of studies finding no association between volume**

**Number of studies finding no association between volume and**

**Number of studies finding no association between hospital**

**type and outcomes**

**Number of studies finding no association between physician specialization and outcomes**

**Total**

**Total**

**Total**

**Total**

**and outcomes**

**outcomes**

Evidence-based guidelines on the surgical care of women with ovarian cancer generally rec‐ ommend hysterectomy, bilateral salpingo-oophorectomy, and omentectomy. In early-stage disease, staging should be performed, including cytology, peritoneal biopsies, and pelvic and para-aortic lymphadenectomy. In late-stage disease, debulking should be performed, including the removal of all macroscopic tumour. This sometimes requires the use of bowel resection, splenectomy, diaphragmatic and peritoneal stripping.[42-44] Adjuvant or neoad‐ juvant chemotherapy with a combination of a platinum and a taxane agent has been the standard of care for epithelial ovarian cancers over the past ten years.[45] Appropriate sur‐ gery and chemotherapy have a demonstrated impact on outcomes for ovarian cancer pa‐ tients and represent processes of care indicating quality.

Next we look at whether the processes evaluated in the literature are related to the four structural variables reported, and whether these impact on survival.


**Number of studies finding an association between specialized hospitals and improved processes**

**Table 8.** Relationship between hospital type and evidence-based processes

**Processes Number of studies finding an**

and adjuvant chemotherapy in accordance with guidelines (Table 8).

**association between increased physician specialization and improved outcomes**

**Table 9.** Relationship between physician specialization and evidence-based processes

Optimal debulking 6 0 6 LND 5 0 5 Re-operation 3 0 3 Adjuvant chemotherapy 4 0 4

Physician specialization (ie., gynaecologic oncologist vs general gynaecologist vs general surgeon) was also associated with appropriate surgery and adjuvant chemotherapy in ac‐

In summary, 13 population-based studies involving 22,255 patients across 3 continents linked processes of care to improved survival. The relationship of important processes of care with survival is so clear that this work that has led to defining quality indicators for the treatment of ovarian cancer care. In Ontario, Canada, Gagliardi and colleagues[40] used the Delphi technique to define quality indicators. More recently, Verleye and the EORTC has defined and set surgical benchmarks for quality care in ovarian cancer (Table

LND: lymph node dissection

LND: lymph node dissection

11., Appendix).[46]

cordance with guidelines (Table 9).

Optimal debulking 3 0 3 LND 6 0 6 Re-operation 1 0 1 Adjuvant chemotherapy 5 0 5

Type of hospital (ie. teaching versus non-teaching, academic versus community) where sur‐ gery for ovarian cancer is performed was clearly associated with more appropriate surgery

**Number of studies finding no association between hospital**

In what Setting Should Women with Ovarian Cancer Receive Care?

**Number of studies finding no association between physician specialization and outcomes**

**Total**

11

http://dx.doi.org/10.5772/52936

**Total**

**type and processes**

**Table 6.** Relationship between hospital volume and evidence-based processes

Higher hospital volumes of ovarian cancer surgery were associated with better compliance to process steps in the optimal care of women with ovarian cancer (Table 6). These processes included: surgery according to guidelines (optimal debulking, lymph node dissection) and use of adjuvant chemotherapy.


**Table 7.** Relationship between physician volume and evidence-based processes

Surgery by physicians with higher volumes of ovarian cancer surgeries was also associated with better compliance to process steps such as surgery according to guidelines and use of adjuvant chemotherapy (Table 7).


**Table 8.** Relationship between hospital type and evidence-based processes

**Number of studies finding an association between higher volume**

Adequate surgery 1 1 2 Optimal debulking 5 1 6 LND 3 0 3 Re-operation 2 0 2 Length of Stay 2 0 2 Complications 0 1 1 Adjuvant chemotherapy 1 0 1

Higher hospital volumes of ovarian cancer surgery were associated with better compliance to process steps in the optimal care of women with ovarian cancer (Table 6). These processes included: surgery according to guidelines (optimal debulking, lymph node dissection) and

Surgery by physicians with higher volumes of ovarian cancer surgeries was also associated with better compliance to process steps such as surgery according to guidelines and use of

**Number of studies finding no association between volume**

**Number of studies finding no association between volume**

**and processes**

**Total**

**Total**

**and processes**

**and improved processes**

**Table 6.** Relationship between hospital volume and evidence-based processes

**Table 7.** Relationship between physician volume and evidence-based processes

**Number of studies finding an association between higher volume and improved processes**

LND 2 0 2 Optimal debulking 1 0 1 Length of stay 1 0 1 Re-operation 2 0 2 Adjuvant chemotherapy 1 0 1 Complications 0 1 1 Length of Stay 1 0 1

LND: lymph node dissection

LND: lymph node dissection

adjuvant chemotherapy (Table 7).

use of adjuvant chemotherapy.

10 Ovarian Cancer - A Clinical and Translational Update

Type of hospital (ie. teaching versus non-teaching, academic versus community) where sur‐ gery for ovarian cancer is performed was clearly associated with more appropriate surgery and adjuvant chemotherapy in accordance with guidelines (Table 8).


**Table 9.** Relationship between physician specialization and evidence-based processes

Physician specialization (ie., gynaecologic oncologist vs general gynaecologist vs general surgeon) was also associated with appropriate surgery and adjuvant chemotherapy in ac‐ cordance with guidelines (Table 9).

In summary, 13 population-based studies involving 22,255 patients across 3 continents linked processes of care to improved survival. The relationship of important processes of care with survival is so clear that this work that has led to defining quality indicators for the treatment of ovarian cancer care. In Ontario, Canada, Gagliardi and colleagues[40] used the Delphi technique to define quality indicators. More recently, Verleye and the EORTC has defined and set surgical benchmarks for quality care in ovarian cancer (Table 11., Appendix).[46]


**5.1. Pathology assessment**

in research.[54]

**5.2. Multidisciplinary care**

When making a diagnosis of ovarian cancer, the histology may be assessed by a pathologist, a pathologist with interest and experience in gynaecologic malignancies, or a subspecialist gynaecologic pathologist. Heatley[53] defines a pathologist as someone who has completed training and passed the appropriate examinations. A pathologist with a special interest (PSI) is a general pathologist who takes the lead in a subspecialty area within their department such as gynaecological pathology, attending meetings of specialist societies, participating in the appropriate subspecialist external quality assurance scheme, providing specialist opin‐ ions for colleagues in the department, and on occasion, neighbouring departments. A sub‐ specialist pathologist is a pathologist with a special interest but who now, possibly after a period working as a general pathologist, devotes all or the vast majority of their time to one area of practice.[53] Subspecialisation leads to standardisation of pathology reports and im‐ proved communication of findings, participation in multidisciplinary tumour board meet‐ ings, enhanced knowledge and standards, decreased turnaround times, quality assurance of diagnoses, improved quality of resident training, ability to distinguish appropriate variation from the standard of care, and advancement of academic knowledge through participation

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In gynaecologic oncology, there are several studies reporting up to a 16.9% discrepancy with the referral diagnosis when a PSI or a subspecialist gynaecologic pathologist provides a re‐ view of the original pathology.[55] In 4.7% -12% of cases there is a change in diagnosis which has a major therapeutic or prognostic implication.[55-58] Although these findings were from studies including all gynaecologic malignancies rather than ovarian cancer spe‐ cifically, they demonstrate subspecialist pathology review has an important role to play in the care of patients with ovarian cancer. Verleye and colleagues[59] found that pathology re‐ ports for ovarian cancer surgery originating from high-volume centres and academic hospi‐ tals are of higher quality than those originating from lower volume or non-academic centres. The availability of subspecialist gynaecologic pathologists may be one structural aspect of care in these centres leading to better outcomes. The impact of expert pathology review in

ovarian cancer needs to be evaluated as a process step that could impact survival.

Multidisciplinary care is an integrated team-based approach to cancer care where medical and allied health care professionals consider all relevant treatment options and collabora‐ tively develop an individual treatment and care plan for each patient. Evidence in oncolo‐ gy suggests that multidisciplinary care leads to improved survival and quality of life, satisfaction with treatment, and mental well-being of clinicians.[60] An important compo‐ nent of multidisciplinary care is availability of regularly scheduled tumour board meet‐ ings[61] with participation of gynaecologic oncologists, pathologists, radiologists, radiation and medical oncologists, and allied health professionals with a special interest in care of gynaecologic oncology patients. Tumour board conferencing in Auckland City Hospital from 2005-2006 led to a 5.9% rate of major changes in patient management.[62] This re‐ sulted from radiologic review (major discrepancy rate 1.4%) and pathology review (major

**Table 10.** Studies reporting on process variables in relation to outcomes in ovarian cancer

#### **5. Processes or structures of care that require further evaluation**

There are many processes considered by experts to be important in the care of women with ovarian cancer. These process variables have face validity but have not yet been clearly eval‐ uated for their impact on outcomes in ovarian cancer. Additionally, the organizational struc‐ ture for care provision is a complex construct; it is unclear what components contribute most positively to outcomes. We wish to focus on three variables that may or may not be related to survival but may impact treatment and decision making.

#### **5.1. Pathology assessment**

**Study Country Data Source Number of**

Chan 2008[47] USA Retrospective

12 Ovarian Cancer - A Clinical and Translational Update

Elit 2006[11] Canada Retrospective

Elit 2008[34] Canada Retrospective

Engelen 2006[26]Netherlands Retrospective

Fairfield 2010[48]USA Retrospective

Grossi 2002[18] Australia Retrospective

Maas 2005[50] Netherlands Retrospective

Hershman 2004[49]

Petignat 2007[51]

Rochon 2011[39,52]

Bailey 2006[24] UK Prospective cohort 361 X

database

database

database

review

database

review

database

database

database

database

**Table 10.** Studies reporting on process variables in relation to outcomes in ovarian cancer

related to survival but may impact treatment and decision making.

**5. Processes or structures of care that require further evaluation**

There are many processes considered by experts to be important in the care of women with ovarian cancer. These process variables have face validity but have not yet been clearly eval‐ uated for their impact on outcomes in ovarian cancer. Additionally, the organizational struc‐ ture for care provision is a complex construct; it is unclear what components contribute most positively to outcomes. We wish to focus on three variables that may or may not be

Paulsen 2006[32] Norway Prospective registry 198 X X

Marth 2009[36] Austria Prospective cohort 1,948 X

USA Retrospective

Switzerland Retrospective

Germany Retrospective

database + chart

database + chart

**patients**

8,372 X

632 X

4,589 X

434 X

50 X

**Which process variables affected**

Surgery Chemo

**survival?**

2,502 X X

1,341 X X

236 X

1,116 X X

476 X X

When making a diagnosis of ovarian cancer, the histology may be assessed by a pathologist, a pathologist with interest and experience in gynaecologic malignancies, or a subspecialist gynaecologic pathologist. Heatley[53] defines a pathologist as someone who has completed training and passed the appropriate examinations. A pathologist with a special interest (PSI) is a general pathologist who takes the lead in a subspecialty area within their department such as gynaecological pathology, attending meetings of specialist societies, participating in the appropriate subspecialist external quality assurance scheme, providing specialist opin‐ ions for colleagues in the department, and on occasion, neighbouring departments. A sub‐ specialist pathologist is a pathologist with a special interest but who now, possibly after a period working as a general pathologist, devotes all or the vast majority of their time to one area of practice.[53] Subspecialisation leads to standardisation of pathology reports and im‐ proved communication of findings, participation in multidisciplinary tumour board meet‐ ings, enhanced knowledge and standards, decreased turnaround times, quality assurance of diagnoses, improved quality of resident training, ability to distinguish appropriate variation from the standard of care, and advancement of academic knowledge through participation in research.[54]

In gynaecologic oncology, there are several studies reporting up to a 16.9% discrepancy with the referral diagnosis when a PSI or a subspecialist gynaecologic pathologist provides a re‐ view of the original pathology.[55] In 4.7% -12% of cases there is a change in diagnosis which has a major therapeutic or prognostic implication.[55-58] Although these findings were from studies including all gynaecologic malignancies rather than ovarian cancer spe‐ cifically, they demonstrate subspecialist pathology review has an important role to play in the care of patients with ovarian cancer. Verleye and colleagues[59] found that pathology re‐ ports for ovarian cancer surgery originating from high-volume centres and academic hospi‐ tals are of higher quality than those originating from lower volume or non-academic centres. The availability of subspecialist gynaecologic pathologists may be one structural aspect of care in these centres leading to better outcomes. The impact of expert pathology review in ovarian cancer needs to be evaluated as a process step that could impact survival.

#### **5.2. Multidisciplinary care**

Multidisciplinary care is an integrated team-based approach to cancer care where medical and allied health care professionals consider all relevant treatment options and collabora‐ tively develop an individual treatment and care plan for each patient. Evidence in oncolo‐ gy suggests that multidisciplinary care leads to improved survival and quality of life, satisfaction with treatment, and mental well-being of clinicians.[60] An important compo‐ nent of multidisciplinary care is availability of regularly scheduled tumour board meet‐ ings[61] with participation of gynaecologic oncologists, pathologists, radiologists, radiation and medical oncologists, and allied health professionals with a special interest in care of gynaecologic oncology patients. Tumour board conferencing in Auckland City Hospital from 2005-2006 led to a 5.9% rate of major changes in patient management.[62] This re‐ sulted from radiologic review (major discrepancy rate 1.4%) and pathology review (major discrepancy rate of 4.5%) which led to identification of major diagnostic discrepancies. However, they could not quantify how the changes in diagnosis and management might impact patient outcomes. Santoso did a comparison of the initial gynecologic cancer diag‐ nosis and management plan to the diagnosis and management plan after discussion at a multidisciplinary tumor board meeting. They showed that 6.9% of cases discussed at tu‐ mor board had changes made to the diagnosis or plan, and in 5% there were major changes in treatment.[63] The most convincing research suggesting care by a multidiscipli‐ nary team is a process that improves outcomes was published by Junor and colleagues us‐ ing population-based data from Scotland. In a retrospective analysis of all 533 cases of ovarian cancer diagnosed in Scotland in 1987, referral to a multidisciplinary team was one of five factors significantly associated with improved 5 yr survival after adjusting for pa‐ tient and disease characteristics (hazard ratio 0.60, p<0.001).[64]

processes by the involvement of highly regarded opinion leaders providing education. More consistent improvement in processes of care has been noted using the audit and feedback system.[68] These approaches have been variously referred to as quality assessment, quality management quality improvement and knowledge translation. In this paper, we refer to quality assessment as the audit process whereby performance is measured and compared with a reference standard. Quality improvement includes the steps taken to actively change

In what Setting Should Women with Ovarian Cancer Receive Care?

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15

Quality assurance and monitoring of outcomes is essential to allow for quality improvement initiatives. Regions, hospitals, and care providers must understand which outcomes are not reaching a targeted standard in order to identify structures and processes which may im‐ prove outcomes. Initiatives such as the International Cancer Benchmarking Partnership[69] have used population-based registry data to identify significant discrepancies in survival for women with ovarian cancer based on geographic location. Striking differences were ob‐ served, with women in Australia and Canada having significantly longer survival than

Measuring the quality of surgical procedures has lagged behind quality-assurance initiatives in other areas because of the difficulty in identifying parameters to evaluate.[71] Early stud‐ ies suggested operative morbidity and mortality, adequacy of resection, local recurrence and survival as parameters to measure surgical quality.[71] However several of these factors are also highly influenced by the use of appropriate adjuvant therapy. Several programs have now begun systematically tracking outcomes for surgical oncology patients in an effort to

One such program is the American College of Surgeons (ACS) National Surgical Quality Improvement Program (NSQIP).[72] This is a nationally validated, multi-specialty, risk-ad‐ justed 30-day outcomes measurement program which originated in the Veterans Health Administration in 1991. Since 2004, NSQIP has been expanding and now includes more than 400 hospitals in the US, Canada, Lebanon and the UAE. The aim of the program is to provide institutions and surgeons with 30-day outcomes which can be used to compare performance to other institutions. Risk adjustment incorporates pre-operative comorbidi‐ ties and intra-operative risk factors using hierarchical modelling. Twice per year, institu‐ tions are given a report with their risk-adjusted outcomes in the form of odds ratios, which can be used for bench-marking. After implementation of NSQIP in 10 Tennessee hospitals, significantly fewer surgical site infections, failed grafts and flaps, episodes of acute renal failure, and prolonged ventilation of more than 48 hours was achieved.[73] The ACS evaluated outcomes across all participating institutions from 2005 to 2007 and found 66% of hospitals showed improvement in 30-day mortality and 82% of hospitals achieved a reduction in complications after enrollment in the NSQIP program.[74] These improvements also led to significant cost savings. The remarkable success of this program

identify areas where quality improvement measures should be implemented.

practice to improve adherence to processes and to improve outcomes.

women in the UK and Denmark after adjusting for stage.[70]

**7. Quality assurance and monitoring**

will likely lead to further expansion.

#### **5.3. Institutional participation in clinical trials**

Several studies have identified clinical trial participation as an institutional marker of quali‐ ty care. In 1994, Stiller published a review of several cancer disease sites and found that across disease sites, patients treated as part of a clinical trial had better outcomes.[65] du Bois and colleagues evaluated outcomes in a population-based cohort of patients diagnosed with ovarian cancer in Germany in 2001.[52] After adjusting for disease stage, patients treat‐ ed in an institution participating in multi-centre clinical trials had improved overall survival (35 months vs 25 months for patients with stage III-IV ovarian cancer treated at participating vs non-participating hospitals).[39,52] Notably, patients treated in participating hospitals had better outcomes even if they were not themselves participating in a trial. Patients treat‐ ed in hospitals participating in trials were more likely to receive care in accordance with clinical practice guidelines including staging, debulking and combination chemotherapy where appropriate.[39] Trial participation at an individual patient level may indicate good performance status that can, in and of itself, lead to better outcomes. However, it appears all patients treated at hospitals participating in trials may benefit from improved outcomes. This is likely due to differences in processes of care at these institutions.

#### **6. What does all this mean?**

When geographic variation in outcomes exist at a population level, there are opportunities to assess whether changes in structures or processes of care could improve outcomes. There have been several strategies to improve outcome. One is to standardize care using evidencebased guidelines and techniques to optimize processes like a structured care path, whether in a paper chart or as part of an electronic medical record. Another approach to try to im‐ prove outcomes at a population level has been to centralize care. In some situations where care requires an experienced surgical team and highly developed perio-operative care, such as for the surgical management of pancreatic cancer, there is evidence that centralization of care to high-volume centres decreases 30-mortality.[66] However, not all reports are consis‐ tent with this finding.[67] Another strategy to improve outcomes is to focus on improving processes by the involvement of highly regarded opinion leaders providing education. More consistent improvement in processes of care has been noted using the audit and feedback system.[68] These approaches have been variously referred to as quality assessment, quality management quality improvement and knowledge translation. In this paper, we refer to quality assessment as the audit process whereby performance is measured and compared with a reference standard. Quality improvement includes the steps taken to actively change practice to improve adherence to processes and to improve outcomes.

#### **7. Quality assurance and monitoring**

discrepancy rate of 4.5%) which led to identification of major diagnostic discrepancies. However, they could not quantify how the changes in diagnosis and management might impact patient outcomes. Santoso did a comparison of the initial gynecologic cancer diag‐ nosis and management plan to the diagnosis and management plan after discussion at a multidisciplinary tumor board meeting. They showed that 6.9% of cases discussed at tu‐ mor board had changes made to the diagnosis or plan, and in 5% there were major changes in treatment.[63] The most convincing research suggesting care by a multidiscipli‐ nary team is a process that improves outcomes was published by Junor and colleagues us‐ ing population-based data from Scotland. In a retrospective analysis of all 533 cases of ovarian cancer diagnosed in Scotland in 1987, referral to a multidisciplinary team was one of five factors significantly associated with improved 5 yr survival after adjusting for pa‐

Several studies have identified clinical trial participation as an institutional marker of quali‐ ty care. In 1994, Stiller published a review of several cancer disease sites and found that across disease sites, patients treated as part of a clinical trial had better outcomes.[65] du Bois and colleagues evaluated outcomes in a population-based cohort of patients diagnosed with ovarian cancer in Germany in 2001.[52] After adjusting for disease stage, patients treat‐ ed in an institution participating in multi-centre clinical trials had improved overall survival (35 months vs 25 months for patients with stage III-IV ovarian cancer treated at participating vs non-participating hospitals).[39,52] Notably, patients treated in participating hospitals had better outcomes even if they were not themselves participating in a trial. Patients treat‐ ed in hospitals participating in trials were more likely to receive care in accordance with clinical practice guidelines including staging, debulking and combination chemotherapy where appropriate.[39] Trial participation at an individual patient level may indicate good performance status that can, in and of itself, lead to better outcomes. However, it appears all patients treated at hospitals participating in trials may benefit from improved outcomes.

When geographic variation in outcomes exist at a population level, there are opportunities to assess whether changes in structures or processes of care could improve outcomes. There have been several strategies to improve outcome. One is to standardize care using evidencebased guidelines and techniques to optimize processes like a structured care path, whether in a paper chart or as part of an electronic medical record. Another approach to try to im‐ prove outcomes at a population level has been to centralize care. In some situations where care requires an experienced surgical team and highly developed perio-operative care, such as for the surgical management of pancreatic cancer, there is evidence that centralization of care to high-volume centres decreases 30-mortality.[66] However, not all reports are consis‐ tent with this finding.[67] Another strategy to improve outcomes is to focus on improving

tient and disease characteristics (hazard ratio 0.60, p<0.001).[64]

This is likely due to differences in processes of care at these institutions.

**5.3. Institutional participation in clinical trials**

14 Ovarian Cancer - A Clinical and Translational Update

**6. What does all this mean?**

Quality assurance and monitoring of outcomes is essential to allow for quality improvement initiatives. Regions, hospitals, and care providers must understand which outcomes are not reaching a targeted standard in order to identify structures and processes which may im‐ prove outcomes. Initiatives such as the International Cancer Benchmarking Partnership[69] have used population-based registry data to identify significant discrepancies in survival for women with ovarian cancer based on geographic location. Striking differences were ob‐ served, with women in Australia and Canada having significantly longer survival than women in the UK and Denmark after adjusting for stage.[70]

Measuring the quality of surgical procedures has lagged behind quality-assurance initiatives in other areas because of the difficulty in identifying parameters to evaluate.[71] Early stud‐ ies suggested operative morbidity and mortality, adequacy of resection, local recurrence and survival as parameters to measure surgical quality.[71] However several of these factors are also highly influenced by the use of appropriate adjuvant therapy. Several programs have now begun systematically tracking outcomes for surgical oncology patients in an effort to identify areas where quality improvement measures should be implemented.

One such program is the American College of Surgeons (ACS) National Surgical Quality Improvement Program (NSQIP).[72] This is a nationally validated, multi-specialty, risk-ad‐ justed 30-day outcomes measurement program which originated in the Veterans Health Administration in 1991. Since 2004, NSQIP has been expanding and now includes more than 400 hospitals in the US, Canada, Lebanon and the UAE. The aim of the program is to provide institutions and surgeons with 30-day outcomes which can be used to compare performance to other institutions. Risk adjustment incorporates pre-operative comorbidi‐ ties and intra-operative risk factors using hierarchical modelling. Twice per year, institu‐ tions are given a report with their risk-adjusted outcomes in the form of odds ratios, which can be used for bench-marking. After implementation of NSQIP in 10 Tennessee hospitals, significantly fewer surgical site infections, failed grafts and flaps, episodes of acute renal failure, and prolonged ventilation of more than 48 hours was achieved.[73] The ACS evaluated outcomes across all participating institutions from 2005 to 2007 and found 66% of hospitals showed improvement in 30-day mortality and 82% of hospitals achieved a reduction in complications after enrollment in the NSQIP program.[74] These improvements also led to significant cost savings. The remarkable success of this program will likely lead to further expansion.

#### **8. Quality improvement**

Quality improvement naturally follows from quality assessment. Review of performance in terms of adherence to best-practices (processes of care) in a methodologically rigorous and transparent manner (quality assessment) can lead to improvement in outcomes if interven‐ tions are undertaken to improve areas of weakness in performance. Interventions based on quality assurance data attempt to improve processes of care in order to improve outcomes. A framework for quality improvement could include the following steps:[75]

2004-2008 and 5-year survival in 24%, 34% and 36% of patients in the three time periods. Changes in both structures and processes of care were achieved using this quality manage‐

In what Setting Should Women with Ovarian Cancer Receive Care?

http://dx.doi.org/10.5772/52936

17

A quality improvement program for the surgical care of patients with advanced ovarian cancer was implemented at the Mayo Clinic using an audit and feedback approach, with the aim of increasing the proportion of patients debulked to microscopic residual disease.[44] A surgical complexity score was developed to categorize the aggressiveness of the surgical ap‐ proach.[80] The quality improvement program consisted of weekly conferences where pa‐ tient outcomes and treatment approaches were discussed, confidential benchmarking allowing individual surgeons to see their rates of complete surgical debulking in compari‐ son to peers, teaching fellows and staff how to perform techniques needed for complete de‐ bulking, and intra-operative mentoring of staff and fellows by surgeons experienced in advanced procedures. After the quality improvement program was implemented, rates of

Knowledge translation is the science of moving knowledge into action.[81] Several studies across various disciplines in medicine have demonstrated many patients do not receive care known to improve outcomes.[81,82] One of the first groups to show this in ovarian cancer was Munstedt and colleagues who found a large proportion of patients treated in Hesse, Germany between 1997 and 2001 did not receive care recommended in national guidelines. [83] Knowledge translation aims to bridge the gap between what is known from research, and implementation of this knowledge in an effort to improve outcomes for patients and ef‐

Knowledge translation has been described as a cycle, where a clinical problem is identified (possibly by quality assurance or monitoring efforts), processes of care are identified from research to address the problem, these processes are adapted to the local context and any barriers to implementation are identified and addressed, and the new processes are imple‐ mented. After implementation, adherence to the process is monitored, and final patient out‐ comes are evaluated.[81] Evaluation of outcomes and monitoring of processes may then identify additional clinical problems. If no evidence-based solution to the problem is identi‐ fied, this leads to a need for additional research. In this way, new research informs clinical

practice, and problems from clinical practice help to identify research priorities.[82]

A major focus of knowledge translation research is finding ways to change clinician and pa‐ tient behaviour given the results of research. Simply publishing new findings in peer-re‐ viewed journals, a method termed 'diffusion', is not adequate for wide-spread adoption of new processes.[84] Other methods that have been investigated include audit and feedback, [85] educational outreach by local opinion leaders,[86] and clinical decision support and re‐ minder systems which can be integrated into computer-based patient-care platforms.[87] Audit and feedback, such as the ACS NSQIP or ASCO QOPI programs, are one of the most

ment system, leading to improved survival for patients.[79]

debulking to microscopic disease increased from 31% to 43%.

**9. Knowledge translation**

ficiency for the health care system.[81]


The American Society of Clinical Oncology (ASCO) initiated the Quality Oncology Practice Initiative (QOPI)[76] for US-based Hematology-Oncology practices in order to improve quality of cancer care by using measurement and feedback and by providing improvement tools. Processes of care indicative of quality were identified by a group of oncologists using consensus and clinical practice guidelines.[77] QOPI provides individual care providers with quality of care benchmarking information twice per year, allowing clinicians to make improvements within their own practices. Implementation of QOPI and sharing results with physicians at one academic oncology centre in the US led to significant improvements in several areas of quality.[78] Although this program is only available to medical oncology practices in the US, it serves as a good example of how measurement and feedback can lead to improvement in quality of care.

A quality management program was implemented in one German academic oncology cen‐ tre in 2001 with the aim of improving the quality of surgery provided to patients with ovari‐ an cancer.[79] The components of the quality management system included establishment of a prospective tumour registry, creation and training of dedicated surgical teams operating on patients with advanced ovarian cancer, inter-disciplinary surgical care, intra-operative second opinion by another gynecologic oncologist if the first surgeon did not believe de‐ bulking to microscopic residual disease was attainable, interdisciplinary management of complications, and quality conferences including assessment and benchmarking of morbidi‐ ty and survival outcomes. This effort, along with a significant increase in the volume of ovarian cancer surgery performed at this centre over time, led to a significant improvement in processes and outcomes. Debulking to microscopic residual disease increased from 33% in 1997-2000, and 47% in 2001-2003, to 62% in 2004-2008. This led to median survival of 26 months for patients treated in 1997-2000, 37 months in 2000-2003 and to 45 months in 2004-2008 and 5-year survival in 24%, 34% and 36% of patients in the three time periods. Changes in both structures and processes of care were achieved using this quality manage‐ ment system, leading to improved survival for patients.[79]

A quality improvement program for the surgical care of patients with advanced ovarian cancer was implemented at the Mayo Clinic using an audit and feedback approach, with the aim of increasing the proportion of patients debulked to microscopic residual disease.[44] A surgical complexity score was developed to categorize the aggressiveness of the surgical ap‐ proach.[80] The quality improvement program consisted of weekly conferences where pa‐ tient outcomes and treatment approaches were discussed, confidential benchmarking allowing individual surgeons to see their rates of complete surgical debulking in compari‐ son to peers, teaching fellows and staff how to perform techniques needed for complete de‐ bulking, and intra-operative mentoring of staff and fellows by surgeons experienced in advanced procedures. After the quality improvement program was implemented, rates of debulking to microscopic disease increased from 31% to 43%.

#### **9. Knowledge translation**

**8. Quality improvement**

16 Ovarian Cancer - A Clinical and Translational Update

**3.** Select team members

**5.** Collect data for selected markers

to improvement in quality of care.

**7.** Re-evaluate, modify and repeat the steps

Quality improvement naturally follows from quality assessment. Review of performance in terms of adherence to best-practices (processes of care) in a methodologically rigorous and transparent manner (quality assessment) can lead to improvement in outcomes if interven‐ tions are undertaken to improve areas of weakness in performance. Interventions based on quality assurance data attempt to improve processes of care in order to improve outcomes.

A framework for quality improvement could include the following steps:[75]

**6.** Select and operationalize interventions to achieve improvements in markers

The American Society of Clinical Oncology (ASCO) initiated the Quality Oncology Practice Initiative (QOPI)[76] for US-based Hematology-Oncology practices in order to improve quality of cancer care by using measurement and feedback and by providing improvement tools. Processes of care indicative of quality were identified by a group of oncologists using consensus and clinical practice guidelines.[77] QOPI provides individual care providers with quality of care benchmarking information twice per year, allowing clinicians to make improvements within their own practices. Implementation of QOPI and sharing results with physicians at one academic oncology centre in the US led to significant improvements in several areas of quality.[78] Although this program is only available to medical oncology practices in the US, it serves as a good example of how measurement and feedback can lead

A quality management program was implemented in one German academic oncology cen‐ tre in 2001 with the aim of improving the quality of surgery provided to patients with ovari‐ an cancer.[79] The components of the quality management system included establishment of a prospective tumour registry, creation and training of dedicated surgical teams operating on patients with advanced ovarian cancer, inter-disciplinary surgical care, intra-operative second opinion by another gynecologic oncologist if the first surgeon did not believe de‐ bulking to microscopic residual disease was attainable, interdisciplinary management of complications, and quality conferences including assessment and benchmarking of morbidi‐ ty and survival outcomes. This effort, along with a significant increase in the volume of ovarian cancer surgery performed at this centre over time, led to a significant improvement in processes and outcomes. Debulking to microscopic residual disease increased from 33% in 1997-2000, and 47% in 2001-2003, to 62% in 2004-2008. This led to median survival of 26 months for patients treated in 1997-2000, 37 months in 2000-2003 and to 45 months in

**1.** Debate and select values and goals that will inform the effort

**2.** Select a clinical area requiring improvement

**4.** Select relevant quality markers for improvement

Knowledge translation is the science of moving knowledge into action.[81] Several studies across various disciplines in medicine have demonstrated many patients do not receive care known to improve outcomes.[81,82] One of the first groups to show this in ovarian cancer was Munstedt and colleagues who found a large proportion of patients treated in Hesse, Germany between 1997 and 2001 did not receive care recommended in national guidelines. [83] Knowledge translation aims to bridge the gap between what is known from research, and implementation of this knowledge in an effort to improve outcomes for patients and ef‐ ficiency for the health care system.[81]

Knowledge translation has been described as a cycle, where a clinical problem is identified (possibly by quality assurance or monitoring efforts), processes of care are identified from research to address the problem, these processes are adapted to the local context and any barriers to implementation are identified and addressed, and the new processes are imple‐ mented. After implementation, adherence to the process is monitored, and final patient out‐ comes are evaluated.[81] Evaluation of outcomes and monitoring of processes may then identify additional clinical problems. If no evidence-based solution to the problem is identi‐ fied, this leads to a need for additional research. In this way, new research informs clinical practice, and problems from clinical practice help to identify research priorities.[82]

A major focus of knowledge translation research is finding ways to change clinician and pa‐ tient behaviour given the results of research. Simply publishing new findings in peer-re‐ viewed journals, a method termed 'diffusion', is not adequate for wide-spread adoption of new processes.[84] Other methods that have been investigated include audit and feedback, [85] educational outreach by local opinion leaders,[86] and clinical decision support and re‐ minder systems which can be integrated into computer-based patient-care platforms.[87] Audit and feedback, such as the ACS NSQIP or ASCO QOPI programs, are one of the most effective methods for behaviour change in clinicians.[68,88] An excellent overview of these methods has been published by Brouwers and colleagues, who performed a review of sys‐ tematic reviews on knowledge translation interventions used in cancer control.[68] The sci‐ ence of knowledge translation is relatively new. As research methods continue to improve, strategies are expected to be refined.

the science of moving knowledge into action, and encompasses both quality assurance and quality improvement. The concepts underlying quality of care are essential information for health care providers caring for women with ovarian cancer given the current global focus

Early-stage epithelial ovarian cancer -Percent of patients with a suspicious ovarian mass undergoing staging

patient-related reason for delay

rupture before or during surgery

patient-related reason for delay

documented in the operation notes

the end of the operation, was achieved

optimal debulking feasible

**Table 11.** EORTC benchmarks for quality surgical care in ovarian cancer[46]

adhesions, percent of dense adhesions biopsied

high risk features

be malignant performed through a vertical incision

laparotomy within 1 month after decision to treat or documented clinical or

In what Setting Should Women with Ovarian Cancer Receive Care?

http://dx.doi.org/10.5772/52936

19


Percent of performed staging laparotomies in which all of the following procedures are included: total hysterectomy, bilateral salpingo-oophorectomy, cytology of the peritoneal cavity, infracolic omentectomy, random peritoneal biopsies and systematic pelvic and para-aortic lymphadenectomy if medium or

Percent of surgery reports with documented presence or absence of cyst

Percent of surgery reports with documented presence or absence of dense

Percent of patients with advanced-stage ovarian cancer undergoing debulking laparotomy within 31 days after decision to treat or documented clinical or

Percent of patients undergoing debulking surgery with the spread of disease

Percent of debulking operations including a hysterectomy, bilateral salpingooophorectomy and infracolic omentectomy when the surgeon considers

Percent of debulking operations for advanced ovarian cancer at the end of which complete cytoreduction, defined as no macroscopic residual disease at

Percent of debulking operations including a pelvic and para-aortic lymphadenectomy when otherwise complete debulking has been achieved percent of debulking operations for which the size and location of residual disease at the end of the operation is documented in the operation notes

fully assessed for operability at the start of study and initial findings

on outcomes and value for money in health care systems.

**Appendix**

Primary debulking surgery in advanced-stage epithelial ovarian

cancer

**Figure 2.** Flow diagram for study selection

#### **10. Conclusion**

Women with ovarian cancer should be treated in institutions providing high quality care. Quality of care can be evaluated by examining the processes and structures of care leading to improved outcomes such as survival and quality of life.

In the US, there is a trend to link reimbursement for hospitals and care providers to clinical outcomes in an effort to improve quality of care.[72] Because of financial pressures in the health care system, this trend is expected to continue, since improvement in several metrics used to identify quality surgical care (such as decreased surgical site infections) can save a significant amount of money. Whether health systems are achieving value for money can only be assessed if performance is measured in a systematic way. Tracking outcomes with the use of population-based registries is an essential component of quality assurance, which allows for comparison of outcomes across jurisdictions.[71] Identifying variations in out‐ comes can then trigger specific quality improvement initiatives. Knowledge translation is the science of moving knowledge into action, and encompasses both quality assurance and quality improvement. The concepts underlying quality of care are essential information for health care providers caring for women with ovarian cancer given the current global focus on outcomes and value for money in health care systems.

### **Appendix**

effective methods for behaviour change in clinicians.[68,88] An excellent overview of these methods has been published by Brouwers and colleagues, who performed a review of sys‐ tematic reviews on knowledge translation interventions used in cancer control.[68] The sci‐ ence of knowledge translation is relatively new. As research methods continue to improve,

Women with ovarian cancer should be treated in institutions providing high quality care. Quality of care can be evaluated by examining the processes and structures of care leading

In the US, there is a trend to link reimbursement for hospitals and care providers to clinical outcomes in an effort to improve quality of care.[72] Because of financial pressures in the health care system, this trend is expected to continue, since improvement in several metrics used to identify quality surgical care (such as decreased surgical site infections) can save a significant amount of money. Whether health systems are achieving value for money can only be assessed if performance is measured in a systematic way. Tracking outcomes with the use of population-based registries is an essential component of quality assurance, which allows for comparison of outcomes across jurisdictions.[71] Identifying variations in out‐ comes can then trigger specific quality improvement initiatives. Knowledge translation is

strategies are expected to be refined.

18 Ovarian Cancer - A Clinical and Translational Update

**Figure 2.** Flow diagram for study selection

to improved outcomes such as survival and quality of life.

**10. Conclusion**


**Table 11.** EORTC benchmarks for quality surgical care in ovarian cancer[46]

#### **Author details**

Laurie Elit1,2 and Clare Reade2,3

1 Dept of Obstetrics and Gynecology, Division of Gynecologic Oncology, McMaster Univer‐ sity, Hamilton, Canada

[11] Elit L, Chartier C, Oza A, Hirte H, Levine M, Paszat L. Outcomes for systemic thera‐ py in women with ovarian cancer. Gynecol Oncol. Nov 2006;103(2):554-558.

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21

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[14] Stockton D, Davies T. Multiple cancer site comparison of adjusted survival by hospi‐ tal of treatment: an East Anglian study. Br J Cancer. Jan 2000;82(1):208-212.

[15] Olaitan A, Weeks J, Mocroft A, Smith J, Howe K, Murdoch J. The surgical manage‐ ment of women with ovarian cancer in the south west of England. Br J Cancer. Dec

[16] Carney ME, Lancaster JM, Ford C, Tsodikov A, Wiggins CL. A population-based study of patterns of care for ovarian cancer: who is seen by a gynecologic oncologist

[17] Elit L, Bondy SJ, Paszat L, Przybysz R, Levine M. Outcomes in surgery for ovarian

[18] Grossi M, Quinn MA, Thursfield VJ, et al. Ovarian cancer: patterns of care in Victoria

[19] Kumpulainen S, Grenman S, Kyyronen P, Pukkala E, Sankila R. Evidence of benefit from centralised treatment of ovarian cancer: a nationwide population-based surviv‐

[20] Cress RD, O'Malley CD, Leiserowitz GS, Campleman SL. Patterns of chemotherapy use for women with ovarian cancer: a population-based study. J Clin Oncol. Apr 15

[21] Harlan LC, Clegg LX, Trimble EL. Trends in surgery and chemotherapy for women diagnosed with ovarian cancer in the United States. J Clin Oncol. Sep 15 2003;21(18):

[22] Ioka A, Tsukuma H, Ajiki W, Oshima A. Influence of hospital procedure volume on ovarian cancer survival in Japan, a country with low incidence of ovarian cancer.

[23] Diaz-Montes TP, Zahurak ML, Giuntoli RL, 2nd, et al. Surgical care of elderly wom‐ en with ovarian cancer: a population-based perspective. Gynecol Oncol. Nov

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2 Department of Clinical Epidemiology and Biostatistics, Health Research Methodology Pro‐ gram, McMaster University, Hamilton, Canada

3 Division of Gynecologic Oncology, University of Toronto, Toronto, Canada

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**Author details**

Laurie Elit1,2 and Clare Reade2,3

20 Ovarian Cancer - A Clinical and Translational Update

gram, McMaster University, Hamilton, Canada

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oncology. Int J Gynecol Cancer. Mar-Apr 2004;14(2):206-209.

plinary team matters. Br J Cancer. Aug 1994;70(2):363-370.

267-271.

Pathol. Sep 2010;63(9):761-767.


[79] Harter P, Muallem ZM, Buhrmann C, et al. Impact of a structured quality manage‐ ment program on surgical outcome in primary advanced ovarian cancer. Gynecol Oncol. Jun 1 2011;121(3):615-619.

**Chapter 2**

**Quality of Life in Ovarian Cancer**

Additional information is available at the end of the chapter

The past two decades have witnessed an unprecedented level of attention devoted to the as‐ sessment of Quality of Life (QOL) in cancer patients. This is a result of a major change that occurred in the way cancer management and its impact has been understood and practiced. Contrary to earlier views, which focused primarily on prolonging the quantity of life of the patient, cancer management recognizes now the potential effects of the diagnosis and treat‐ ment on the overall functioning and well-being of the patient. QOL issues and its measure‐ ment became particularly important in oncology throughout the different phases of the cancer trajectory. In this context, the National Cancer Institute (NCI) has recommended that cancer research focus on both survival and QOL [1]. Many instruments have been devel‐ oped and used in clinical and research settings. It is noteworthy the inclusion of QOL as one of the main endpoints in important randomized clinical trials [2]. The benefits of studying QOL outcomes are evident. Primarily, QOL measurement has the potential to provide infor‐ mation to guide clinical decision making [3]. The knowledge about the impact of the illness and its treatment on cancer patients can help clinicians and patients to make decisions re‐ garding treatment options and choose appropriate supportive therapy adjusted to the pa‐ tient's needs. The toxicity and tolerability of a given treatment can be as important as its efficacy, as is the ability to help decrease or prevent associated toxicities that have a negative impact on QOL [4]. Furthermore, QOL data can foster patient-clinician interactions in rou‐ tine practice, identify problems that have a significant impact on QOL, prioritize problems, develop interventions to deal with these problems and evaluate the impact of palliative and rehabilitative efforts [5]. Additionally, it can help to shape public policy and health care de‐ cisions made by governmental and private institutions [6] and allow the economic evalua‐

> © 2013 Gonçalves; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

**Treatment and Survivorship**

Vânia Gonçalves

**1. Introduction**

http://dx.doi.org/10.5772/54542

tion of healthcare provision [7].


**Chapter 2**

### **Quality of Life in Ovarian Cancer Treatment and Survivorship**

Vânia Gonçalves

[79] Harter P, Muallem ZM, Buhrmann C, et al. Impact of a structured quality manage‐ ment program on surgical outcome in primary advanced ovarian cancer. Gynecol

[80] Aletti GD, Santillan A, Eisenhauer EL, et al. A new frontier for quality of care in gy‐ necologic oncology surgery: multi-institutional assessment of short-term outcomes for ovarian cancer using a risk-adjusted model. Gynecol Oncol. Oct 2007;107(1):

[81] Graham ID, Logan J, Harrison MB, et al. Lost in knowledge translation: time for a

[82] MacDermid JC, Graham ID. Knowledge translation: putting the "practice" in evi‐

[83] Munstedt K, von Georgi R, Zygmunt M, Misselwitz B, Stillger R, Kunzel W. Short‐ comings and deficits in surgical treatment of gynecological cancers: a German prob‐

[84] Farmer AP, Légaré F, Turcot L, et al. Printed educational materials: effects on profes‐ sional practice and health care outcomes. Cochrane Database of Systematic Reviews. 2008(3). http://www.mrw.interscience.wiley.com/cochrane/clsysrev/articles/

[85] Ivers N, Jamtvedt G, Flottorp S, et al. Audit and feedback: effects on professional practice and healthcare outcomes. Cochrane Database of Systematic Reviews. 2012(6). http://www.mrw.interscience.wiley.com/cochrane/clsysrev/articles/

[86] Flodgren G, Parmelli E, Doumit G, et al. Local opinion leaders: effects on professio‐ nal practice and health care outcomes. Cochrane Database of Systematic Reviews. 2011(8). http://www.mrw.interscience.wiley.com/cochrane/clsysrev/articles/

[87] Shojania KG, Jennings A, Mayhew A, Ramsay CR, Eccles MP, Grimshaw J. The ef‐ fects of on-screen, point of care computer reminders on processes and outcomes of care. Cochrane Database of Systematic Reviews. 2009(3). http://www.mrw.inter‐

[88] Grimshaw JM, Thomas RE, MacLennan G, et al. Effectiveness and efficiency of guideline dissemination and implementation strategies. Health Technol Assess. Feb

science.wiley.com/cochrane/clsysrev/articles/CD001096/frame.html.

map? J Contin Educ Health Prof. Winter 2006;26(1):13-24.

lem only? Gynecol Oncol. Sep 2002;86(3):337-343.

dence-based practice. Hand Clin. Feb 2009;25(1):125-143, viii.

Oncol. Jun 1 2011;121(3):615-619.

26 Ovarian Cancer - A Clinical and Translational Update

99-106.

CD004398/frame.html.

CD000259/frame.html.

CD000125/frame.html.

2004;8(6):iii-iv, 1-72.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54542

#### **1. Introduction**

The past two decades have witnessed an unprecedented level of attention devoted to the as‐ sessment of Quality of Life (QOL) in cancer patients. This is a result of a major change that occurred in the way cancer management and its impact has been understood and practiced. Contrary to earlier views, which focused primarily on prolonging the quantity of life of the patient, cancer management recognizes now the potential effects of the diagnosis and treat‐ ment on the overall functioning and well-being of the patient. QOL issues and its measure‐ ment became particularly important in oncology throughout the different phases of the cancer trajectory. In this context, the National Cancer Institute (NCI) has recommended that cancer research focus on both survival and QOL [1]. Many instruments have been devel‐ oped and used in clinical and research settings. It is noteworthy the inclusion of QOL as one of the main endpoints in important randomized clinical trials [2]. The benefits of studying QOL outcomes are evident. Primarily, QOL measurement has the potential to provide infor‐ mation to guide clinical decision making [3]. The knowledge about the impact of the illness and its treatment on cancer patients can help clinicians and patients to make decisions re‐ garding treatment options and choose appropriate supportive therapy adjusted to the pa‐ tient's needs. The toxicity and tolerability of a given treatment can be as important as its efficacy, as is the ability to help decrease or prevent associated toxicities that have a negative impact on QOL [4]. Furthermore, QOL data can foster patient-clinician interactions in rou‐ tine practice, identify problems that have a significant impact on QOL, prioritize problems, develop interventions to deal with these problems and evaluate the impact of palliative and rehabilitative efforts [5]. Additionally, it can help to shape public policy and health care de‐ cisions made by governmental and private institutions [6] and allow the economic evalua‐ tion of healthcare provision [7].

© 2013 Gonçalves; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

When considering ovarian cancer in particular, researchers follow the general trend by re‐ garding QOL as one of the most important outcomes. Several reasons make the study of QOL in ovarian cancer patients especially worthy and relevant. First, ovarian cancer is an aggressive illness which is associated with very poor survival and high recurrence rates. It is the most fatal malignancy of the female genital tract and the fourth most common cause of female cancer death [8]. Generally, it is detected at an advance stage, with a 5-year survival rate of 46% for all the stages and 31% for advanced stages [8]. The management of ovarian cancer normally includes radical pelvic surgery and multiple aggressive courses of chemo‐ therapy. The stress of receiving the diagnosis of such an aggressive and life threatening ill‐ ness, which can be unexpected for many women, may be associated with uncertainty and anxiety about the future. This may be regarded as an immediate threat to a woman's life and an associated fear of death. Additionally, women may suffer disease-related symptoms, which may be very difficult to cope with. These include weight loss, bloating and ascites, fatigue and pain. Women may also experience a wide range of sequalae related to their treatment that do not dissipate with time and may persist for a long-term period [9, 10]. Ex‐ amples include neutropenia, body distortion, hair loss, bowel and bladder incontinence, loss of taste and appetite, premature menopause, infertility, decrease physical functioning, poor sleep, edema and sexual problems [9, 10]. Another burden involves the amount of time spend in treatments that is lost from family and work [11]. Second, research carried out, spe‐ cifically, with ovarian cancer patients has shown that a substantial proportion of women ex‐ perience psychological disorders. Anxiety, depression [9, 12-16] and Post-Traumatic Stress Disorder (PTSD) [17] have been found among different studies. Reports have also highlight‐ ed the occurrence of impairments in physical, vocational, social, familial and sexual func‐ tioning. Those are not confined to the diagnosis and treatment periods, but have been also observed in short and long-term ovarian cancer survivors. Lastly, advances in Medicine fu‐ elled the development of new treatments for ovarian cancer. However, these treatments have associated side-effects and toxicities that may impact on the QOL of the women. There‐ fore, when considering a treatment plan, risks and benefits must be balanced in order to achieve an optimal QOL [11]. Improvements in survival in ovarian cancer have been rela‐ tively reduced [18]. The ability of chemotherapeutic regimens in slowing the progression of disease to prolong life with active disease has been responsible for those improvements in survival [19]. Undoubtedly, QOL is a fundamental consideration for patients with ovarian cancer.

of different definitions and, inevitably, means of measurement. This makes difficult the comparison of findings among studies and to establish more definite conclusions. Issues of definition and measurement continue to be, in fact, the subject of ongoing debate. Despite lack of consensus in its definition, it is widely accepted that QOL is a multidimensional con‐ struct that includes several important dimensions (any area of behavior or experience) [4, 7, 20, 21]. These encompass physical functioning (physical well-being, mobility, ability to per‐ form self-care activities, physical activities, role activities such as work or housework, appe‐ tite, comorbidities, fatigue/sleep, symptoms, side-effects), cognitive and psychological functioning (emotional well-being, anxiety, depression, coping, perceptions, prior experi‐ ence, enjoyment, optimism), social functioning (family interactions, time with friends, lei‐ sure activities), disease and treatment related symptoms (such as pain and fatigue), spiritual or existential concerns, sexual functioning, body image, patient's satisfaction with health care, control of the disease [7, 21]. According to the WHO [22], QOL is defined as 'an indi‐ vidual's perception of their position in life in the context of the culture and value systems in which they live and in relation to their goals, expectations, standards and concerns. It is a broad ranging concept affected in a complex way by the person's physical health, psycho‐ logical state, level of independence, social relationships, and their relationships to salient features of their environment". Following these lines, QOL includes all aspects of the indi‐

Quality of Life in Ovarian Cancer Treatment and Survivorship

http://dx.doi.org/10.5772/54542

29

vidual well-being and must be evaluated from the individual's perspective.

treatment, and policy [25].

When QOL is considered in the context of health, it is often referred to as health-related QOL (HRQOL). HRQOL is a more specific concept, which reflects the effect of the illness and illness treatment on general well-being. Bowling defined HRQOL as 'optimum levels of mental, physical, role (e.g. work, parent, career, etc) and social functioning, including relationships, and perceptions of health, fitness, life satisfaction and well-being. It should also include some assessment of patient´s level of satisfaction with treatment, outcome and health status and with future prospects. It is distinct from QOL as a whole, which would also include adequacy of housing, income and perceptions of immediate environ‐ ment' [23]. HRQOL is a dynamic concept, as health status deteriorates, experiences, roles and relationships change [24]. Furthermore, It may be modified by impairments, func‐ tional status, perceptions, and social opportunities and may be influenced by disease,

Particularly in ovarian cancer literature, the term QOL is much more extensively used in‐ stead of HRQOL. In general, QOL assessment in ovarian cancer patients has been focusing more on the acute phase of the treatment. Of interest is the evaluation of QOL under treat‐ ment conditions in randomized clinical trials, focusing on different treatment options. The measurement of QOL in screening and early diagnosis of ovarian cancer is very scarce. It of note that, in fact, screening and early detection of ovarian cancer are very limited in clinical practice, existing narrow useful technologies to assist in early diagnosis. The majority of the QOL measurement in ovarian cancer screening evaluates populations at high risk, such as women with genetic mutations undergoing risk-reducing salpingo-oophorectomy [26]. However, for individuals undergoing risk-reducing salpingo-oophorectomy, screening is more a process of early detection or diagnosis rather than a true screening test [26]. Regard‐

This chapter addresses the most recent knowledge regarding the impact of the treatment on QOL of ovarian cancer patients. Additionally, QOL in ovarian cancer survivors is al‐ so discussed.

#### **2. Quality of life: Brief overview**

Central to this particular subject, is the question: What is QOL? Although, it is somehow consensual by the clinical and research communities the importance of studying QOL, it is much less consensual what exactly QOL means. This lack of consensus fuels the appearance of different definitions and, inevitably, means of measurement. This makes difficult the comparison of findings among studies and to establish more definite conclusions. Issues of definition and measurement continue to be, in fact, the subject of ongoing debate. Despite lack of consensus in its definition, it is widely accepted that QOL is a multidimensional con‐ struct that includes several important dimensions (any area of behavior or experience) [4, 7, 20, 21]. These encompass physical functioning (physical well-being, mobility, ability to per‐ form self-care activities, physical activities, role activities such as work or housework, appe‐ tite, comorbidities, fatigue/sleep, symptoms, side-effects), cognitive and psychological functioning (emotional well-being, anxiety, depression, coping, perceptions, prior experi‐ ence, enjoyment, optimism), social functioning (family interactions, time with friends, lei‐ sure activities), disease and treatment related symptoms (such as pain and fatigue), spiritual or existential concerns, sexual functioning, body image, patient's satisfaction with health care, control of the disease [7, 21]. According to the WHO [22], QOL is defined as 'an indi‐ vidual's perception of their position in life in the context of the culture and value systems in which they live and in relation to their goals, expectations, standards and concerns. It is a broad ranging concept affected in a complex way by the person's physical health, psycho‐ logical state, level of independence, social relationships, and their relationships to salient features of their environment". Following these lines, QOL includes all aspects of the indi‐ vidual well-being and must be evaluated from the individual's perspective.

When considering ovarian cancer in particular, researchers follow the general trend by re‐ garding QOL as one of the most important outcomes. Several reasons make the study of QOL in ovarian cancer patients especially worthy and relevant. First, ovarian cancer is an aggressive illness which is associated with very poor survival and high recurrence rates. It is the most fatal malignancy of the female genital tract and the fourth most common cause of female cancer death [8]. Generally, it is detected at an advance stage, with a 5-year survival rate of 46% for all the stages and 31% for advanced stages [8]. The management of ovarian cancer normally includes radical pelvic surgery and multiple aggressive courses of chemo‐ therapy. The stress of receiving the diagnosis of such an aggressive and life threatening ill‐ ness, which can be unexpected for many women, may be associated with uncertainty and anxiety about the future. This may be regarded as an immediate threat to a woman's life and an associated fear of death. Additionally, women may suffer disease-related symptoms, which may be very difficult to cope with. These include weight loss, bloating and ascites, fatigue and pain. Women may also experience a wide range of sequalae related to their treatment that do not dissipate with time and may persist for a long-term period [9, 10]. Ex‐ amples include neutropenia, body distortion, hair loss, bowel and bladder incontinence, loss of taste and appetite, premature menopause, infertility, decrease physical functioning, poor sleep, edema and sexual problems [9, 10]. Another burden involves the amount of time spend in treatments that is lost from family and work [11]. Second, research carried out, spe‐ cifically, with ovarian cancer patients has shown that a substantial proportion of women ex‐ perience psychological disorders. Anxiety, depression [9, 12-16] and Post-Traumatic Stress Disorder (PTSD) [17] have been found among different studies. Reports have also highlight‐ ed the occurrence of impairments in physical, vocational, social, familial and sexual func‐ tioning. Those are not confined to the diagnosis and treatment periods, but have been also observed in short and long-term ovarian cancer survivors. Lastly, advances in Medicine fu‐ elled the development of new treatments for ovarian cancer. However, these treatments have associated side-effects and toxicities that may impact on the QOL of the women. There‐ fore, when considering a treatment plan, risks and benefits must be balanced in order to achieve an optimal QOL [11]. Improvements in survival in ovarian cancer have been rela‐ tively reduced [18]. The ability of chemotherapeutic regimens in slowing the progression of disease to prolong life with active disease has been responsible for those improvements in survival [19]. Undoubtedly, QOL is a fundamental consideration for patients with ovarian

This chapter addresses the most recent knowledge regarding the impact of the treatment on QOL of ovarian cancer patients. Additionally, QOL in ovarian cancer survivors is al‐

Central to this particular subject, is the question: What is QOL? Although, it is somehow consensual by the clinical and research communities the importance of studying QOL, it is much less consensual what exactly QOL means. This lack of consensus fuels the appearance

cancer.

so discussed.

**2. Quality of life: Brief overview**

28 Ovarian Cancer - A Clinical and Translational Update

When QOL is considered in the context of health, it is often referred to as health-related QOL (HRQOL). HRQOL is a more specific concept, which reflects the effect of the illness and illness treatment on general well-being. Bowling defined HRQOL as 'optimum levels of mental, physical, role (e.g. work, parent, career, etc) and social functioning, including relationships, and perceptions of health, fitness, life satisfaction and well-being. It should also include some assessment of patient´s level of satisfaction with treatment, outcome and health status and with future prospects. It is distinct from QOL as a whole, which would also include adequacy of housing, income and perceptions of immediate environ‐ ment' [23]. HRQOL is a dynamic concept, as health status deteriorates, experiences, roles and relationships change [24]. Furthermore, It may be modified by impairments, func‐ tional status, perceptions, and social opportunities and may be influenced by disease, treatment, and policy [25].

Particularly in ovarian cancer literature, the term QOL is much more extensively used in‐ stead of HRQOL. In general, QOL assessment in ovarian cancer patients has been focusing more on the acute phase of the treatment. Of interest is the evaluation of QOL under treat‐ ment conditions in randomized clinical trials, focusing on different treatment options. The measurement of QOL in screening and early diagnosis of ovarian cancer is very scarce. It of note that, in fact, screening and early detection of ovarian cancer are very limited in clinical practice, existing narrow useful technologies to assist in early diagnosis. The majority of the QOL measurement in ovarian cancer screening evaluates populations at high risk, such as women with genetic mutations undergoing risk-reducing salpingo-oophorectomy [26]. However, for individuals undergoing risk-reducing salpingo-oophorectomy, screening is more a process of early detection or diagnosis rather than a true screening test [26]. Regard‐ ing survivorship, recently, there is a growing interest in the study of QOL in ovarian cancer survivors. The following section focuses on the instruments designed to capture QOL that are more commonly used in this specific population.

three symptom scales (fatigue, pain, nausea and vomiting) (7 items), and six single items, assessing additional symptoms commonly reported by cancer patients (breathlessness, diffi‐ culty sleeping, appetite loss, constipation, diarrhea, and financial difficulties). Each scale is scored separately. Seven questions have a dichotomous yes/no response. For the two global QOL items, respondents have to answer by using a 7-point scale, where '1 = very poor' and '7 = excellent'. The remaining questions have a four-point Likert scale, ranging from '1 = Not at all' to '4 = Very much'. No timeframe is specified in the seven dichotomous questions. In the remaining questions, the patient has to answer according to the past week. Each dimen‐ sion score for each patient is the sum of that patient's item responses for that dimension, transformed, so that the minimum possible value is zero and the maximum possible value is 100. Each scale has a limited set of possible values, determined by the number of items and the range of response options for each item. For the functional scales and the global QOL scale, a higher score corresponds to a better QOL. For the symptom scales and the single items, a higher score indicates more frequent and/or intense symptom experience and thus a lower QOL. Finally, there are two items that ask respondents to rate their overall physical condition. The EORTC QOL-C30 has established reliability and validity [28]. This scale is easy to complete, acceptable to patients and has been translated into several languages. The EORTC QOL-OV28 is the ovarian cancer module designed to supplement the EORTC QOL-C30, for the assessment of QOL in ovarian cancer patients in clinical trials and related stud‐ ies. It consists of 7 subscales and a total of 28 items, which assess abdominal symptoms (abdominal pain, feeling bloated, clothes too tight, changed bowel habit, flatulence, fullness when eating, indigestion), peripheral neuropathy (tingling, numbness, and weakness), other chemotherapy related side effects (hair loss and upset by hair loss, taste change, muscle pain, hearing problem, urinary frequency, and skin problem), hormonal/menopausal symp‐ toms (hot flushes and night sweat), body image (less attractive, dissatisfied with body), atti‐ tude to disease and treatment (disease burden, treatment burden, and worry about future) and sexual functioning (interest in sex, sexual activity, enjoyment of sex and dry vagina) [29, 30]. Each scale is scored separately. For symptom scales, a higher score means a lower QOL, while for function scales, such as body image and sexual function, a higher score means a better QOL. The EORTC QOL-OV28 is a valid and reliable measure to be used in ovarian

Quality of Life in Ovarian Cancer Treatment and Survivorship

http://dx.doi.org/10.5772/54542

31

The FACT-G was developed by Cella et al to evaluate QOL in oncology settings [20]. This is the core scale of the instrument system and consists of four dimensions, comprising a total of 27 items. The dimensions include functional well-being (7 questions), emotional well-be‐ ing (6 questions), social/family well-being (7 questions) and physical well-being (7 ques‐ tions). These four dimensions can be analyzed separately or aggregated to produce a total QOL score. Response categories for all items range from 0 (not at all) to 4 (very much). Higher scores are associated with increased satisfaction with QOL. The timeframe for this instrument is the past 7 days. FACT-G is tested and validated in large international samples, showing reliability, validity and responsiveness to change over time [20]. This instrument is commonly used in ovarian cancer clinical trials and it is available in many languages. The

cancer populations [30].

**3.2. FACT-G**

#### **3. Measurement of QOL in patients with ovarian cancer**

Many instruments have been developed and validated to capture important QOL issues in cancer patients. These instruments comprise four main groups: generic measures of QOL (used to assess non-cancer medical patients), cancer condition-specific (used in general can‐ cer populations), cancer site and treatment-specific instruments. QOL measures are often supplemented by questionnaires designed to evaluate specific dimensions of QOL, for ex‐ ample depression. The use of generic questionnaires allows comparisons of QOL among conditions [7]; however, they lack specificity necessary to understand particular problems inherent to a specific condition, such as cancer. This specificity can be found when disease – and site-specific instruments are used. These are more likely to be responsive to change but are not comprehensive [7]. The Medical Outcome Study (Short Form) MOS SF-36 [27] is an example of a QOL generic instrument used in oncology. The European Organization for Re‐ search and Treatment of Cancer QOL Core Questionnaire (EORTC QOL-C30) [28] and the Functional Assessment of Cancer Therapy – General (FACT-G) [20] are examples of condi‐ tion (cancer) specific instruments. All are self-administered questionnaires, multidimension‐ al, relatively brief, acceptable to patients and have good psychometric properties [7]. The EORTC QOL-C30 and the FACT-G comprise ovarian cancer modules that constitute exam‐ ples of site specific QOL instruments.

Particularly in ovarian cancer, the most commonly used measures are the EORTC QOL-C30 and the FACT-G [18]. The EORTC QOL-C30 and the FACT-G have a similar format: a core QOL questionnaire applicable to cancer patients in general and specific modules, applicable to specific cancer sites. These instruments have been developed primarily from research en‐ vironments; however, they will be extremely helpful if they assist physicians in detecting clinically significant differences or changes in a patient condition.

#### **3.1. EORTC QOL-C30**

This cancer-specific questionnaire was developed by the Study Group on Quality of Life from the European Organization for Research on Treatment of Cancer comprising a core set of questions applicable to all cancer patients and modules to be used to specific cancer sides, such as ovarian cancer [28, 29]. This instrument was designed to be used in international randomized clinical trials. It is based on a multidimensional model of QOL, covering cancerspecific symptoms of the disease, psychological distress, treatment side-effects, social inter‐ action, physical functioning, body image, sexuality, global health and QOL, and satisfaction with medical care. The core QOL instrument is composed by 30 items, comprising nine scales of QOL: one global QOL scale (2 items), five functional scales (physical functioning, role functioning, cognitive functioning, emotional functioning, social functioning) (15 iems), three symptom scales (fatigue, pain, nausea and vomiting) (7 items), and six single items, assessing additional symptoms commonly reported by cancer patients (breathlessness, diffi‐ culty sleeping, appetite loss, constipation, diarrhea, and financial difficulties). Each scale is scored separately. Seven questions have a dichotomous yes/no response. For the two global QOL items, respondents have to answer by using a 7-point scale, where '1 = very poor' and '7 = excellent'. The remaining questions have a four-point Likert scale, ranging from '1 = Not at all' to '4 = Very much'. No timeframe is specified in the seven dichotomous questions. In the remaining questions, the patient has to answer according to the past week. Each dimen‐ sion score for each patient is the sum of that patient's item responses for that dimension, transformed, so that the minimum possible value is zero and the maximum possible value is 100. Each scale has a limited set of possible values, determined by the number of items and the range of response options for each item. For the functional scales and the global QOL scale, a higher score corresponds to a better QOL. For the symptom scales and the single items, a higher score indicates more frequent and/or intense symptom experience and thus a lower QOL. Finally, there are two items that ask respondents to rate their overall physical condition. The EORTC QOL-C30 has established reliability and validity [28]. This scale is easy to complete, acceptable to patients and has been translated into several languages. The EORTC QOL-OV28 is the ovarian cancer module designed to supplement the EORTC QOL-C30, for the assessment of QOL in ovarian cancer patients in clinical trials and related stud‐ ies. It consists of 7 subscales and a total of 28 items, which assess abdominal symptoms (abdominal pain, feeling bloated, clothes too tight, changed bowel habit, flatulence, fullness when eating, indigestion), peripheral neuropathy (tingling, numbness, and weakness), other chemotherapy related side effects (hair loss and upset by hair loss, taste change, muscle pain, hearing problem, urinary frequency, and skin problem), hormonal/menopausal symp‐ toms (hot flushes and night sweat), body image (less attractive, dissatisfied with body), atti‐ tude to disease and treatment (disease burden, treatment burden, and worry about future) and sexual functioning (interest in sex, sexual activity, enjoyment of sex and dry vagina) [29, 30]. Each scale is scored separately. For symptom scales, a higher score means a lower QOL, while for function scales, such as body image and sexual function, a higher score means a better QOL. The EORTC QOL-OV28 is a valid and reliable measure to be used in ovarian cancer populations [30].

#### **3.2. FACT-G**

ing survivorship, recently, there is a growing interest in the study of QOL in ovarian cancer survivors. The following section focuses on the instruments designed to capture QOL that

Many instruments have been developed and validated to capture important QOL issues in cancer patients. These instruments comprise four main groups: generic measures of QOL (used to assess non-cancer medical patients), cancer condition-specific (used in general can‐ cer populations), cancer site and treatment-specific instruments. QOL measures are often supplemented by questionnaires designed to evaluate specific dimensions of QOL, for ex‐ ample depression. The use of generic questionnaires allows comparisons of QOL among conditions [7]; however, they lack specificity necessary to understand particular problems inherent to a specific condition, such as cancer. This specificity can be found when disease – and site-specific instruments are used. These are more likely to be responsive to change but are not comprehensive [7]. The Medical Outcome Study (Short Form) MOS SF-36 [27] is an example of a QOL generic instrument used in oncology. The European Organization for Re‐ search and Treatment of Cancer QOL Core Questionnaire (EORTC QOL-C30) [28] and the Functional Assessment of Cancer Therapy – General (FACT-G) [20] are examples of condi‐ tion (cancer) specific instruments. All are self-administered questionnaires, multidimension‐ al, relatively brief, acceptable to patients and have good psychometric properties [7]. The EORTC QOL-C30 and the FACT-G comprise ovarian cancer modules that constitute exam‐

Particularly in ovarian cancer, the most commonly used measures are the EORTC QOL-C30 and the FACT-G [18]. The EORTC QOL-C30 and the FACT-G have a similar format: a core QOL questionnaire applicable to cancer patients in general and specific modules, applicable to specific cancer sites. These instruments have been developed primarily from research en‐ vironments; however, they will be extremely helpful if they assist physicians in detecting

This cancer-specific questionnaire was developed by the Study Group on Quality of Life from the European Organization for Research on Treatment of Cancer comprising a core set of questions applicable to all cancer patients and modules to be used to specific cancer sides, such as ovarian cancer [28, 29]. This instrument was designed to be used in international randomized clinical trials. It is based on a multidimensional model of QOL, covering cancerspecific symptoms of the disease, psychological distress, treatment side-effects, social inter‐ action, physical functioning, body image, sexuality, global health and QOL, and satisfaction with medical care. The core QOL instrument is composed by 30 items, comprising nine scales of QOL: one global QOL scale (2 items), five functional scales (physical functioning, role functioning, cognitive functioning, emotional functioning, social functioning) (15 iems),

clinically significant differences or changes in a patient condition.

are more commonly used in this specific population.

30 Ovarian Cancer - A Clinical and Translational Update

ples of site specific QOL instruments.

**3.1. EORTC QOL-C30**

**3. Measurement of QOL in patients with ovarian cancer**

The FACT-G was developed by Cella et al to evaluate QOL in oncology settings [20]. This is the core scale of the instrument system and consists of four dimensions, comprising a total of 27 items. The dimensions include functional well-being (7 questions), emotional well-be‐ ing (6 questions), social/family well-being (7 questions) and physical well-being (7 ques‐ tions). These four dimensions can be analyzed separately or aggregated to produce a total QOL score. Response categories for all items range from 0 (not at all) to 4 (very much). Higher scores are associated with increased satisfaction with QOL. The timeframe for this instrument is the past 7 days. FACT-G is tested and validated in large international samples, showing reliability, validity and responsiveness to change over time [20]. This instrument is commonly used in ovarian cancer clinical trials and it is available in many languages. The supplement of the FACT-G with a set of twelve items specific to ovarian cancer is referred as the Functional Assessment of Cancer Therapy – Ovarian (FACT-O). Items include stomach swelling, losing weight, bowels control, vomiting, hair loss, appetite, appearance, getting around, feeling like a woman, stomach cramping, interest in sex and concerns about ability to have children. The ovarian cancer specific subscale assesses severity of problems that can be targeted by proper disease management. The FACT-O is a valid instrument to be used in ovarian cancer patients [31]. This questionnaire has been commonly used in clinical trials and other descriptive studies. The FACT-O can be used alone or in combination with other scales or subscales of the FACT, such as the FACT/GOG neurotoxicity subscale, or the Ane‐ mia (FACT-An) or Fatigue (FACT-F) subscale, if the research interest is these specific issues. The physical well-being and the functional well-being scales of the FACT-G plus the ovarian cancer subscale can be combined to represent the Trial Outcome Index (TOI). This index has excellent psychometric properties [31].

It is paramount to understand how ovarian cancer and its treatment may disrupt the overall well-being and QOL of patients. A recent systematic review and meta-analysis, carried out to assess and summarize QOL data before, during and after chemotherapy among ovarian cancer patients, found that baseline QOL may significantly improve, particularly after com‐ pletion of chemotherapy treatment [18]. Authors identified a total of 139 studies; of those, 48 were randomized clinical trials. However, it was only possible to synthesize data from a subset of studies, due to inconsistencies in the way the data was reported across studies. Pooled data showed that QOL as measured by the EORTC QOL C-30 was found to improve during the treatment period and ovarian cancer specific concerns as measured by the FACT-O subscale, were improved during the treatment period [18]. The EORTC QOL C-30, FACT-G and FACT-O found significant improvements in QOL after completion of primary therapy, despite the lack of measurable improvements during treatment as measured by the FACT-G [18]. Following these lines, a recent longitudinal study evaluated the course of QOL, depressive symptoms, anxiety symptoms and fatigue over the course of chemothera‐ py until 6 months follow-up [35]. Results demonstrated a significant improvement of QOL, as measured by the EORTC QOL C-30 and EORTC QOL OV-28, from the start of chemother‐ apy and post-surgery period (QOL was severely impaired and high levels of anxiety symp‐ toms, depressive symptoms and fatigue were found), until after care (symptoms reach nearly general population symptom levels). Although, this was a small study of 23 patients, it highlighted the importance of understanding QOL over the course of treatment [35]. Simi‐ lar results were obtained by other investigators, reporting improvements of QOL in ovarian cancer during chemotherapy until one year follow-up. Von Gruenigen et al [36] in a sample of 42 ovarian cancer patients found that QOL, as measured by the FACT-G and SF-36, mark‐ edly decreased after surgery with a slow improvement during adjuvant chemotherapy, mainly in the physical, functional and fatigue domains. Physical functioning decreased dur‐ ing chemotherapy but increased to perioperative levels following treatment. Functional well-being increased following chemotherapy, while emotional and social scores did not change over time [36]. Collectively, these findings highlight that, in addition to chemothera‐ peutic treatments, surgery may have a negative impact on QOL. Although several factors may influence this impact, tumour stage, and therefore, the extent of the surgical interven‐ tion and the existence of intra – or postoperative complications may be crucial [35]. Minig et al [37] found in a study of 181 women with gynaecological cancers, of which 116 had ovari‐ an cancer, that postoperative complications, surgical complexity, advanced stage were asso‐ ciated with lower levels of postsurgical QOL specifically in ovarian cancer patients. The strongest predictor of postsurgical QOL was preoperative QOL, closely followed by surgical complications. Investigators stressed that postoperative complications may be difficult to avoid due to the aggressiveness of the surgery performed in order to achieve maximum cy‐ toreduction in ovarian cancer; however, attention needs to be paid intraoperatively and postoperatively to the early detection of complications to optimize QOL whenever possible in this group of patients [37]. Consequences of surgery are well documented, including loss of fertility, sexual dysfunction, surgical menopause and bowel obstruction. For women at re‐

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33

productive age, premature menopause and loss of fertility may be devastating [34].

#### **4. Quality of life in ovarian cancer patients**

How is the QOL of ovarian cancer patients? Do patients with ovarian cancer experience a good QOL? These are questions that researchers have been attempting to answer in the many studies available dedicated to this subject. The number of studies carried out in‐ creased significantly in recent years [18], and collectively, these studies captured ongoing is‐ sues and concerns resulting from the ovarian cancer diagnosis and treatment [9]. However, there is a difficulty in drawing definite conclusions to answer the above questions. This is due to the lack of consistency in the types and format of QOL data collected in ovarian can‐ cer patients [18]. The accumulated knowledge about QOL issues in patients undergoing treatment and in survivors of ovarian cancer is presented below.

#### **4.1. QOL during ovarian cancer treatment**

The management of ovarian cancer generally requires a multimodal approach. Surgery has always been the cornerstone, which plays an essential role in both diagnosis and treatment. The aim of which is to leave no residual deposits greater than 1–2 cm in diameter. In cases of apparent early stage disease, proper surgical management involves comprehensive surgical staging. Advanced-stage disease frequently requires aggressive surgical debulking [32]. The standard approach is to follow surgery by either intravenous or intraperitoneal chemothera‐ py. Two classes of cytotoxic components, the plantinums and the taxanes are key compo‐ nents of chemotherapeutic regimens for advanced disease [33]. Both treatment modalities can impact negatively on the QOL of patients [34]. In recurrent disease, a variety of treat‐ ment regimens are used, including re-treatment with a platinum and/or taxane agent, and second line agents such as liposomal doxorubicin, topotecan, and gemcitabine. Chemothera‐ py side effects may be temporary (e.g. hair loss, nausea and vomiting) or cumulative and/or permanent (e.g. fatigue, neurotoxicity) [34].

It is paramount to understand how ovarian cancer and its treatment may disrupt the overall well-being and QOL of patients. A recent systematic review and meta-analysis, carried out to assess and summarize QOL data before, during and after chemotherapy among ovarian cancer patients, found that baseline QOL may significantly improve, particularly after com‐ pletion of chemotherapy treatment [18]. Authors identified a total of 139 studies; of those, 48 were randomized clinical trials. However, it was only possible to synthesize data from a subset of studies, due to inconsistencies in the way the data was reported across studies. Pooled data showed that QOL as measured by the EORTC QOL C-30 was found to improve during the treatment period and ovarian cancer specific concerns as measured by the FACT-O subscale, were improved during the treatment period [18]. The EORTC QOL C-30, FACT-G and FACT-O found significant improvements in QOL after completion of primary therapy, despite the lack of measurable improvements during treatment as measured by the FACT-G [18]. Following these lines, a recent longitudinal study evaluated the course of QOL, depressive symptoms, anxiety symptoms and fatigue over the course of chemothera‐ py until 6 months follow-up [35]. Results demonstrated a significant improvement of QOL, as measured by the EORTC QOL C-30 and EORTC QOL OV-28, from the start of chemother‐ apy and post-surgery period (QOL was severely impaired and high levels of anxiety symp‐ toms, depressive symptoms and fatigue were found), until after care (symptoms reach nearly general population symptom levels). Although, this was a small study of 23 patients, it highlighted the importance of understanding QOL over the course of treatment [35]. Simi‐ lar results were obtained by other investigators, reporting improvements of QOL in ovarian cancer during chemotherapy until one year follow-up. Von Gruenigen et al [36] in a sample of 42 ovarian cancer patients found that QOL, as measured by the FACT-G and SF-36, mark‐ edly decreased after surgery with a slow improvement during adjuvant chemotherapy, mainly in the physical, functional and fatigue domains. Physical functioning decreased dur‐ ing chemotherapy but increased to perioperative levels following treatment. Functional well-being increased following chemotherapy, while emotional and social scores did not change over time [36]. Collectively, these findings highlight that, in addition to chemothera‐ peutic treatments, surgery may have a negative impact on QOL. Although several factors may influence this impact, tumour stage, and therefore, the extent of the surgical interven‐ tion and the existence of intra – or postoperative complications may be crucial [35]. Minig et al [37] found in a study of 181 women with gynaecological cancers, of which 116 had ovari‐ an cancer, that postoperative complications, surgical complexity, advanced stage were asso‐ ciated with lower levels of postsurgical QOL specifically in ovarian cancer patients. The strongest predictor of postsurgical QOL was preoperative QOL, closely followed by surgical complications. Investigators stressed that postoperative complications may be difficult to avoid due to the aggressiveness of the surgery performed in order to achieve maximum cy‐ toreduction in ovarian cancer; however, attention needs to be paid intraoperatively and postoperatively to the early detection of complications to optimize QOL whenever possible in this group of patients [37]. Consequences of surgery are well documented, including loss of fertility, sexual dysfunction, surgical menopause and bowel obstruction. For women at re‐ productive age, premature menopause and loss of fertility may be devastating [34].

supplement of the FACT-G with a set of twelve items specific to ovarian cancer is referred as the Functional Assessment of Cancer Therapy – Ovarian (FACT-O). Items include stomach swelling, losing weight, bowels control, vomiting, hair loss, appetite, appearance, getting around, feeling like a woman, stomach cramping, interest in sex and concerns about ability to have children. The ovarian cancer specific subscale assesses severity of problems that can be targeted by proper disease management. The FACT-O is a valid instrument to be used in ovarian cancer patients [31]. This questionnaire has been commonly used in clinical trials and other descriptive studies. The FACT-O can be used alone or in combination with other scales or subscales of the FACT, such as the FACT/GOG neurotoxicity subscale, or the Ane‐ mia (FACT-An) or Fatigue (FACT-F) subscale, if the research interest is these specific issues. The physical well-being and the functional well-being scales of the FACT-G plus the ovarian cancer subscale can be combined to represent the Trial Outcome Index (TOI). This index has

How is the QOL of ovarian cancer patients? Do patients with ovarian cancer experience a good QOL? These are questions that researchers have been attempting to answer in the many studies available dedicated to this subject. The number of studies carried out in‐ creased significantly in recent years [18], and collectively, these studies captured ongoing is‐ sues and concerns resulting from the ovarian cancer diagnosis and treatment [9]. However, there is a difficulty in drawing definite conclusions to answer the above questions. This is due to the lack of consistency in the types and format of QOL data collected in ovarian can‐ cer patients [18]. The accumulated knowledge about QOL issues in patients undergoing

The management of ovarian cancer generally requires a multimodal approach. Surgery has always been the cornerstone, which plays an essential role in both diagnosis and treatment. The aim of which is to leave no residual deposits greater than 1–2 cm in diameter. In cases of apparent early stage disease, proper surgical management involves comprehensive surgical staging. Advanced-stage disease frequently requires aggressive surgical debulking [32]. The standard approach is to follow surgery by either intravenous or intraperitoneal chemothera‐ py. Two classes of cytotoxic components, the plantinums and the taxanes are key compo‐ nents of chemotherapeutic regimens for advanced disease [33]. Both treatment modalities can impact negatively on the QOL of patients [34]. In recurrent disease, a variety of treat‐ ment regimens are used, including re-treatment with a platinum and/or taxane agent, and second line agents such as liposomal doxorubicin, topotecan, and gemcitabine. Chemothera‐ py side effects may be temporary (e.g. hair loss, nausea and vomiting) or cumulative and/or

excellent psychometric properties [31].

32 Ovarian Cancer - A Clinical and Translational Update

**4.1. QOL during ovarian cancer treatment**

permanent (e.g. fatigue, neurotoxicity) [34].

**4. Quality of life in ovarian cancer patients**

treatment and in survivors of ovarian cancer is presented below.

Several clinical trials evaluating ovarian cancer treatments have been carried out, in which QOL is one of the outcomes evaluated. Table 1 describes recent clinical trials that have in‐ cluded QOL as an outcome. QOL measurement in clinical trials has been useful to argue in favor or against novel therapies. Furthermore, there is some evidence demonstrating that QOL is a prognostic indicator for treatment outcomes [26] and future survival [38-41].

standard care as first-line chemotherapy for advanced ovarian cancer. However, paclitax‐ el is associated with several toxicities (e.g. anemia, thrombocytopenia) that overlap the toxicities of the plantinums, and the co-administration of paclitaxel and a platinum com‐ pound can potentially increase the frequency and/or severity of shared toxicities. By it‐ self, paclitaxel is associated with peripheral neuropathy that can add to the disease burden of the patient [4]. Therefore, studies have been conducted to find the least toxic combination of medications used in chemotherapy in order to improve treatment tolera‐ bility and QOL [49]. For example, a Phase III Trial conducted by the Scotish Gynaecolog‐ ical Cancer Trials Group (SCOTROC Trial), which included 1077 patients, compared carboplatin docetaxel with carboplatin paclitaxel for first line therapy. Results demon‐ strated a clear advantage for docetaxel in terms of neurotoxicity [43]. Concurrent with the developments in intravenous treatment, intraperitoneal treatment has also been shown a valuable strategy. The Gynecologic Oncology Group published data from the GOG randomized phase III trial (GOG 172) pertaining QOL outcomes associated with the use of intravenous paclitaxel plus intraperitoneal cisplatin plus paclitaxel, versus in‐ travenous paclitaxel plus cisplatin, for advanced stage cancer [42]. This was the first Phase III GOG ovarian cancer that proposed a change in route for the administration of front-line chemotherapy. In the intraperitoneal arm, overall survival was improved by approximately 16 months; however, during active treatment, patients reported more QOL disruptions, abdominal discomfort and neurotoxicity compared to those patients re‐ ceiving conventional intravenous chemotherapy. However, only neurotoxicity remained significantly higher for patients in the intraperitoneal arm 12 months post-treatment. Fu‐ ture studies to lessen the added burden associated with intraperitoneal therapy are going [42]. Recently, Vergote et al. [44] reported the results of a Gynaecologic Cancer Inter‐ group Collaboration Trial which compared upfront debulking followed by chemotherapy to neoadjuvant chemotherapy. This was the first randomized Phase III Trial of neoadju‐ vant chemotherapy in ovarian cancer using QOL as an endpoint. The two groups report‐ ed similar survival outcomes. QOL scores did not differ among the two groups [44].

Quality of Life in Ovarian Cancer Treatment and Survivorship

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35

The majority of ovarian cancer patients will eventually relapse. In fact, it is not uncommon for ovarian cancer patients to undergo numerous chemotherapeutics treatments. In this con‐ text, the evaluation of QOL is of utmost importance. In the management of recurrent ovari‐ an cancer, tumour control without compromising QOL should be the goal of the therapy [50]. However, there are deficits in the measurement of general QOL data in the recurrent setting, in terms of QOL disruptions and number of studies including QOL measurements [26]. A recent trial published data pertaining the impact of early versus delayed treatment of recurrent ovarian cancer based on Ca125 measurements exceeding twice the upper limit of normal. Results showed that women did not live longer if chemotherapy was initiated earli‐ er based on Ca125, as opposed to delaying treatment until symptoms developed. In addi‐ tion, QOL was higher in women who underwent treatment at the time of clinical recurrence [47]. Despite the limitations of this study, these findings may have potential impact on clini‐

cal practice.


**Table 1.** Some recent clinical trials that have included QOL as an outcome

The aggressiveness of treatments in advanced ovarian cancer patients place more atten‐ tion upon their QOL than patients diagnosed at an early stage. Several randomized clini‐ cal trials have been conducted in the first-line treatment of ovarian cancer. Clinical trials focus in important issues concerning the combination of surgery and chemotherapy, the identification of new targeted therapeutics and the route and timing of chemotherapy ad‐ ministration [48]. Paclitaxel in combination with a platinum compound is considered a standard care as first-line chemotherapy for advanced ovarian cancer. However, paclitax‐ el is associated with several toxicities (e.g. anemia, thrombocytopenia) that overlap the toxicities of the plantinums, and the co-administration of paclitaxel and a platinum com‐ pound can potentially increase the frequency and/or severity of shared toxicities. By it‐ self, paclitaxel is associated with peripheral neuropathy that can add to the disease burden of the patient [4]. Therefore, studies have been conducted to find the least toxic combination of medications used in chemotherapy in order to improve treatment tolera‐ bility and QOL [49]. For example, a Phase III Trial conducted by the Scotish Gynaecolog‐ ical Cancer Trials Group (SCOTROC Trial), which included 1077 patients, compared carboplatin docetaxel with carboplatin paclitaxel for first line therapy. Results demon‐ strated a clear advantage for docetaxel in terms of neurotoxicity [43]. Concurrent with the developments in intravenous treatment, intraperitoneal treatment has also been shown a valuable strategy. The Gynecologic Oncology Group published data from the GOG randomized phase III trial (GOG 172) pertaining QOL outcomes associated with the use of intravenous paclitaxel plus intraperitoneal cisplatin plus paclitaxel, versus in‐ travenous paclitaxel plus cisplatin, for advanced stage cancer [42]. This was the first Phase III GOG ovarian cancer that proposed a change in route for the administration of front-line chemotherapy. In the intraperitoneal arm, overall survival was improved by approximately 16 months; however, during active treatment, patients reported more QOL disruptions, abdominal discomfort and neurotoxicity compared to those patients re‐ ceiving conventional intravenous chemotherapy. However, only neurotoxicity remained significantly higher for patients in the intraperitoneal arm 12 months post-treatment. Fu‐ ture studies to lessen the added burden associated with intraperitoneal therapy are going [42]. Recently, Vergote et al. [44] reported the results of a Gynaecologic Cancer Inter‐ group Collaboration Trial which compared upfront debulking followed by chemotherapy to neoadjuvant chemotherapy. This was the first randomized Phase III Trial of neoadju‐ vant chemotherapy in ovarian cancer using QOL as an endpoint. The two groups report‐ ed similar survival outcomes. QOL scores did not differ among the two groups [44].

Several clinical trials evaluating ovarian cancer treatments have been carried out, in which QOL is one of the outcomes evaluated. Table 1 describes recent clinical trials that have in‐ cluded QOL as an outcome. QOL measurement in clinical trials has been useful to argue in favor or against novel therapies. Furthermore, there is some evidence demonstrating that QOL is a prognostic indicator for treatment outcomes [26] and future survival [38-41].

**Study Comparison Group QOL measures QOL findings**

FACT-TOI Neurotoxicity and abdominal discomfort subscales

EORTC QOL-C30 EORTC QOL-OV28

EORTC QOL-C30 EORTC QOL-OV28 During active treatment, patients on IP had more QOL disruptions when compared to IV therapy

Global QOL scores did not differ between treatment arms. Less neurotoxicity was found in the docetaxel group

No differences in global health scores

carboplatin/paclitaxel

pegylated doxorubicin arm

the early treatment arm; significant disadvantages in role, emotional, social and fatigue subscales

EORTC QOL-C30 Higher QOL with

EORTC QOL-C30 Higher QOL in the

EORTC QOL-C30 QOL decreased shorter in

GOG-17242 Intraperitoneal (IP) versus

34 Ovarian Cancer - A Clinical and Translational Update

SCOTROC43 Carboplatin docetaxel

OVAR 345 Cisplatin/paclitaxel versus

OV-0547 Early versus delayed

**Table 1.** Some recent clinical trials that have included QOL as an outcome

Vergote (2010)44 a Gynecologic Cancer Intergroup Collaboration Trial

Ferrandina (2008)46 Multicenter Italian Trials in ovarian Cancer group

intravenous (IV) therapy for first line therapy

compared with carboplatin paclitaxel for first line therapy

Neoadjuvant chemotherapy versus primary surgery in stages IIIC or IV

carboplatin/paclitaxel for first line therapy

Pegylated doxorubicin versus gemcitabine for progressive or recurrent disease

treatment for recurrent disease

The aggressiveness of treatments in advanced ovarian cancer patients place more atten‐ tion upon their QOL than patients diagnosed at an early stage. Several randomized clini‐ cal trials have been conducted in the first-line treatment of ovarian cancer. Clinical trials focus in important issues concerning the combination of surgery and chemotherapy, the identification of new targeted therapeutics and the route and timing of chemotherapy ad‐ ministration [48]. Paclitaxel in combination with a platinum compound is considered a The majority of ovarian cancer patients will eventually relapse. In fact, it is not uncommon for ovarian cancer patients to undergo numerous chemotherapeutics treatments. In this con‐ text, the evaluation of QOL is of utmost importance. In the management of recurrent ovari‐ an cancer, tumour control without compromising QOL should be the goal of the therapy [50]. However, there are deficits in the measurement of general QOL data in the recurrent setting, in terms of QOL disruptions and number of studies including QOL measurements [26]. A recent trial published data pertaining the impact of early versus delayed treatment of recurrent ovarian cancer based on Ca125 measurements exceeding twice the upper limit of normal. Results showed that women did not live longer if chemotherapy was initiated earli‐ er based on Ca125, as opposed to delaying treatment until symptoms developed. In addi‐ tion, QOL was higher in women who underwent treatment at the time of clinical recurrence [47]. Despite the limitations of this study, these findings may have potential impact on clini‐ cal practice.

#### **4.2. QOL in ovarian cancer survivors**

Despite the considerable increase in the number of QOL studies carried out in ovarian can‐ cer patients, few studies have focused, particularly, in assessing QOL in ovarian cancer sur‐ vivors. Although, ovarian cancer patients do not belong to the most prevalent survivor population due to the aggressiveness of the disease and relatively low survival rates, it is of utmost importance to understand the QOL of those women who live years after the diagno‐ sis without symptoms of the disease [9, 51, 52]. QOL has been evaluated namely among small samples of survivors by using mostly the EORTC QOL-C30, EORTC QOL-OV28 and supplemented by several other questionnaires to assess specific dimensions of QOL.

survivors had good physical QOL, with few long-term physical symptoms (such as abdomi‐ nal complaints and neurotoxicity) and few unmet needs. However, survivors reported emo‐ tional problems, such as psychological distress (40%), anxiety about Ca125 testing (54%), fear of recurrence (56%) and 26% had scores suggestive of PTSD. Better mental health was associated with less fatigue and pain, fewer stressful life events and higher social support. The authors reported as well sexual problems, namely pain during sexual intercourse (52%). Less than 10% of participants were interested in sex or were sexually active. Additionally, it was noted that younger survivors presented greater sexual problems. Similarly, Mirabeau-Beale et al [57] who conducted the first comparison between early stage (58 women) and ad‐ vanced stage (42 women) survivors on QOL (> 3 years), physical, sexual and mental function, reported that survivors experienced positive overall QOL and long-term adjust‐ ment. Investigators reported no differences between early stage and advanced stage survi‐ vors on overall QOL, unmet needs, social support, complementary therapy use, physical symptoms (neurotoxicity, fatigue and comorbidities), functioning (cognitive, sexual, physi‐ cal, role, emotional and sexuality), spirituality, hopelessness and psychological state. How‐ ever, advanced stage survivors experienced better social functioning. Although, the majority of survivors had a good emotional functioning, scores suggestive of PTSD were noted in 7% of early stage survivors. Diagnosable PTSD scores were not found in the advance stage sur‐ vivors group. Decreased sexual interest attributed to cancer, physical comorbidities, such as degenerative joint disease, gastrointestinal distress and thyroid disease, fear of recurrence, use of complementary and alternative medicines (exercise, vitamins, prayer and massage) in order to improve their QOL were reported by survivors. The most recent account on QOL in ovarian cancer survivors was given by Greimel et al [52], who attempted to fill a gap in the literature by conducting a prospective study on QOL in long-term survivors (> 10 years). This longitudinal study examined survivors at three time points: pre-treatment (baseline), 1 year after diagnosis and 10 years post-treatment using the EORTC QOL-C30. At the base‐ line, 33 survivors were included; of those, 22 died within 5 years post diagnosis and 11 survived beyond 10 years. In general, results corroborated previous findings reporting that survivors experienced a good physical, psychological, social and spiritual health. Despite no differences at baseline in FIGO stage, residual tumour, performance status and treatment characteristics between short-term and long-term survivors, the latter group experienced better physical functioning, role functioning, cognitive functioning and less symptoms than short-term survivors. Higher levels of symptoms and intra operative ascites were also more prevalent in the short-term survivors group. One year after treatment, the majority of the QOL dimensions were comparable among the two groups; however, long-term survivors re‐ ported better global QOL but more insomnia. Emotional functioning and global QOL im‐ proved significantly from baseline to 1 year after diagnosis and remained relatively stable in the 10 year follow-up evaluation. Long-term survivors did not experience more sleeping

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37

problems 10 years after their diagnosis than women from a general population [52].

services when compared to age-adjusted controls from the general population.

Contradicting the trend described above, Liaavaaq et al [53] evaluated 189 ovarian cancer survivors (> 18 months after primary treatment) and found that survivors experienced poor‐ er QOL, had more chronic fatigue and mental morbidity, used more medication and health

Overall, with the exception of the study conducted by Liaavaaq et al [53], available data sug‐ gests that ovarian cancer survivors have generally good QOL; however, specific deficits are reported and these are more prevalent in ovarian cancer survivors that in women without a history of cancer [52, 54-57]. Results concerning psychological functioning are inconsistent, ranging from good emotional status to psychological distress, including PTSD and depres‐ sion. Below are described with more detail findings from recent studies examining QOL in ovarian cancer survivors.

Results from the study conducted by Steward et al [54] support the view that this group of survivors experiences overall good QOL. These investigators assessed 200 ovarian cancer survivors, who were at the time of the study without active disease and not on treatment, on physical, psychological and social well-being. On average, women had been diagnosed with ovarian cancer in the previous 7 years. Results showed that the majority of the survivors (89%) regarded their health as good or excellent. Participants also reported a better mental health and equivalent energy levels comparing to the general population. However, the ma‐ jority of the women suffered from pelvic pain and discomfort (54%). Study findings also demonstrated that although 57% of the survivors referred that their sexual life had been negatively affected by the cancer and its treatment, their general sense of loss regarding sex‐ ual functioning was perceived as moderate to low. Unsurprisingly, women under 55 years of age reported a greater sense of loss about sexual functioning and fertility. According to these authors, the experience of surviving ovarian cancer appeared to have enriched these women, altering their life priorities and developing on them an impressive resilience [54]. Furthermore, authors highlighted that these survivors showed in general a great pleasure in life and relationships [54]. Similar findings were obtained in the study conducted by Wenzel et al [55], who examined 49 early stage ovarian cancer survivors (> 5 years). Findings re‐ vealed that survivors enjoyed a good QOL, with physical, emotional and social well-being comparable to other survivors and same aged samples without a history of cancer. Few defi‐ cits were reported, such as problems related to abdominal and gynaecological symptoms, and neurotoxicity. In the emotional domain, scores were more variable, with only one third of the survivors experiencing an excellent emotional well-being. Fears of future diagnostic tests (30%) and recurrence (20%) were also found. Investigators emphasised the resilience and growth that survivors reported in their study as a result of their ovarian cancer experi‐ ence [55]. Another attempt to understand QOL in ovarian cancer survivors was carried out by Matulonis et al [56], who evaluated 55 early stage survivors. Findings demonstrated that survivors had good physical QOL, with few long-term physical symptoms (such as abdomi‐ nal complaints and neurotoxicity) and few unmet needs. However, survivors reported emo‐ tional problems, such as psychological distress (40%), anxiety about Ca125 testing (54%), fear of recurrence (56%) and 26% had scores suggestive of PTSD. Better mental health was associated with less fatigue and pain, fewer stressful life events and higher social support. The authors reported as well sexual problems, namely pain during sexual intercourse (52%). Less than 10% of participants were interested in sex or were sexually active. Additionally, it was noted that younger survivors presented greater sexual problems. Similarly, Mirabeau-Beale et al [57] who conducted the first comparison between early stage (58 women) and ad‐ vanced stage (42 women) survivors on QOL (> 3 years), physical, sexual and mental function, reported that survivors experienced positive overall QOL and long-term adjust‐ ment. Investigators reported no differences between early stage and advanced stage survi‐ vors on overall QOL, unmet needs, social support, complementary therapy use, physical symptoms (neurotoxicity, fatigue and comorbidities), functioning (cognitive, sexual, physi‐ cal, role, emotional and sexuality), spirituality, hopelessness and psychological state. How‐ ever, advanced stage survivors experienced better social functioning. Although, the majority of survivors had a good emotional functioning, scores suggestive of PTSD were noted in 7% of early stage survivors. Diagnosable PTSD scores were not found in the advance stage sur‐ vivors group. Decreased sexual interest attributed to cancer, physical comorbidities, such as degenerative joint disease, gastrointestinal distress and thyroid disease, fear of recurrence, use of complementary and alternative medicines (exercise, vitamins, prayer and massage) in order to improve their QOL were reported by survivors. The most recent account on QOL in ovarian cancer survivors was given by Greimel et al [52], who attempted to fill a gap in the literature by conducting a prospective study on QOL in long-term survivors (> 10 years). This longitudinal study examined survivors at three time points: pre-treatment (baseline), 1 year after diagnosis and 10 years post-treatment using the EORTC QOL-C30. At the base‐ line, 33 survivors were included; of those, 22 died within 5 years post diagnosis and 11 survived beyond 10 years. In general, results corroborated previous findings reporting that survivors experienced a good physical, psychological, social and spiritual health. Despite no differences at baseline in FIGO stage, residual tumour, performance status and treatment characteristics between short-term and long-term survivors, the latter group experienced better physical functioning, role functioning, cognitive functioning and less symptoms than short-term survivors. Higher levels of symptoms and intra operative ascites were also more prevalent in the short-term survivors group. One year after treatment, the majority of the QOL dimensions were comparable among the two groups; however, long-term survivors re‐ ported better global QOL but more insomnia. Emotional functioning and global QOL im‐ proved significantly from baseline to 1 year after diagnosis and remained relatively stable in the 10 year follow-up evaluation. Long-term survivors did not experience more sleeping problems 10 years after their diagnosis than women from a general population [52].

**4.2. QOL in ovarian cancer survivors**

36 Ovarian Cancer - A Clinical and Translational Update

ovarian cancer survivors.

Despite the considerable increase in the number of QOL studies carried out in ovarian can‐ cer patients, few studies have focused, particularly, in assessing QOL in ovarian cancer sur‐ vivors. Although, ovarian cancer patients do not belong to the most prevalent survivor population due to the aggressiveness of the disease and relatively low survival rates, it is of utmost importance to understand the QOL of those women who live years after the diagno‐ sis without symptoms of the disease [9, 51, 52]. QOL has been evaluated namely among small samples of survivors by using mostly the EORTC QOL-C30, EORTC QOL-OV28 and

supplemented by several other questionnaires to assess specific dimensions of QOL.

Overall, with the exception of the study conducted by Liaavaaq et al [53], available data sug‐ gests that ovarian cancer survivors have generally good QOL; however, specific deficits are reported and these are more prevalent in ovarian cancer survivors that in women without a history of cancer [52, 54-57]. Results concerning psychological functioning are inconsistent, ranging from good emotional status to psychological distress, including PTSD and depres‐ sion. Below are described with more detail findings from recent studies examining QOL in

Results from the study conducted by Steward et al [54] support the view that this group of survivors experiences overall good QOL. These investigators assessed 200 ovarian cancer survivors, who were at the time of the study without active disease and not on treatment, on physical, psychological and social well-being. On average, women had been diagnosed with ovarian cancer in the previous 7 years. Results showed that the majority of the survivors (89%) regarded their health as good or excellent. Participants also reported a better mental health and equivalent energy levels comparing to the general population. However, the ma‐ jority of the women suffered from pelvic pain and discomfort (54%). Study findings also demonstrated that although 57% of the survivors referred that their sexual life had been negatively affected by the cancer and its treatment, their general sense of loss regarding sex‐ ual functioning was perceived as moderate to low. Unsurprisingly, women under 55 years of age reported a greater sense of loss about sexual functioning and fertility. According to these authors, the experience of surviving ovarian cancer appeared to have enriched these women, altering their life priorities and developing on them an impressive resilience [54]. Furthermore, authors highlighted that these survivors showed in general a great pleasure in life and relationships [54]. Similar findings were obtained in the study conducted by Wenzel et al [55], who examined 49 early stage ovarian cancer survivors (> 5 years). Findings re‐ vealed that survivors enjoyed a good QOL, with physical, emotional and social well-being comparable to other survivors and same aged samples without a history of cancer. Few defi‐ cits were reported, such as problems related to abdominal and gynaecological symptoms, and neurotoxicity. In the emotional domain, scores were more variable, with only one third of the survivors experiencing an excellent emotional well-being. Fears of future diagnostic tests (30%) and recurrence (20%) were also found. Investigators emphasised the resilience and growth that survivors reported in their study as a result of their ovarian cancer experi‐ ence [55]. Another attempt to understand QOL in ovarian cancer survivors was carried out by Matulonis et al [56], who evaluated 55 early stage survivors. Findings demonstrated that

Contradicting the trend described above, Liaavaaq et al [53] evaluated 189 ovarian cancer survivors (> 18 months after primary treatment) and found that survivors experienced poor‐ er QOL, had more chronic fatigue and mental morbidity, used more medication and health services when compared to age-adjusted controls from the general population.

Recent studies attempted to improve methodological deficits observed in previous research, for example, by using more standardized and validate measures to assess QOL in this can‐ cer population. However, small sample sizes, heterogeneity of samples, timing of assess‐ ment are among the difficulties posed by current research, which make problematic to reach definite conclusions. Despite this, collectively, existing studies highlight important issues and concerns experienced by ovarian cancer survivors. Beyond the expected physical and sexual sequalaes of the illness and treatment, studies highlighted, particularly, psychological difficulties faced by survivors, which may adversely affect their psychological adjustment and well-being. Findings from survivorship research are paramount to provide critical infor‐ mation to guide the development and design of interventions to assist survivors at risk.

ther larger and rigorous studies are needed to fully understand QOL issues in ovarian can‐ cer patients. Longitudinal studies examining QOL across the different phases of ovarian

Quality of Life in Ovarian Cancer Treatment and Survivorship

http://dx.doi.org/10.5772/54542

39

As new treatment regimens for ovarian cancer continue to be developed and investigated in the hope of improving survival of patients, it is paramount that QOL is regarded as one of the most important endpoints in clinical trials. However, this is not sufficient. It is as well important to routinely assess QOL disruptions in patients in clinical settings in order to screen and identify patients at risk. Therefore, efforts should also be targeted to the develop‐ ment of interventions to be used in women at need, to prevent or ameliorate the negative impact of the illness on QOL. The assessment of QOL in clinical settings also allows the

Ovarian cancer patients may experience QOL disruptions and a wide range of sequalae that do not dissipate with time and may persist for a long-term period. Measuring QOL in ovari‐ an cancer patients during the illness trajectory is of utmost importance. This is of great value to develop and design interventions to assist ovarian cancer patients at need, and as well to

Relationships, Development and Health Research Line, Institute of Cognitive Psychology, Vocational and Social Development, Faculty of Psychology and Educational Sciences, Uni‐

[1] Arriba LN, Fader AN Frasure HE, von Gruenigen. A review of issues surrounding quality of life among women with ovarian cancer. Gynecologic Oncology 2010; 119

[2] Bottomley A. The cancer patient and quality of life. Oncologist 2002; 7(2):120−125.

[3] Higginson I. Using quality of life measures in the clinical setting. British Medical

cancer trajectory would give valuable insights into the QOL of these patients.

identification of QOL needs throughout the cancer trajectory.

Address all correspondence to: vmo.goncalves@hotmail.com

**6. Conclusion**

**Author details**

Vânia Gonçalves

**References**

(2): 390-396.

assist in the therapeutic decision process.

versity of Coimbra, Coimbra, Portugal

Journal 2001; 322 (7297): 1297-300.

The care provided to the cancer patient does not cease when the treatment ends. Survivor‐ ship is now recognized as a phase in the cancer trajectory that requires special attention and ongoing specialized care. In 2006, the Institute of Medicine (IOM) published a report on can‐ cer survivorship entitled: 'From cancer patient to cancer survivor: Lost in transition' [58], identifying unique concerns for cancer survivors, recommending the development of a sur‐ vivorship plan to be developed at the end of treatment for all people treated for cancer of any type. Examples of requirements of the survivorship care plan as recommended by the IOM include, among others, information on possible late and long term effects of treatments and symptoms of such effects, information on the possible effects of cancer on marital/part‐ ner relationship, sexual functioning, work and parenting and the potential future need for psychosocial support, referrals to specific follow-up care providers (e.g. rehabilitation, psy‐ chology), support groups, and/or the patient's primary care provider.

#### **5. QOL in ovarian cancer: The challenges**

Definitely, one of the main challenges in QOL research is to translate and apply the findings obtained in research settings to clinical practice. In fact, in order to fully take advantage of all the benefits offered by QOL research, it is imperative that QOL research provides health care professionals with clinically relevant and interpretable information that can guide treat‐ ment decisions. However, routine use of QOL measures has been limited in clinical settings [6]. Challenges of using QOL data to inform clinical practice may include the use of some‐ what arbitrary cutoff points or magnitude of change in QOL scores to determine when ther‐ apeutic change is needed [26]. To optimize treatment decisions for patients with ovarian cancer, it is paramount that health care professionals are familiar with differences between treatment regimens regarding toxicity, dosage and administration but also findings from QOL measurements [11].

From the research perspective, there is a need for standardized collection and reporting of QOL data from ovarian cancer patients, such as use of common instruments that demon‐ strate the most sensitivity to the study hypothesis and outcomes of interest, common data collection time points, minimum expectations for data analysis and publication reporting guidelines. These would allow comparative effectiveness research to be carried out [18]. Fur‐ ther larger and rigorous studies are needed to fully understand QOL issues in ovarian can‐ cer patients. Longitudinal studies examining QOL across the different phases of ovarian cancer trajectory would give valuable insights into the QOL of these patients.

As new treatment regimens for ovarian cancer continue to be developed and investigated in the hope of improving survival of patients, it is paramount that QOL is regarded as one of the most important endpoints in clinical trials. However, this is not sufficient. It is as well important to routinely assess QOL disruptions in patients in clinical settings in order to screen and identify patients at risk. Therefore, efforts should also be targeted to the develop‐ ment of interventions to be used in women at need, to prevent or ameliorate the negative impact of the illness on QOL. The assessment of QOL in clinical settings also allows the identification of QOL needs throughout the cancer trajectory.

#### **6. Conclusion**

Recent studies attempted to improve methodological deficits observed in previous research, for example, by using more standardized and validate measures to assess QOL in this can‐ cer population. However, small sample sizes, heterogeneity of samples, timing of assess‐ ment are among the difficulties posed by current research, which make problematic to reach definite conclusions. Despite this, collectively, existing studies highlight important issues and concerns experienced by ovarian cancer survivors. Beyond the expected physical and sexual sequalaes of the illness and treatment, studies highlighted, particularly, psychological difficulties faced by survivors, which may adversely affect their psychological adjustment and well-being. Findings from survivorship research are paramount to provide critical infor‐ mation to guide the development and design of interventions to assist survivors at risk.

The care provided to the cancer patient does not cease when the treatment ends. Survivor‐ ship is now recognized as a phase in the cancer trajectory that requires special attention and ongoing specialized care. In 2006, the Institute of Medicine (IOM) published a report on can‐ cer survivorship entitled: 'From cancer patient to cancer survivor: Lost in transition' [58], identifying unique concerns for cancer survivors, recommending the development of a sur‐ vivorship plan to be developed at the end of treatment for all people treated for cancer of any type. Examples of requirements of the survivorship care plan as recommended by the IOM include, among others, information on possible late and long term effects of treatments and symptoms of such effects, information on the possible effects of cancer on marital/part‐ ner relationship, sexual functioning, work and parenting and the potential future need for psychosocial support, referrals to specific follow-up care providers (e.g. rehabilitation, psy‐

Definitely, one of the main challenges in QOL research is to translate and apply the findings obtained in research settings to clinical practice. In fact, in order to fully take advantage of all the benefits offered by QOL research, it is imperative that QOL research provides health care professionals with clinically relevant and interpretable information that can guide treat‐ ment decisions. However, routine use of QOL measures has been limited in clinical settings [6]. Challenges of using QOL data to inform clinical practice may include the use of some‐ what arbitrary cutoff points or magnitude of change in QOL scores to determine when ther‐ apeutic change is needed [26]. To optimize treatment decisions for patients with ovarian cancer, it is paramount that health care professionals are familiar with differences between treatment regimens regarding toxicity, dosage and administration but also findings from

From the research perspective, there is a need for standardized collection and reporting of QOL data from ovarian cancer patients, such as use of common instruments that demon‐ strate the most sensitivity to the study hypothesis and outcomes of interest, common data collection time points, minimum expectations for data analysis and publication reporting guidelines. These would allow comparative effectiveness research to be carried out [18]. Fur‐

chology), support groups, and/or the patient's primary care provider.

**5. QOL in ovarian cancer: The challenges**

38 Ovarian Cancer - A Clinical and Translational Update

QOL measurements [11].

Ovarian cancer patients may experience QOL disruptions and a wide range of sequalae that do not dissipate with time and may persist for a long-term period. Measuring QOL in ovari‐ an cancer patients during the illness trajectory is of utmost importance. This is of great value to develop and design interventions to assist ovarian cancer patients at need, and as well to assist in the therapeutic decision process.

#### **Author details**

#### Vânia Gonçalves

Address all correspondence to: vmo.goncalves@hotmail.com

Relationships, Development and Health Research Line, Institute of Cognitive Psychology, Vocational and Social Development, Faculty of Psychology and Educational Sciences, Uni‐ versity of Coimbra, Coimbra, Portugal

#### **References**


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**Chapter 3**

**Preventive Strategies for Ovarian Cancer**

According to the American Cancer Society, in 2012 ovarian cancer is expected to account for 3% (22,280) of all new cases and 6% (15,500) of all female cancer deaths in the United States. The proportion of ovarian cancer among gynaecological cancers is increasing, also because of the decrease in cervical cancer as a result of pap smear screening programmes. On the other hand, survival from ovarian cancer is the poorest of all gynaecological cancers, with a fiveyear relative survival rate of 44% for all stages [1,2]. The main reasons for this poor survival are the lack of early detection strategies and an unfavourable anatomical situation. Thus, the vast majority of ovarian cancer is diagnosed at an advanced stage and therapy for this pathology is very complex [3-5]. Reduction in mortality rates could be gained both with new screening strategies and with ameliorations in surgical and medical treatments. However, neither of these approaches will affect cancer incidence, thus, it is clear that the prospects for making a major impact on the mortality from ovarian cancer lie more in the area of prevention.

The purpose of this chapter is to identify the evidence for the appropriate practical strategies to prevent ovarian cancer or the detection of cancer in the early stages in order to improve the overall survival. The search was restricted to full reports and guidelines published in English between 2000 and May 2012, in an attempt to summarize the principal findings regarding

Primary prevention aims to prevent the disease before its biological onset, thus it is based on

and reproduction in any medium, provided the original work is properly cited.

© 2013 Cortesi et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

**2. Primary prevention for ovarian cancer in general population**

L. Cortesi, A. Toss and E. De Matteis

http://dx.doi.org/10.5772/54686

**1. Introduction**

Additional information is available at the end of the chapter

primary and secondary ovarian cancer prevention.

avoiding risk factors and increasing protective factors.


### **Preventive Strategies for Ovarian Cancer**

L. Cortesi, A. Toss and E. De Matteis

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54686

#### **1. Introduction**

[52] Greimel E, Daghofer F, Petru E. Prospective assessment of quality of life in long-term ovarian cancer survivors. International Journal of Cancer 2011; 128(12):3005-3011

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[56] Matulonis UA, Kornblith A, Lee H, Bryan J, Gibson C, Wells C, Lee J, Sullivan L, Pen‐ son R. Long-term adjustment of early-stage ovarian cancer survivors. International

[57] Mirabeau-Beale KL, Kornblith AB, Penson RT, Lee H, Goodman A, Campos SM, Duska L, Pereira L, Bryan J, Matulonis UA. Comparison of the quality of life of early and advanced stage ovarian cancer survivors. Gynecologic Oncology 2009;114(2):

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transition. Washington, DC: National Academic Press; 2006.

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353-359.

2001; 83(3):537-542.

44 Ovarian Cancer - A Clinical and Translational Update

According to the American Cancer Society, in 2012 ovarian cancer is expected to account for 3% (22,280) of all new cases and 6% (15,500) of all female cancer deaths in the United States. The proportion of ovarian cancer among gynaecological cancers is increasing, also because of the decrease in cervical cancer as a result of pap smear screening programmes. On the other hand, survival from ovarian cancer is the poorest of all gynaecological cancers, with a fiveyear relative survival rate of 44% for all stages [1,2]. The main reasons for this poor survival are the lack of early detection strategies and an unfavourable anatomical situation. Thus, the vast majority of ovarian cancer is diagnosed at an advanced stage and therapy for this pathology is very complex [3-5]. Reduction in mortality rates could be gained both with new screening strategies and with ameliorations in surgical and medical treatments. However, neither of these approaches will affect cancer incidence, thus, it is clear that the prospects for making a major impact on the mortality from ovarian cancer lie more in the area of prevention.

The purpose of this chapter is to identify the evidence for the appropriate practical strategies to prevent ovarian cancer or the detection of cancer in the early stages in order to improve the overall survival. The search was restricted to full reports and guidelines published in English between 2000 and May 2012, in an attempt to summarize the principal findings regarding primary and secondary ovarian cancer prevention.

### **2. Primary prevention for ovarian cancer in general population**

Primary prevention aims to prevent the disease before its biological onset, thus it is based on avoiding risk factors and increasing protective factors.

© 2013 Cortesi et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

A summary of the most significant risk and protective factors, with relative hazard ratio, for epithelial ovarian cancer is summarized in Tables 1a and1b,

ences [21]. In particular, regarding dietary habits, a comprehensive meta-analysis of the observational studies published up to September 2011 provided no evidence of a material association between alcohol drinking and epithelial ovarian cancer risk [22]. Finally, a recent study provided some suggestion that soy and phytoestrogen consumption may decrease

Preventive Strategies for Ovarian Cancer http://dx.doi.org/10.5772/54686 47

Exposure to radiation may increase the risk of ovarian cancer and the risk increases with increasing dose. The Life Span Study incidence data for ovarian cancer demonstrated a borderline significant association [24], and mortality data showed a significant positive

With regards to reproductive factors, early age at menarche and late menopause have been consistently associated with an increased risk of ovarian cancer, likely due to an increase in ovulation and in oestrogen exposure [26]. The effect of combined hormonal contraceptive use on the risk of ovarian cancer has been long discussed. In 2007, the IARC review concluded that women who had at least for a period used combined hormonal contraceptives orally had an overall reduced risk for ovarian cancer, which persists for at least 20 years after cessation of use, and an inverse relationship was observed with duration of use [27]. These results have been confirmed by the Collaborative Group on Epidemiological Studies of Ovarian Cancer [16] that reported an overall reduction in ovarian cancer risk in users versus non-users of 27%, which was not confined to any particular type of oral formulation nor to any histological type of ovarian cancer, although it was less consistent for mucinous than for other types of ovarian cancer. On this basis, the "incessant menstruation" hypothesis was postulated, which con‐ cludes that the use of oral contraceptives (OC) should be favoured for prolonged periods of time, especially in women with endometriosis, a population at doubled risk of ovarian cancer [28]. On the other hand, in the Million Women Study HRT after menopause was shown to

Women who have never had children are at increased risk of developing ovarian cancer [29]. Regarding fertility drug use, previous studies have provided conflicting results. Recent data demonstrated that fertility drug use does not significantly contribute to ovarian cancer risk among the majority of women. However, women who despite their use remain nulliparous may have an increased risk [30]. The role of breastfeeding as a protective factor against ovarian cancer has been long discussed. Finally, the risk of ovarian cancer decreases in women who underwent bilateral tube ligation or hysterectomy, probably because these surgical interven‐ tions do not allow the carcinogenic agents to enter the body from the vagina and reach the ovaries [19, 31]. For instance, a number of observational studies (largely case-control) con‐ ducted over the last two decades suggested an association between use of talc powders on the female perineum and increased risk of ovarian cancer, although the weak statistical associa‐ tions observed in a number of epidemiological studies do not support a causal association

Endometriosis represents another considerable risk factor for epithelial ovarian cancer. In particular, self-reported endometriosis was associated with a significantly increased risk of clear-cell, low-grade serous and endometrioid invasive ovarian cancers. No association was noted between endometriosis and risk of mucinous or high-grade serous invasive ovarian

ovarian cancer risk, although the results did not reach statistical significance [23].

association between exposure to radiation and ovarian cancer [25].

increase the risk of ovarian cancer [11].

between cosmetic talc use and ovarian cancer [32,33].


**Table 1.** (a) Main significant risk factors for ovarian cancer. (b) Main significant protective factors for ovarian cancer.

The average age at diagnosis is approximately 60 years, but the overall incidence of ovarian cancer rises with increasing age up to 75-84 years, due to the accumulation of random genetic alterations, before declining slightly among women beyond 84 years [20]. Women residing in North America, Northern Europe or in any industrialized Western country have a higher risk of developing ovarian cancer. Conversely, women residing in developing countries have shown the lowest rate [6]. The exact reasons for this distribution are unknown but discrepan‐ cies in parity, rates of gynaecologic surgery and dietary habits may account for some differ‐ ences [21]. In particular, regarding dietary habits, a comprehensive meta-analysis of the observational studies published up to September 2011 provided no evidence of a material association between alcohol drinking and epithelial ovarian cancer risk [22]. Finally, a recent study provided some suggestion that soy and phytoestrogen consumption may decrease ovarian cancer risk, although the results did not reach statistical significance [23].

A summary of the most significant risk and protective factors, with relative hazard ratio, for

**AUTHORS RISK FACTORS RR (95% CI)** Schorge JO et al. [6] White race 1.35 (1.08-1.50) Schouten LJ et al. [7] Height≥160 cm 1.38 (1.16-1.65) Lahmann PH et al. [8] BMI≥25 1.33 (1.05-1.68) Camargo MC et al. [9] Asbestos exposure 1.77 (1.37-2.28) Cramer DW [10] early age at menarche 1.74 (1.28-2.18) Cramer DW [10] late menopause 1.61 (1.15-2.08) Beral V et al. [11] HRT 1.20 (0.98-1.32) Melin A et al. [12] Endometriosis 1.43 (1.19-1.71)

> BRCA1 BRCA2

Watson P et al. [14] MMR 19 (5.0-30.0) (a) **AUTHORS PROTECTIVE FACTORS HR (95% CI)** Trudel D et al. [15] green tea components 0.66 (0.54-0.80)

Ness RB et al. [17] Multiparity(≥ 4 pregnancies) 0.40 (0.30-0.50)

Danforth KN et al. [18] breastfeeding 0.98 per month

Hankinson SE et al. [19] bilateral tube ligation 0.33 (0.16-0.64) Hankinson SE et al. [19] hysterectomy 0.67 (0.45-1.00) (b)

**Table 1.** (a) Main significant risk factors for ovarian cancer. (b) Main significant protective factors for ovarian cancer.

The average age at diagnosis is approximately 60 years, but the overall incidence of ovarian cancer rises with increasing age up to 75-84 years, due to the accumulation of random genetic alterations, before declining slightly among women beyond 84 years [20]. Women residing in North America, Northern Europe or in any industrialized Western country have a higher risk of developing ovarian cancer. Conversely, women residing in developing countries have shown the lowest rate [6]. The exact reasons for this distribution are unknown but discrepan‐ cies in parity, rates of gynaecologic surgery and dietary habits may account for some differ‐

hormonal contraceptive use 0.73 (0.70-0.76)

42.4 (15-119.6) 20.6 (7.75-57.2)

( 0.97-1.00)

epithelial ovarian cancer is summarized in Tables 1a and1b,

46 Ovarian Cancer - A Clinical and Translational Update

Chen S et al. [13]

Collaborative Group on Epidemiological Studies of Ovarian Cancer [16]

Exposure to radiation may increase the risk of ovarian cancer and the risk increases with increasing dose. The Life Span Study incidence data for ovarian cancer demonstrated a borderline significant association [24], and mortality data showed a significant positive association between exposure to radiation and ovarian cancer [25].

With regards to reproductive factors, early age at menarche and late menopause have been consistently associated with an increased risk of ovarian cancer, likely due to an increase in ovulation and in oestrogen exposure [26]. The effect of combined hormonal contraceptive use on the risk of ovarian cancer has been long discussed. In 2007, the IARC review concluded that women who had at least for a period used combined hormonal contraceptives orally had an overall reduced risk for ovarian cancer, which persists for at least 20 years after cessation of use, and an inverse relationship was observed with duration of use [27]. These results have been confirmed by the Collaborative Group on Epidemiological Studies of Ovarian Cancer [16] that reported an overall reduction in ovarian cancer risk in users versus non-users of 27%, which was not confined to any particular type of oral formulation nor to any histological type of ovarian cancer, although it was less consistent for mucinous than for other types of ovarian cancer. On this basis, the "incessant menstruation" hypothesis was postulated, which con‐ cludes that the use of oral contraceptives (OC) should be favoured for prolonged periods of time, especially in women with endometriosis, a population at doubled risk of ovarian cancer [28]. On the other hand, in the Million Women Study HRT after menopause was shown to increase the risk of ovarian cancer [11].

Women who have never had children are at increased risk of developing ovarian cancer [29]. Regarding fertility drug use, previous studies have provided conflicting results. Recent data demonstrated that fertility drug use does not significantly contribute to ovarian cancer risk among the majority of women. However, women who despite their use remain nulliparous may have an increased risk [30]. The role of breastfeeding as a protective factor against ovarian cancer has been long discussed. Finally, the risk of ovarian cancer decreases in women who underwent bilateral tube ligation or hysterectomy, probably because these surgical interven‐ tions do not allow the carcinogenic agents to enter the body from the vagina and reach the ovaries [19, 31]. For instance, a number of observational studies (largely case-control) con‐ ducted over the last two decades suggested an association between use of talc powders on the female perineum and increased risk of ovarian cancer, although the weak statistical associa‐ tions observed in a number of epidemiological studies do not support a causal association between cosmetic talc use and ovarian cancer [32,33].

Endometriosis represents another considerable risk factor for epithelial ovarian cancer. In particular, self-reported endometriosis was associated with a significantly increased risk of clear-cell, low-grade serous and endometrioid invasive ovarian cancers. No association was noted between endometriosis and risk of mucinous or high-grade serous invasive ovarian cancers or borderline tumours of either subtype [34]. Also, pelvic inflammatory disease has been suggested to double the risk of epithelial ovarian cancer [35], but few studies have been done and the conclusions are inconsistent.

The most important risk factor still remains a family history of breast or ovarian cancer. Up to 10% of ovarian cancer patients may have inherited a germline mutation that places them at in‐ creased risk of the disease. Mutations in the breast and ovarian cancer-susceptibility genes *BRCA1* and *BRCA2* confer an increased lifetime risk of ovarian cancer. *BRCA1* and *BRCA2* are tumour suppressor genes involved in many cellular functions to prevent carcinogenesis [Fig.1]. The most important risk factor still remains a family history of breast or ovarian cancer. Up to 10% of ovarian cancer patients may have inherited a germline mutation that places them at increased risk of the disease. Mutations in the breast and ovarian cancer-susceptibility genes *BRCA1* and *BRCA2* confer an increased lifetime risk of ovarian cancer. *BRCA1*  and *BRCA2* are tumour suppressor genes involved in many cellular functions to prevent carcinogenesis [Fig.1].

**Figure 1.** BrCa1 protein functions.

ovarian cancer.

Figure 1. BrCa1 protein functions. The mechanism to repair the double-strand DNA breaks is shown in Fig.2.

**Figure 2**: *Repair of double-strand DNA breaks by BRCA1 and BRCA2 genes.*

Heterozygous germline mutation leads to genetic instability as shown in the Fig.3, modified by Brodie *et al.* [36]

Heterozygous germline mutation leads to genetic instability as shown in the Fig.3, modified by Brodie *et al.* [36]

However, *BRCA* mutations do not account for the entire range of hereditary ovarian cancer syndromes. Other hereditary epithelial ovarian cancers are attributed to Lynch syndrome. Lynch syndrome is an autosomal dominant disorder, which predisposes to colorectal cancer, endometrial cancer, ovarian, gastric, small bowel, biliary/pancreatic, urothelial, skin, and central nervous system cancers. The cumulative risk of ovarian cancer is estimated to be 8–10%, with an average age at onset of 42 years [14]. Moreover, other genes often associated with rare cancer syndromes such as *TP53* and *PTEN*, or *CHEK2* and *PALB2* confer a low to moderate risk of breast and ovarian cancer [37-39]. Recent technological advances have aided in the recognition of additional tumour suppressor genes potentially associated with hereditary breast cancer, such as *RAD51* and *BARD1* [40]. To date, at least 16 genes have been associated with hereditary ovarian cancer, mostly involved in the *FA-BRCA* pathway and the mismatch repair system. However, many families with suspicious pedigrees do not have a specific mutation identified through clinical testing, due to a currently undetectable *BRCA1/2* mutation or a mutation in another susceptibility gene. Although their cancer risks are not as well defined, these families should be considered as part of the hereditary breast and/or ovarian cancer spectrum [13].

However, most of the common risk and protective factors only slightly influence the risk of developing ovarian cancer, thus, to date; the knowledge of these factors has still not been translated into practical strategies to prevent

The mechanism to repair the double-strand DNA breaks is shown in Fig.2.

3

Heterozygous germline mutation leads to genetic instability as shown in the Fig.3, modified

Preventive Strategies for Ovarian Cancer http://dx.doi.org/10.5772/54686 49

Heterozygous germline mutation leads to genetic instability as shown in the Fig.3, modified by Brodie *et al.* [36]

However, *BRCA* mutations do not account for the entire range of hereditary ovarian cancer syndromes. Other hereditary epithelial ovarian cancers are attributed to Lynch syndrome. Lynch syndrome is an autosomal dominant disorder, which predisposes to colorectal cancer, endometrial cancer, ovarian, gastric, small bowel, biliary/pancreatic, urothelial, skin, and central nervous system cancers. The cumulative risk of ovarian cancer is estimated to be 8–10%, with an average age at onset of 42 years [14]. Moreover, other genes often associated with rare cancer syndromes such as *TP53* and *PTEN*, or *CHEK2* and *PALB2* confer a low to moderate risk of breast and ovarian cancer [37-39]. Recent technological advances have aided in the recognition of additional tumour suppressor genes potentially associated with hereditary breast cancer, such as *RAD51* and *BARD1* [40]. To date, at least 16 genes have been associated with hereditary ovarian cancer, mostly involved in the *FA-BRCA* pathway and the mismatch repair system. However, many families with suspicious pedigrees do not have a specific mutation identified through clinical testing, due to a currently undetectable *BRCA1/2* mutation or a mutation in another susceptibility gene. Although their cancer risks are not as well defined, these families should be considered as part of the hereditary breast and/or ovarian cancer spectrum [13].

However, *BRCA* mutations do not account for the entire range of hereditary ovarian can‐ cer syndromes. Other hereditary epithelial ovarian cancers are attributed to Lynch syn‐ drome. Lynch syndrome is an autosomal dominant disorder, which predisposes to colorectal cancer, endometrial cancer, ovarian, gastric, small bowel, biliary/pancreatic, uro‐ thelial, skin, and central nervous system cancers. The cumulative risk of ovarian cancer is estimated to be 8–10%, with an average age at onset of 42 years [14]. Moreover, other genes often associated with rare cancer syndromes such as *TP53* and *PTEN*, or *CHEK2* and *PALB2* confer a low to moderate risk of breast and ovarian cancer [37-39]. Recent technological advances have aided in the recognition of additional tumour suppressor genes potentially associated with hereditary breast cancer, such as *RAD51* and *BARD1* [40]. To date, at least 16 genes have been associated with hereditary ovarian cancer, most‐ ly involved in the *FA-BRCA* pathway and the mismatch repair system. However, many families with suspicious pedigrees do not have a specific mutation identified through clin‐ ical testing, due to a currently undetectable *BRCA1/2* mutation or a mutation in another susceptibility gene. Although their cancer risks are not as well defined, these families should be considered as part of the hereditary breast and/or ovarian cancer spectrum [13]. However, most of the common risk and protective factors only slightly influence the risk of developing ovarian cancer, thus, to date; the knowledge of these factors has still not been

However, most of the common risk and protective factors only slightly influence the risk of developing ovarian cancer, thus, to date; the knowledge of these factors has still not been translated into practical strategies to prevent

**Figure 2**: *Repair of double-strand DNA breaks by BRCA1 and BRCA2 genes.*

**Figure 3:** *Genetic instability in the germline and somatic BrCa1 mutations [36].* 

**Figure 3.** Genetic instability in the germline and somatic BrCa1 mutations [36].

translated into practical strategies to prevent ovarian cancer.

4

by Brodie *et al.* [36]

ovarian cancer.

4

Figure 2. Repair of double-strand DNA breaks by BRCA1 and BRCA2 genes. **Figure 2.** Repair of double-strand DNA breaks by BRCA1 and BRCA2 genes.

**Figure 3:** *Genetic instability in the germline and somatic BrCa1 mutations [36].*

4

Heterozygous germline mutation leads to genetic instability as shown in the Fig.3, modified by Brodie *et al.* [36]

Heterozygous germline mutation leads to genetic instability as shown in the Fig.3, modified by Brodie *et al.* [36]

**Figure 2**: *Repair of double-strand DNA breaks by BRCA1 and BRCA2 genes.*

**Figure 3:** *Genetic instability in the germline and somatic BrCa1 mutations [36].* 

However, *BRCA* mutations do not account for the entire range of hereditary ovarian cancer syndromes. Other hereditary epithelial ovarian cancers are attributed to Lynch syndrome. Lynch syndrome is an autosomal dominant

with an average age at onset of 42 years [14]. Moreover, other genes often associated with rare cancer syndromes such

disorder, which predisposes to colorectal cancer, endometrial cancer, ovarian, gastric, small bowel, biliary/pancreatic, urothelial, skin, and central nervous system cancers. The cumulative risk of ovarian cancer is estimated to be 8–10%, **Figure 3.** Genetic instability in the germline and somatic BrCa1 mutations [36].

3

4

cancers or borderline tumours of either subtype [34]. Also, pelvic inflammatory disease has been suggested to double the risk of epithelial ovarian cancer [35], but few studies have been

The most important risk factor still remains a family history of breast or ovarian cancer. Up to 10% of ovarian cancer patients may have inherited a germline mutation that places them at in‐ creased risk of the disease. Mutations in the breast and ovarian cancer-susceptibility genes *BRCA1* and *BRCA2* confer an increased lifetime risk of ovarian cancer. *BRCA1* and *BRCA2* are tumour suppressor genes involved in many cellular functions to prevent carcinogenesis [Fig.1].

> BRCA1 tumor suppressor protein

> > Prevention of tumorigenesis

The mechanism to repair the double-strand DNA breaks is shown in Fig.2.

Figure 2. Repair of double-strand DNA breaks by BRCA1 and BRCA2 genes.

**Figure 3:** *Genetic instability in the germline and somatic BrCa1 mutations [36].*

**Figure 2.** Repair of double-strand DNA breaks by BRCA1 and BRCA2 genes.

Heterozygous germline mutation leads to genetic instability as shown in the Fig.3, modified by Brodie *et al.* [36]

Heterozygous germline mutation leads to genetic instability as shown in the Fig.3, modified by Brodie *et al.* [36]

However, *BRCA* mutations do not account for the entire range of hereditary ovarian cancer syndromes. Other hereditary epithelial ovarian cancers are attributed to Lynch syndrome. Lynch syndrome is an autosomal dominant disorder, which predisposes to colorectal cancer, endometrial cancer, ovarian, gastric, small bowel, biliary/pancreatic, urothelial, skin, and central nervous system cancers. The cumulative risk of ovarian cancer is estimated to be 8–10%, with an average age at onset of 42 years [14]. Moreover, other genes often associated with rare cancer syndromes such as *TP53* and *PTEN*, or *CHEK2* and *PALB2* confer a low to moderate risk of breast and ovarian cancer [37-39]. Recent technological advances have aided in the recognition of additional tumour suppressor genes potentially associated with hereditary breast cancer, such as *RAD51* and *BARD1* [40]. To date, at least 16 genes have been associated with hereditary ovarian cancer, mostly involved in the *FA-BRCA* pathway and the mismatch repair system. However, many families with suspicious pedigrees do not have a specific mutation identified through clinical testing, due to a currently undetectable *BRCA1/2* mutation or a mutation in another susceptibility gene. Although their cancer risks are not as well defined, these families should be considered as part of the hereditary breast and/or ovarian cancer spectrum [13].

However, most of the common risk and protective factors only slightly influence the risk of developing ovarian cancer, thus, to date; the knowledge of these factors has still not been translated into practical strategies to prevent

Regulation of transcription

Modulates gene expression in response to cell stress P21DNA damage

The most important risk factor still remains a family history of breast or ovarian cancer. Up to 10% of ovarian cancer patients may have inherited a germline mutation that places them at increased risk of the disease. Mutations in the breast and ovarian cancer-susceptibility genes *BRCA1* and *BRCA2* confer an increased lifetime risk of ovarian cancer. *BRCA1*  and *BRCA2* are tumour suppressor genes involved in many cellular functions to prevent carcinogenesis [Fig.1].

> Regulation of cell cycle

Induces cell cycle arrest at the G1/S, S and G2/M checkpoints

done and the conclusions are inconsistent.

48 Ovarian Cancer - A Clinical and Translational Update

DNA repair Protein

Figure 1. BrCa1 protein functions.

Repair of double strand DNA breaks/ DNA adducts

**Figure 1.** BrCa1 protein functions.

ovarian cancer.

Ubiquitination

Mediates E2-dependent ubiquitination

The mechanism to repair the double-strand DNA breaks is shown in Fig.2.

**Figure 2**: *Repair of double-strand DNA breaks by BRCA1 and BRCA2 genes.*

as *TP53* and *PTEN*, or *CHEK2* and *PALB2* confer a low to moderate risk of breast and ovarian cancer [37-39]. Recent technological advances have aided in the recognition of additional tumour suppressor genes potentially associated with hereditary breast cancer, such as *RAD51* and *BARD1* [40]. To date, at least 16 genes have been associated with hereditary ovarian cancer, mostly involved in the *FA-BRCA* pathway and the mismatch repair system. However, many families with suspicious pedigrees do not have a specific mutation identified through clinical testing, due to a currently undetectable *BRCA1/2* mutation or a mutation in another susceptibility gene. Although their cancer risks are not as well defined, these families should be considered as part of the hereditary breast and/or ovarian cancer spectrum [13]. However, most of the common risk and protective factors only slightly influence the risk of developing ovarian cancer, thus, to date; the knowledge of these factors has still not been translated into practical strategies to prevent ovarian cancer. However, *BRCA* mutations do not account for the entire range of hereditary ovarian can‐ cer syndromes. Other hereditary epithelial ovarian cancers are attributed to Lynch syn‐ drome. Lynch syndrome is an autosomal dominant disorder, which predisposes to colorectal cancer, endometrial cancer, ovarian, gastric, small bowel, biliary/pancreatic, uro‐ thelial, skin, and central nervous system cancers. The cumulative risk of ovarian cancer is estimated to be 8–10%, with an average age at onset of 42 years [14]. Moreover, other genes often associated with rare cancer syndromes such as *TP53* and *PTEN*, or *CHEK2* and *PALB2* confer a low to moderate risk of breast and ovarian cancer [37-39]. Recent technological advances have aided in the recognition of additional tumour suppressor genes potentially associated with hereditary breast cancer, such as *RAD51* and *BARD1* [40]. To date, at least 16 genes have been associated with hereditary ovarian cancer, most‐ ly involved in the *FA-BRCA* pathway and the mismatch repair system. However, many families with suspicious pedigrees do not have a specific mutation identified through clin‐ ical testing, due to a currently undetectable *BRCA1/2* mutation or a mutation in another susceptibility gene. Although their cancer risks are not as well defined, these families should be considered as part of the hereditary breast and/or ovarian cancer spectrum [13].

However, most of the common risk and protective factors only slightly influence the risk of developing ovarian cancer, thus, to date; the knowledge of these factors has still not been translated into practical strategies to prevent ovarian cancer.

#### **3. Primary prevention for ovarian cancer in high risk women**

Some women have a high risk of developing ovarian cancer due to hereditary conditions associated to BRCA syndrome and Lynch syndrome. Thus, when one of these forms of hereditary or familial breast and/or ovarian cancer is suspected in clinical practice, the general practitioner should refer the patient to a cancer centre specialising in cancer-specific genetic counselling for the identification, definition and management of risk. Genetic counselling, defined by the American Society of Human Genetics as 'a communication process which deals with the human problems associated with the occurrence or risk of occurrence of a genetic disorder in a family', involves one or more professional figures to help the affected individuals or families [41-44]. Genetic counselling in the oncological setting (cancer-specific genetic counselling) should also provide sufficient information to enable the user to make a fully informed choice as to course of action, particularly with regards to prevention, in the case of the identification of a mutation or of a familial cancer risk [45, 46].

RRSO has also been demonstrated to decrease the risk of both breast and ovarian cancer in *BRCA1/2* mutation carriers [55-60]. However, the studies examining the extent of risk reduction have used different designs; some are retrospective case–control studies, while others used a prospective cohort design. In a large, retrospective analysis of 551 *BRCA* carriers, RRSO was found to reduce the risk of ovarian cancer by 96% and breast cancer by 53% at a mean followup of 9 years [55]. A multicentre prospective study, Kauff *et al*. [56] found that, during a 3-year follow-up, RRSO was associated with an 85% reduction in BRCA1-associated gynaecologic cancer risk and a 72% reduction in BRCA2-associated breast cancer risk. Although protection against BRCA1-associated breast cancer and BRCA2-associated gynaecologic cancer was suggested, neither effect reached statistical significance. The authors postulate that the protection conferred by RRSO against breast and gynaecologic cancers may differ between the

Similar findings were observed in a prospective study of 170 *BRCA* carriers. During a mean fol‐ low-up of 2 years, the incidence of ovarian or peritoneal cancer and breast cancer was significant‐ ly greater amongst those women who selected surveillance than amongst those who chose to undergo RRSO [59.] Even among prospective studies, the inclusion criteria and the definitions of follow-up time differ. In some studies, only unaffected mutation-positive women are included and followed up. In others, particularly when examining ovarian cancer risk, women with breast cancer are included. Such differences in study design can introduce biases (such as the survival bias) and can have an impact on risk reduction estimates. For example, the reported efficacy of RRSO in reducing the risk of ovarian/fallopian tube cancers varies from 71% to 96% [55-61]. Al‐ though these estimates imply a substantial reduction in risk, this variability may affect the deci‐ sions of premenopausal women who are making a decision about whether to undergo a treatment that will cause abrupt and premature menopause. Patients and their physicians need as much information as possible regarding the efficacy of RRSO in reducing cancer risk to bal‐

ance this benefit with the health risks caused by premature entry into menopause.

**With/Without RRSO**

Rebbeck et al., 2002 [55] RC 261/292 8.5 96 53 Kauff et al., 2008 [56] PC 509/283 3.2 85 72 Finch et al., 2006 [57] RC 1041/779 3.5 80 NA Chang-Claude et al., 2007 [58] RC 55/1601 65,675 PY NA 44 Rutter et al., 2003 [60] RC 5/223 NA 67 NA Kauff et al., 2002 [61] PC 98/72 2.0 85 68

PC = prospective cohort; RC = retrospective cohort; MYFU = mean years of follow-up; PY = person-years; NR = not reported;

**Table 2.** Published studies on risk-reducing salpingo-oophorectomy in BrCa1and/ or BrCa2 mutation carriers.

**MYFU**

**OC Risk Reduction (%)**

Preventive Strategies for Ovarian Cancer http://dx.doi.org/10.5772/54686 51

**BC Risk Reduction (%)**

A summary of published studies on RRSO is presented in Table 2.

**design**

and NA = not applicable; RRSO = risk-reducing salpingo-oophorectomy.

carriers of *BRCA1* and *BRCA2* mutations.

**Reference Study**

A recent review investigated the impact of cancer genetic risk assessment on outcomes, including perceived risk of inherited cancer and psychological distress. The review found favourable outcomes for patients after risk assessment for familial breast cancer, suggesting that cancer-specific genetic risk assessment services help to reduce distress, improve the accuracy of the perception of risk of ovarian cancer, and increase the knowledge of ovarian cancer and genetics. However, there were too few papers to make any significant conclusions on how best to deliver cancer genetic risk assessment services. Further research is needed, assessing the best means of delivering cancer risk evaluation, by different health professionals, in different ways and in alternative locations [47].

Women at increased risk of breast and ovarian cancer are advised to consider risk-reducing strategies; however, such methods vary in their effectiveness. These strategies include chemoprevention and prophylactic surgery (risk-reducing salpingo-oophorectomy, RRSO). Risk-reducing strategies have been shown to have associations with a lengthening of life expectancy in *BRCA1/2* carriers.

#### **3.1. Risk-reducing salpingo-oophorectomy (RRSO)**

Women who have inherited mutations in the *BRCA1* or *BRCA2* genes have substantially elevated risks of breast and ovarian cancer, with a lifetime risk of breast cancer of 56%–84% [48-51]. Breast cancer in *BRCA1/2* mutation carriers also occurs at an earlier age, particularly among the *BRCA1* mutation carriers, than in non-carriers. The risk for ovarian cancer depends on whether the mutation has occurred in *BRCA1* or *BRCA2*, with estimated risks ranging from 34% to 44% for *BRCA1* mutation carriers and from 12% to 25% for *BRCA2* mutation carriers [48, 49, 52-54]. Carriers of *BRCA1/2* mutations are counselled to help them interpret the implications of these elevated risks, choose strategies to reduce these risks, and maximize early detection of cancers. The risk of breast cancer can be reduced either with RRSO and/or mastectomy or non-surgically (i.e. with chemoprevention). However, due to the lack of effective screening for ovarian cancer, RRSO is usually strongly recommended to *BRCA1*/*2* mutation carriers once childbearing is complete.

RRSO has also been demonstrated to decrease the risk of both breast and ovarian cancer in *BRCA1/2* mutation carriers [55-60]. However, the studies examining the extent of risk reduction have used different designs; some are retrospective case–control studies, while others used a prospective cohort design. In a large, retrospective analysis of 551 *BRCA* carriers, RRSO was found to reduce the risk of ovarian cancer by 96% and breast cancer by 53% at a mean followup of 9 years [55]. A multicentre prospective study, Kauff *et al*. [56] found that, during a 3-year follow-up, RRSO was associated with an 85% reduction in BRCA1-associated gynaecologic cancer risk and a 72% reduction in BRCA2-associated breast cancer risk. Although protection against BRCA1-associated breast cancer and BRCA2-associated gynaecologic cancer was suggested, neither effect reached statistical significance. The authors postulate that the protection conferred by RRSO against breast and gynaecologic cancers may differ between the carriers of *BRCA1* and *BRCA2* mutations.

Similar findings were observed in a prospective study of 170 *BRCA* carriers. During a mean fol‐ low-up of 2 years, the incidence of ovarian or peritoneal cancer and breast cancer was significant‐ ly greater amongst those women who selected surveillance than amongst those who chose to undergo RRSO [59.] Even among prospective studies, the inclusion criteria and the definitions of follow-up time differ. In some studies, only unaffected mutation-positive women are included and followed up. In others, particularly when examining ovarian cancer risk, women with breast cancer are included. Such differences in study design can introduce biases (such as the survival bias) and can have an impact on risk reduction estimates. For example, the reported efficacy of RRSO in reducing the risk of ovarian/fallopian tube cancers varies from 71% to 96% [55-61]. Al‐ though these estimates imply a substantial reduction in risk, this variability may affect the deci‐ sions of premenopausal women who are making a decision about whether to undergo a treatment that will cause abrupt and premature menopause. Patients and their physicians need as much information as possible regarding the efficacy of RRSO in reducing cancer risk to bal‐ ance this benefit with the health risks caused by premature entry into menopause.


A summary of published studies on RRSO is presented in Table 2.

**3. Primary prevention for ovarian cancer in high risk women**

the identification of a mutation or of a familial cancer risk [45, 46].

in different ways and in alternative locations [47].

**3.1. Risk-reducing salpingo-oophorectomy (RRSO)**

mutation carriers once childbearing is complete.

expectancy in *BRCA1/2* carriers.

50 Ovarian Cancer - A Clinical and Translational Update

Some women have a high risk of developing ovarian cancer due to hereditary conditions associated to BRCA syndrome and Lynch syndrome. Thus, when one of these forms of hereditary or familial breast and/or ovarian cancer is suspected in clinical practice, the general practitioner should refer the patient to a cancer centre specialising in cancer-specific genetic counselling for the identification, definition and management of risk. Genetic counselling, defined by the American Society of Human Genetics as 'a communication process which deals with the human problems associated with the occurrence or risk of occurrence of a genetic disorder in a family', involves one or more professional figures to help the affected individuals or families [41-44]. Genetic counselling in the oncological setting (cancer-specific genetic counselling) should also provide sufficient information to enable the user to make a fully informed choice as to course of action, particularly with regards to prevention, in the case of

A recent review investigated the impact of cancer genetic risk assessment on outcomes, including perceived risk of inherited cancer and psychological distress. The review found favourable outcomes for patients after risk assessment for familial breast cancer, suggesting that cancer-specific genetic risk assessment services help to reduce distress, improve the accuracy of the perception of risk of ovarian cancer, and increase the knowledge of ovarian cancer and genetics. However, there were too few papers to make any significant conclusions on how best to deliver cancer genetic risk assessment services. Further research is needed, assessing the best means of delivering cancer risk evaluation, by different health professionals,

Women at increased risk of breast and ovarian cancer are advised to consider risk-reducing strategies; however, such methods vary in their effectiveness. These strategies include chemoprevention and prophylactic surgery (risk-reducing salpingo-oophorectomy, RRSO). Risk-reducing strategies have been shown to have associations with a lengthening of life

Women who have inherited mutations in the *BRCA1* or *BRCA2* genes have substantially elevated risks of breast and ovarian cancer, with a lifetime risk of breast cancer of 56%–84% [48-51]. Breast cancer in *BRCA1/2* mutation carriers also occurs at an earlier age, particularly among the *BRCA1* mutation carriers, than in non-carriers. The risk for ovarian cancer depends on whether the mutation has occurred in *BRCA1* or *BRCA2*, with estimated risks ranging from 34% to 44% for *BRCA1* mutation carriers and from 12% to 25% for *BRCA2* mutation carriers [48, 49, 52-54]. Carriers of *BRCA1/2* mutations are counselled to help them interpret the implications of these elevated risks, choose strategies to reduce these risks, and maximize early detection of cancers. The risk of breast cancer can be reduced either with RRSO and/or mastectomy or non-surgically (i.e. with chemoprevention). However, due to the lack of effective screening for ovarian cancer, RRSO is usually strongly recommended to *BRCA1*/*2*

PC = prospective cohort; RC = retrospective cohort; MYFU = mean years of follow-up; PY = person-years; NR = not reported; and NA = not applicable; RRSO = risk-reducing salpingo-oophorectomy.

**Table 2.** Published studies on risk-reducing salpingo-oophorectomy in BrCa1and/ or BrCa2 mutation carriers.

A synopsis of different management strategies available for *BRCA1* and *BRCA2* mutation carriers is shown in Table 3.

The significantly reduced risk of breast cancer by RRSO seems to be higher in *BRCA2* mutation carriers than in *BRCA1* carriers. Several reports have addressed this question although additional research is required [56]. Short-term HRT after RRSO seems not to decrease the

However, it should be noted that the NCCN and other institutions couch these recommenda‐ tions within a multidisciplinary consultative process in which reproductive desires, assess‐ ment of cancer risk, and the pros and cons of surgery along with the potential sequelae of

The recommendations of different organizations regarding surgical primary prevention for

**ACOG Committee on Genetics and the Society of Gynecologic Oncologists [63]**

By the age of 40 years or when childbearing is complete

**Table 4.** Recommendations of several organizations regarding primary prevention for BRCA mutation carriers*.*

Women at increased risk, based on their personal or family history of breast and/or ovarian cancer including *BRCA1/2* mutation carriers, may join a cancer prevention clinical trial or a chemoprevention trial. OC have been the most widely studied chemopreventive agents in ovarian cancer. Recently, Iodice *et al.* conducted a meta-analysis updated to March 2010 on the association between OC use and breast or ovarian cancer in *BRCA1/2* mutation carriers [69]. Based on 18 studies a total of 2855 breast cancer cases and 1503 ovarian cancer cases carrying an ascertained *BRCA1/2* mutation were included. As previously noted, use of OC at any point during one's life was associated with a 50% reduction in relative risk of developing ovarian cancer for *BRCA1/2* mutation carriers. Looking specifically at duration of use, each 10-year period of OC use resulted in a 36% relative risk reduction in the development of ovarian cancer. However, the meta-analysis showed no evidence of a significant association between OC use and breast cancer risk. Notably, formulations used before 1975 correlated with an increased risk of breast cancer, but there was no correlation with the use of more recent formulations. A summary of the association between OC use and ovarian cancer risk in mutation carriers is

**National Cancer Institute (NCI) [64]**

Preventive Strategies for Ovarian Cancer http://dx.doi.org/10.5772/54686 53

Considered but age is not indicated

Considered but age is not indicated

**ESMO [65]**

After age 35 and when childbearing decisions are complete


overall benefit of this strategy for breast cancer risk reduction [68].

Between 35 and 40 years or upon completion of child bearing

Bilateral salpingectomy - -

surgery are fully discussed.

RRSO

**3.2. Chemoprevention**

shown in Table 5.

*BRCA1/2* mutation carriers are shown in Table 4.

**Management options NCCN [62]**


**Table 3.** Synopsis of different prevention strategies for BRCA1 and BRCA2 mutation carriers.

The National Comprehensive Cancer Network (NCCN) guidelines and other institutions concerning this method, recommend RRSO "for women with a known BRCA1/2 mutation, ideally between 35 and 40 years or upon completion of child bearing" or at an adjusted age based on earliest age of ovarian cancer diagnosis in the family" [62].

Also ACOG, the Committee on Genetics and the Society of Gyneacologic Oncologists, recommends RRSO for women with *BRCA1/2* mutations, by the age of 40 years or when childbearing is complete [63].

The National Cancer Institute (NCI) [64] on the clinical management of *BRCA* mutation carriers considers, besides salpingo-oophorectomy, bilateral salpingectomy as an interim procedure to reduce risk in *BRCA* mutation carriers. There are no data available on the efficacy of salpingectomy as a risk-reducing procedure. The procedure preserves ovarian function and spares the premenopausal patient the adverse effects of a premature menopause. It can be performed using a minimally invasive approach, and a subsequent bilateral oophorectomy could be deferred until the patient approaches menopause. While the data make the compel‐ ling argument that some pelvic serous cancers in *BRCA* mutation carriers originate in the fallopian tube, clearly, some cancers arise in the ovary. Furthermore, bilateral salpingectomy could give patients a false sense of security that they have eliminated their cancer risk as completely as if they had undergone a bilateral salpingo-oophorectomy. A small study of 14 young *BRCA* mutation carriers documented the procedure as feasible [65]. However, efficacy and impact on ovarian function was not assessed in this study. Future prospective trials are needed to establish the validity of the procedure as a risk-reducing intervention.

For the European Society of Medical Oncology ESMO [66], RRSO is associated with a reduction in risk of breast cancer in premenopausal *BRCA* mutation carriers, a reduction in risk of ovarian cancer, and there is evidence of a reduction in overall mortality [67]. RRSO is recommended after the age of 35 and when childbearing decisions are complete.

The significantly reduced risk of breast cancer by RRSO seems to be higher in *BRCA2* mutation carriers than in *BRCA1* carriers. Several reports have addressed this question although additional research is required [56]. Short-term HRT after RRSO seems not to decrease the overall benefit of this strategy for breast cancer risk reduction [68].

However, it should be noted that the NCCN and other institutions couch these recommenda‐ tions within a multidisciplinary consultative process in which reproductive desires, assess‐ ment of cancer risk, and the pros and cons of surgery along with the potential sequelae of surgery are fully discussed.

The recommendations of different organizations regarding surgical primary prevention for *BRCA1/2* mutation carriers are shown in Table 4.


**Table 4.** Recommendations of several organizations regarding primary prevention for BRCA mutation carriers*.*

#### **3.2. Chemoprevention**

A synopsis of different management strategies available for *BRCA1* and *BRCA2* mutation

**Gynecologic cancer Strategy Advantage Limitation**

Screening TVUS, CA 125 Avoids RRSO Unproven efficacy

The National Comprehensive Cancer Network (NCCN) guidelines and other institutions concerning this method, recommend RRSO "for women with a known BRCA1/2 mutation, ideally between 35 and 40 years or upon completion of child bearing" or at an adjusted age

Also ACOG, the Committee on Genetics and the Society of Gyneacologic Oncologists, recommends RRSO for women with *BRCA1/2* mutations, by the age of 40 years or when

The National Cancer Institute (NCI) [64] on the clinical management of *BRCA* mutation carriers considers, besides salpingo-oophorectomy, bilateral salpingectomy as an interim procedure to reduce risk in *BRCA* mutation carriers. There are no data available on the efficacy of salpingectomy as a risk-reducing procedure. The procedure preserves ovarian function and spares the premenopausal patient the adverse effects of a premature menopause. It can be performed using a minimally invasive approach, and a subsequent bilateral oophorectomy could be deferred until the patient approaches menopause. While the data make the compel‐ ling argument that some pelvic serous cancers in *BRCA* mutation carriers originate in the fallopian tube, clearly, some cancers arise in the ovary. Furthermore, bilateral salpingectomy could give patients a false sense of security that they have eliminated their cancer risk as completely as if they had undergone a bilateral salpingo-oophorectomy. A small study of 14 young *BRCA* mutation carriers documented the procedure as feasible [65]. However, efficacy and impact on ovarian function was not assessed in this study. Future prospective trials are

needed to establish the validity of the procedure as a risk-reducing intervention.

after the age of 35 and when childbearing decisions are complete.

For the European Society of Medical Oncology ESMO [66], RRSO is associated with a reduction in risk of breast cancer in premenopausal *BRCA* mutation carriers, a reduction in risk of ovarian cancer, and there is evidence of a reduction in overall mortality [67]. RRSO is recommended

in ovarian cancer risk

Substantial decrease in risk of ovarian and fallopian tube cancers

Potential increase in risk of breast cancer

Premature menopause and iatrogenic infertility

Chemoprevention OC Likely 30-60% reduction

oophorectomy

**Table 3.** Synopsis of different prevention strategies for BRCA1 and BRCA2 mutation carriers.

based on earliest age of ovarian cancer diagnosis in the family" [62].

TVUS= Transvaginal Ultrasound; RRSO = risk-reducing salpingo-oophorectomy

carriers is shown in Table 3.

52 Ovarian Cancer - A Clinical and Translational Update

**Management options**

childbearing is complete [63].

Risk-reducing surgery Bilateral salpingo-

Women at increased risk, based on their personal or family history of breast and/or ovarian cancer including *BRCA1/2* mutation carriers, may join a cancer prevention clinical trial or a chemoprevention trial. OC have been the most widely studied chemopreventive agents in ovarian cancer. Recently, Iodice *et al.* conducted a meta-analysis updated to March 2010 on the association between OC use and breast or ovarian cancer in *BRCA1/2* mutation carriers [69]. Based on 18 studies a total of 2855 breast cancer cases and 1503 ovarian cancer cases carrying an ascertained *BRCA1/2* mutation were included. As previously noted, use of OC at any point during one's life was associated with a 50% reduction in relative risk of developing ovarian cancer for *BRCA1/2* mutation carriers. Looking specifically at duration of use, each 10-year period of OC use resulted in a 36% relative risk reduction in the development of ovarian cancer. However, the meta-analysis showed no evidence of a significant association between OC use and breast cancer risk. Notably, formulations used before 1975 correlated with an increased risk of breast cancer, but there was no correlation with the use of more recent formulations. A summary of the association between OC use and ovarian cancer risk in mutation carriers is shown in Table 5.


**STUDY DESIGN TREATMENT END POINTS OUTCOMES**

Tolerability

Pharmacokinetic

Second breast cancer prevention

Second breast cancer prevention Recommended dose for chemoprevention trials of HPR

Preventive Strategies for Ovarian Cancer http://dx.doi.org/10.5772/54686 55

HPR treatment lowers retinol

concentrations. Effect related to HPR levels and reversible on

No statistically significant effect but a possible benefit in premenopausal women.

4-HPR induces a significant reduction of risk of second

premenopausal women, which is remarkable at younger ages, and persists several years after

is 200mg/die.

and RPB plasma

cessation of HPR administration.

breast cancer in

treatment cessation.

Orally: 100, 200 and 300mg x 6 months subsequently at 200mg for another 6 months.

Orally: 100, 200 and 300mg x 6 months subsequently at 200mg for another 6 months.

Orally 200mg versus no treatment x 5 years.

Orally 200mg versus no treatment x 5 years: 15-years followup.

A recent population-based case-control study, carried out in Denmark in the period 1995-1999, analysed the association between analgesic drug use and ovarian cancer risk using multiple logistic regression models. The study showed that regular use of non-aspirin non-steroidal anti-inflammatory drugs (NA-NSAID), paracetamol or other analgesics did not decrease ovarian cancer risk. In contrast, use of any analgesics (OR = 0.72; 95% CI 0.53-0.98) or aspirin (OR = 0.60; 95% CI 0.36-1.00) resulted in a statistically significant decreased risk of serous ovarian cancer but not mucinous or other ovarian tumours [79]. On the other hand, recent data reported by the Multiethnic Cohort Study did not find compelling evidence to support an association between use of NSAID and risk of ovarian and endometrial cancers in a multiethnic population. The RR (95% CI) for ovarian cancer associated with aspirin, non-aspirin NSAID, and acetaminophen were 0.87 (0.68, 1.14), 0.97 (0.74, 1.26), and 0.86 (0.67, 1.12), respectively. No heterogeneity across ethnic groups (P's ≥0.29) or dose-response relation with increased duration of use (P's for trend ≥0.16) was observed [80]. Finally, in an attempt to review and summarize the evidence provided by longitudinal studies on the association between NSAID use and ovarian cancer risk, a comprehensive literature search for articles published up to December 2011 was performed (Table 7). The meta-analysis found no evidence of an associa‐ tion between aspirin or NA-NSAID use and ovarian cancer risk, based on a random-effects

Costa et al., 1989 [75]

Formelli et al., 1989 [76]

Veronesi et al., 1999 [77]

Veronesi et al., 2006 [78]

Phase I, Randomized, Placebo controlled (60)

Phase II, Randomized, Placebo controlled (60)

> Phase III, Randomized (2867)

Phase III, Randomized, 15 year follow-up (1879)

HPR: fenretinide, RBP: retinol-binding protein.

**Table 6.** Clinical trials with 4-HPR [74].

**Table 5.** Summary of the association between OC use and ovarian cancer risk in mutation carriers

Another meta-analysis of cohort, case-control and case-case studies published in English up to December 2009 confirmed a significantly decreased ovarian cancer risk in *BRCA1/2* mutation carriers associated with the use of OC, while a significantly increased risk in breast cancer was only shown in a subset of cohort studies on *BRCA1* mutation carriers. To conclude, OC use can be considered as an alternative strategy in the chemoprevention of ovarian cancer in *BRCA1* mutation carriers who do not accept RRSO above the age of 30 years [70].

Other chemopreventative agents such as retinoids, vitamin D, cyclo-oxygenase inhibitors and peroxisome proliferator activated receptor-gamma ligands have shown promise in early investigations of disease prevention [71].

Retinoids, a class of compounds comprising vitamin A, its natural derivatives, and synthetic analogs, have been extensively studied in both the prevention and treatment of gynaecologic malignancies [72]. One of the most promising retinoids to be used in chemoprevention trials is the synthetic amide of retinoic acid fenretinide, *N*-4-hydroxyphenyl retinamide (4-HPR). 4- HPR has been found to have significant chemopreventive action in a large variety of in vitro and in vivo systems. Since both fenretinide and its major metabolite, 4-metoxyphenyl retina‐ mide (MPR), selectively accumulate in the human breast, evaluation of 4-HPR as a chemopre‐ ventive agent in breast cancer has been particularly attractive [73]. The most important clinical trials with 4-HPR are mentioned in Table 6.

The most important study where 4-HPR was administrated was a multicentric phase III randomized trial, coordinated by the Istituto Nazionale dei Tumori in Milan, which started in 1987. Most notably, the younger the women were, the greater the benefit of 4-HPR. Such a benefit was associated with a remarkable 50% risk reduction in women aged 40 years or younger, whereas it disappeared after 55 years of age. Interestingly, the incidence of ovarian cancer during the 5-year intervention period was significantly lower in the treatment arm [74].

The role of analgesic drug use in the development of ovarian cancer is still widely discussed.


**Table 6.** Clinical trials with 4-HPR [74].

**EXPOSURE**

OC USERS

DURATION OF USE BRCA 1/2

investigations of disease prevention [71].

trials with 4-HPR are mentioned in Table 6.

**GENES WITH MUTATION**

54 Ovarian Cancer - A Clinical and Translational Update

BRCA 1/2

**CATEGORIES OF EXPOSURE**

Users

1 year

**Table 5.** Summary of the association between OC use and ovarian cancer risk in mutation carriers

*BRCA1* mutation carriers who do not accept RRSO above the age of 30 years [70].

Another meta-analysis of cohort, case-control and case-case studies published in English up to December 2009 confirmed a significantly decreased ovarian cancer risk in *BRCA1/2* mutation carriers associated with the use of OC, while a significantly increased risk in breast cancer was only shown in a subset of cohort studies on *BRCA1* mutation carriers. To conclude, OC use can be considered as an alternative strategy in the chemoprevention of ovarian cancer in

Other chemopreventative agents such as retinoids, vitamin D, cyclo-oxygenase inhibitors and peroxisome proliferator activated receptor-gamma ligands have shown promise in early

Retinoids, a class of compounds comprising vitamin A, its natural derivatives, and synthetic analogs, have been extensively studied in both the prevention and treatment of gynaecologic malignancies [72]. One of the most promising retinoids to be used in chemoprevention trials is the synthetic amide of retinoic acid fenretinide, *N*-4-hydroxyphenyl retinamide (4-HPR). 4- HPR has been found to have significant chemopreventive action in a large variety of in vitro and in vivo systems. Since both fenretinide and its major metabolite, 4-metoxyphenyl retina‐ mide (MPR), selectively accumulate in the human breast, evaluation of 4-HPR as a chemopre‐ ventive agent in breast cancer has been particularly attractive [73]. The most important clinical

The most important study where 4-HPR was administrated was a multicentric phase III randomized trial, coordinated by the Istituto Nazionale dei Tumori in Milan, which started in 1987. Most notably, the younger the women were, the greater the benefit of 4-HPR. Such a benefit was associated with a remarkable 50% risk reduction in women aged 40 years or younger, whereas it disappeared after 55 years of age. Interestingly, the incidence of ovarian cancer during the 5-year intervention period was significantly lower in the treatment arm [74]. The role of analgesic drug use in the development of ovarian cancer is still widely discussed.

BRCA 1 5 1251 0.51 (0.40-0.65)

BRCA 2 4 286 0.52 (0.31-0.87)

4 1336

10 years 0.64 (0.53-0.78)

5 years 0.80 (0.73-0.88)

**No. of STUDIES No. of CASES HR (95% CI)**

5 1503 0.50 (0.33-0.75)

0.96 (0.94-0.97)

A recent population-based case-control study, carried out in Denmark in the period 1995-1999, analysed the association between analgesic drug use and ovarian cancer risk using multiple logistic regression models. The study showed that regular use of non-aspirin non-steroidal anti-inflammatory drugs (NA-NSAID), paracetamol or other analgesics did not decrease ovarian cancer risk. In contrast, use of any analgesics (OR = 0.72; 95% CI 0.53-0.98) or aspirin (OR = 0.60; 95% CI 0.36-1.00) resulted in a statistically significant decreased risk of serous ovarian cancer but not mucinous or other ovarian tumours [79]. On the other hand, recent data reported by the Multiethnic Cohort Study did not find compelling evidence to support an association between use of NSAID and risk of ovarian and endometrial cancers in a multiethnic population. The RR (95% CI) for ovarian cancer associated with aspirin, non-aspirin NSAID, and acetaminophen were 0.87 (0.68, 1.14), 0.97 (0.74, 1.26), and 0.86 (0.67, 1.12), respectively. No heterogeneity across ethnic groups (P's ≥0.29) or dose-response relation with increased duration of use (P's for trend ≥0.16) was observed [80]. Finally, in an attempt to review and summarize the evidence provided by longitudinal studies on the association between NSAID use and ovarian cancer risk, a comprehensive literature search for articles published up to December 2011 was performed (Table 7). The meta-analysis found no evidence of an associa‐ tion between aspirin or NA-NSAID use and ovarian cancer risk, based on a random-effects model or a fixed-effects model. Furthermore, the analysis did not show strong association between frequency or duration of NA-NSAID use and ovarian cancer, leading to the conclu‐ sion that there is no strong evidence of an association between aspirin/NA-NSAID use and ovarian cancer [81].

**4.1. Transvaginal ultrasound**

**4.2. Serum CA125**

section [84-90].

In the general population, TVUS appears to be superior to transabdominal ultrasound in the pre‐ operative diagnosis of adnexal masses. Both techniques have lower specificity in premenopaus‐ al women than in postmenopausal women due to the cyclic menstrual changes in premenopausal ovaries (e.g., transient corpus luteum cysts) that can cause difficulty in the inter‐ pretation. The randomized prospective Prostate, Lung, Colorectal, and Ovarian Cancer Screen‐ ing Trial found no reduction in mortality with the annual use of combined TVUS and CA125 in

Preventive Strategies for Ovarian Cancer http://dx.doi.org/10.5772/54686 57

Data are limited regarding the potential benefit of TVUS in screening women at inherited risk of ovarian cancer. A number of retrospective studies have reported experiments with ovarian

However, there is little uniformity in the definition of high-risk criteria and compliance with screening, and in whether the cancers detected were incident or prevalent. One of the largest re‐ ported studies included 888 *BRCA1/BRCA2* mutation carriers who were annually screened with TVUS and CA125. Ten women developed ovarian cancer; five of the ten developed inter‐ val cancers after normal screening results within 3 to 10 months before diagnosis. Five of the ten ovarian cancers were screen-detected incident cases, which had had normal screening results within 6 to 14 months before diagnosis. Out of these five cases, four were stage IIIB or IV [85].

A similar study reported the results of annual TVUS and CA125 combined-screening in a cohort of 312 high-risk women (152 *BRCA1/BRCA2* mutation carriers) [86]. Out of four cancers detected because of abnormal TVUS and CA125, all cases were symptomatic, and three had an advanced-stage disease. Annual screening of *BRCA1/BRCA2* mutation carriers with pelvic ultrasound, TVUS, and CA125 failed to detect early-stage ovarian cancer among 241 women in a study from the Netherlands [87]. Three cancers were detected over the course of the study, all advanced stage IIIC disease. Finally, a study of 1,100 moderate- and high-risk women who underwent annual TVUS and CA125 combined screening reported that ten out of 13 ovarian tumours were detected due to screening. Only five out of ten were stage I or II [88]. There are limited data related to the efficacy of semiannual screening with TVUS and CA125 [89].

The first prospective study of TVUS and CA125 with survival as the primary outcome was com‐ pleted in 2009. Out of 3,532 high-risk women screened, 981 were *BRCA* mutation carriers, of which 49 developed ovarian cancer. The 5- and 10-year survival was 58.6% (95% CI, 50.9–66.3) and 36% (95% CI, 27–45), respectively, and there was no difference in survival between carriers and non-carriers. A major limitation of the study was the absence of a control group. Despite these limitations, this study suggests that annual surveillance by TVUS and CA125 level appears to be ineffective in detecting tumours at an early stage to substantially influence survival [90].

Serum CA125 screening for ovarian cancer in high-risk women has been evaluated in combination with TVUS in a number of retrospective studies, as described in the previous

screening asymptomatic, postmenopausal women at average risk of ovarian cancer [83].

cancer screening in high-risk women using TVUS with or without CA125 [84-90].


NA-NSAID: non aspirin- non-steroidal anti-inflammatory drugs

**Table 7.** Metanalysis of longitudinal studies on the association between NSAID use and ovarian cancer risk

#### **4. Secondary prevention for ovarian cancer**

This is based on diagnosing and treating extant disease in the early stages before it causes signif‐ icant morbidity. CA125 (or MUC16) glycoprotein is the most studied tumour marker, alone and/or in combination with other biomarkers, for ovarian cancer screening. However, false pos‐ itive CA125 levels can occur in women with benign conditions, including menstruation, appen‐ dicitis, benign ovarian cysts, endometriosis and pelvic inflammatory disease, as well as with other malignancies, including breast, lung, endometrial and pancreatic cancers. Thus, a large number of false-positive screening tests can occur, potentially leading to unnecessary surgeries and subsequent issues of morbidity and cost [82]. Consequently, multimodal strategies, in par‐ ticular the combination of CA125 with pelvic ultrasound, have been examined, in order to im‐ prove sensitivity and positive predictive value of ovarian cancer screening.

#### **4.1. Transvaginal ultrasound**

model or a fixed-effects model. Furthermore, the analysis did not show strong association between frequency or duration of NA-NSAID use and ovarian cancer, leading to the conclu‐ sion that there is no strong evidence of an association between aspirin/NA-NSAID use and

**ASPIRIN USE** All studies 17 0,94 (0,87-1,01) 0,91 (0,82-1,01) 0,046 C-C studies 14 0,94 (0,87-1,02) 0,90 (0,79-1,03) 0,015 Cohort studies 3 0,92 (0,77-1,09) 0,92 (0,77-1,10) 0,456 Regular Use 7 0,86 (0,73-1,03) 0,83 (0,65-1,05) 0,119 Irregular Use 7 1,07 (0,96-1,20) 1,07 (0,96-1,21) 0,421 Duration > 5 yrs 5 0,91 (0,67-1,24) 0,89 (0,63-1,25) 0,332 **NA-NSAID use** All studies 7 0,86 (0,76-0,98) 0,89 (0,74-1,08) 0,089 C-C studies 4 0,88 (0,75-1,03) 0,97 (0,73-1,28) 0,042 Cohort studies 3 0,82 (0,64-1,04) 0,89 (0,74-1,08) 0,283 Regular Use 3 1,45 (1,07-1,98) 1,47 (0,95-2,27) 0,153 Irregular Use 3 0,96 (0,69-1,33) 0,93 (0,49-1,76) 0,038 Duration > 5 yrs 3 1,65 (1,13-2,41) 1,56 (0,92-2,65) 0,21

**Table 7.** Metanalysis of longitudinal studies on the association between NSAID use and ovarian cancer risk

prove sensitivity and positive predictive value of ovarian cancer screening.

This is based on diagnosing and treating extant disease in the early stages before it causes signif‐ icant morbidity. CA125 (or MUC16) glycoprotein is the most studied tumour marker, alone and/or in combination with other biomarkers, for ovarian cancer screening. However, false pos‐ itive CA125 levels can occur in women with benign conditions, including menstruation, appen‐ dicitis, benign ovarian cysts, endometriosis and pelvic inflammatory disease, as well as with other malignancies, including breast, lung, endometrial and pancreatic cancers. Thus, a large number of false-positive screening tests can occur, potentially leading to unnecessary surgeries and subsequent issues of morbidity and cost [82]. Consequently, multimodal strategies, in par‐ ticular the combination of CA125 with pelvic ultrasound, have been examined, in order to im‐

**Fixed-effects model Random-effects model**

RR (95% CI) RR (95% CI)

**P-value**

ovarian cancer [81].

56 Ovarian Cancer - A Clinical and Translational Update

**No. Of studies**

NA-NSAID: non aspirin- non-steroidal anti-inflammatory drugs

**4. Secondary prevention for ovarian cancer**

In the general population, TVUS appears to be superior to transabdominal ultrasound in the pre‐ operative diagnosis of adnexal masses. Both techniques have lower specificity in premenopaus‐ al women than in postmenopausal women due to the cyclic menstrual changes in premenopausal ovaries (e.g., transient corpus luteum cysts) that can cause difficulty in the inter‐ pretation. The randomized prospective Prostate, Lung, Colorectal, and Ovarian Cancer Screen‐ ing Trial found no reduction in mortality with the annual use of combined TVUS and CA125 in screening asymptomatic, postmenopausal women at average risk of ovarian cancer [83].

Data are limited regarding the potential benefit of TVUS in screening women at inherited risk of ovarian cancer. A number of retrospective studies have reported experiments with ovarian cancer screening in high-risk women using TVUS with or without CA125 [84-90].

However, there is little uniformity in the definition of high-risk criteria and compliance with screening, and in whether the cancers detected were incident or prevalent. One of the largest re‐ ported studies included 888 *BRCA1/BRCA2* mutation carriers who were annually screened with TVUS and CA125. Ten women developed ovarian cancer; five of the ten developed inter‐ val cancers after normal screening results within 3 to 10 months before diagnosis. Five of the ten ovarian cancers were screen-detected incident cases, which had had normal screening results within 6 to 14 months before diagnosis. Out of these five cases, four were stage IIIB or IV [85].

A similar study reported the results of annual TVUS and CA125 combined-screening in a cohort of 312 high-risk women (152 *BRCA1/BRCA2* mutation carriers) [86]. Out of four cancers detected because of abnormal TVUS and CA125, all cases were symptomatic, and three had an advanced-stage disease. Annual screening of *BRCA1/BRCA2* mutation carriers with pelvic ultrasound, TVUS, and CA125 failed to detect early-stage ovarian cancer among 241 women in a study from the Netherlands [87]. Three cancers were detected over the course of the study, all advanced stage IIIC disease. Finally, a study of 1,100 moderate- and high-risk women who underwent annual TVUS and CA125 combined screening reported that ten out of 13 ovarian tumours were detected due to screening. Only five out of ten were stage I or II [88]. There are limited data related to the efficacy of semiannual screening with TVUS and CA125 [89].

The first prospective study of TVUS and CA125 with survival as the primary outcome was com‐ pleted in 2009. Out of 3,532 high-risk women screened, 981 were *BRCA* mutation carriers, of which 49 developed ovarian cancer. The 5- and 10-year survival was 58.6% (95% CI, 50.9–66.3) and 36% (95% CI, 27–45), respectively, and there was no difference in survival between carriers and non-carriers. A major limitation of the study was the absence of a control group. Despite these limitations, this study suggests that annual surveillance by TVUS and CA125 level appears to be ineffective in detecting tumours at an early stage to substantially influence survival [90].

#### **4.2. Serum CA125**

Serum CA125 screening for ovarian cancer in high-risk women has been evaluated in combination with TVUS in a number of retrospective studies, as described in the previous section [84-90].

The National Institutes of Health (NIH) Consensus Statement on Ovarian Cancer recommend‐ ed against routine screening of the general population for ovarian cancer with serum CA125. The NIH Consensus Statement did, however, recommend that women at inherited risk of ovarian cancer undergo TVUS and serum CA125 screening every 6 to 12 months, beginning at the age of 35 years [91]. The Cancer Genetics Studies Consortium task force recommends that female carriers of a deleterious *BRCA1* mutation undergo annual or semi-annual screening using TVUS and serum CA125 levels, beginning at age of 25 to 35 years [92]. Both recommen‐ dations are based solely on expert opinion and best clinical judgment.

as RRSO. The guidelines, summarized in Table 8, include age ranges for which these options

**Surveillance**

**Surgery**

**Chemoprevention** Considered Considered Considered Not considered

Additional potential serum biomarkers have been studied for the detection of ovarian cancer. For instance, human epididymis protein 4 (HE4) is a secreted glycoprotein over-expressed by serous and endometrioid ovarian cancers and expressed by 32% of ovarian cancers lacking

To define the clinical utility of HE4, a comprehensive assessment of HE4 protein expression in benign and malignant ovarian and non-ovarian tissues by immunohistochemistry was performed and published in 2005. In comparison with normal surface epithelium, which does not express the protein, HE4 was widely found in cortical inclusion cysts lined by metaplastic Mullerian epithelium. These findings suggested that the formation of Mullerian epithelium is a prerequisite step in the development of some types of epithelial ovarian cancer. Moreover, the expression was restricted to certain histologic subtypes: 93% of serous and 100% of endometrioid epithelial ovarian cancers expressed HE4, while only 50% and 0% of clear cell carcinomas and mucinous tumours, respectively, were positive. HE4 protein expression is restricted in normal tissue to the reproductive tracts and respiratory epithelium. In fact, tissue microarrays revealed that the majority of non-ovarian carcinomas do not express HE4 [100].

In 2008 the Food and Drug Administration (FDA) approved HE4 to monitor disease recurrence and this marker was recently incorporated into the clinical evaluation of ovarian cancer

**Table 8.** Published Guidelines/Consensus Statements for the management of BRCA mutation carriers.

**ACOG Committee on Genetics and the Society of Gynecologic Oncologists [63]**

Periodic screening beginning between the ages 30 years and 35years

> By age 40 years or when childbearing is complete

Considered Considered Considered Not considered

**National Cancer Institute (NCI) [64]**

Preventive Strategies for Ovarian Cancer http://dx.doi.org/10.5772/54686

Every 6 to 12 months, beginning at age 35 years

Considered but age is not indicated

Considered but age is not indicated

**ESMO [65]**

59

Not considered

After age 35 and when childbearing decisions are complete


should be begun and how often screening should take place. [53].

Every 6 months starting at age 30 years

Between 35 and 40 years or upon completion of child bearing

Bilateral salpingectomy - -

**4.4. Human Epididymis Protein 4 (HE4)**

**Management options NCCN [62]**

TVU+CA125

RRSO

**Investigational imaging and screening studies**

CA125 expression.

NCCN for those patients who have not chosen RRSO, consider concurrent TVUS (preferably day 1-10 menstrual cycle women in premenopausal women) + CA125 (preferably after day 5 of menstrual cycle women in premenopausal women) every 6 months starting at the age of 30 years or 5-10 years before the earliest age of first diagnosis of ovarian cancer in the family [93].

Although there are retrospective data indicating that annual ovarian cancer screening using TVUS and measurement of serum CA125 levels is neither an effective strategy for the early detection of ovarian tumours nor a reasonable substitute for a bilateral RRSO, the effectiveness of these interventions is limited to six-monthly screening. Investigational imaging and screening studies may be considered for this population.

#### **4.3. Proton Magnetic Resonance Spectroscopy (MRS)**

MRS has proved to be a reliable technique for probing metabolic patterns, biochemical effects of tumour microenvironment, and the action of therapy in cancer cells, both in vivo and in vitro [94]. In particular, an increase in the total choline-containing compounds (tCho) content allows to distinguish malignant from benign lesions in the breast [95].

Moreover, some studies have also shown alterations of the phospholipid metabolism in vitro using epithelial ovarian carcinoma cell lines [96,97], and demonstrated the feasibility of 3D CSI MRS to detect a choline peak in ovarian lesions in vivo at 1.5 T.

Then, the metabolic meaning of a high concentration of choline in ovarian tumours merits some consideration. This topic has been extensively reviewed by Podo *et al.* in 2007 [98]. The high choline concentration of ovarian tumours can be considered as the result of an inappro‐ priate storage attributable to metabolic deregulation associated with clinical indicators of increased malignancy. The possibility of using a spatially resolved approach for MRS of ovarian masses opens an intriguing prospect for the diagnosis of early-stage tumours, with potential impact on the overall survival. This is especially true for carriers of a *BRCA* mutation, with a lifetime risk of 39% to 46% among women with the *BRCA1* mutation and a risk of 12% to 20% among those with the *BRCA2* mutation [99].

Based on peer-reviewed published data, several institutions established the Guidelines to facilitate clinical management of patients with a suggestive personal or family history of breast and/or ovarian cancer, in particular individuals from a family with a known deleterious BRCA1/2 mutation. Screening options include transvaginal ultrasonography (TVUS), and serum CA125, while prevention options include medical therapy with drugs and surgery such as RRSO. The guidelines, summarized in Table 8, include age ranges for which these options should be begun and how often screening should take place. [53].


**Table 8.** Published Guidelines/Consensus Statements for the management of BRCA mutation carriers.

#### **4.4. Human Epididymis Protein 4 (HE4)**

The National Institutes of Health (NIH) Consensus Statement on Ovarian Cancer recommend‐ ed against routine screening of the general population for ovarian cancer with serum CA125. The NIH Consensus Statement did, however, recommend that women at inherited risk of ovarian cancer undergo TVUS and serum CA125 screening every 6 to 12 months, beginning at the age of 35 years [91]. The Cancer Genetics Studies Consortium task force recommends that female carriers of a deleterious *BRCA1* mutation undergo annual or semi-annual screening using TVUS and serum CA125 levels, beginning at age of 25 to 35 years [92]. Both recommen‐

NCCN for those patients who have not chosen RRSO, consider concurrent TVUS (preferably day 1-10 menstrual cycle women in premenopausal women) + CA125 (preferably after day 5 of menstrual cycle women in premenopausal women) every 6 months starting at the age of 30 years or 5-10 years before the earliest age of first diagnosis of ovarian cancer in the family [93].

Although there are retrospective data indicating that annual ovarian cancer screening using TVUS and measurement of serum CA125 levels is neither an effective strategy for the early detection of ovarian tumours nor a reasonable substitute for a bilateral RRSO, the effectiveness of these interventions is limited to six-monthly screening. Investigational imaging and

MRS has proved to be a reliable technique for probing metabolic patterns, biochemical effects of tumour microenvironment, and the action of therapy in cancer cells, both in vivo and in vitro [94]. In particular, an increase in the total choline-containing compounds (tCho) content

Moreover, some studies have also shown alterations of the phospholipid metabolism in vitro using epithelial ovarian carcinoma cell lines [96,97], and demonstrated the feasibility of 3D CSI

Then, the metabolic meaning of a high concentration of choline in ovarian tumours merits some consideration. This topic has been extensively reviewed by Podo *et al.* in 2007 [98]. The high choline concentration of ovarian tumours can be considered as the result of an inappro‐ priate storage attributable to metabolic deregulation associated with clinical indicators of increased malignancy. The possibility of using a spatially resolved approach for MRS of ovarian masses opens an intriguing prospect for the diagnosis of early-stage tumours, with potential impact on the overall survival. This is especially true for carriers of a *BRCA* mutation, with a lifetime risk of 39% to 46% among women with the *BRCA1* mutation and a risk of 12%

Based on peer-reviewed published data, several institutions established the Guidelines to facilitate clinical management of patients with a suggestive personal or family history of breast and/or ovarian cancer, in particular individuals from a family with a known deleterious BRCA1/2 mutation. Screening options include transvaginal ultrasonography (TVUS), and serum CA125, while prevention options include medical therapy with drugs and surgery such

dations are based solely on expert opinion and best clinical judgment.

allows to distinguish malignant from benign lesions in the breast [95].

MRS to detect a choline peak in ovarian lesions in vivo at 1.5 T.

to 20% among those with the *BRCA2* mutation [99].

screening studies may be considered for this population.

58 Ovarian Cancer - A Clinical and Translational Update

**4.3. Proton Magnetic Resonance Spectroscopy (MRS)**

Additional potential serum biomarkers have been studied for the detection of ovarian cancer. For instance, human epididymis protein 4 (HE4) is a secreted glycoprotein over-expressed by serous and endometrioid ovarian cancers and expressed by 32% of ovarian cancers lacking CA125 expression.

To define the clinical utility of HE4, a comprehensive assessment of HE4 protein expression in benign and malignant ovarian and non-ovarian tissues by immunohistochemistry was performed and published in 2005. In comparison with normal surface epithelium, which does not express the protein, HE4 was widely found in cortical inclusion cysts lined by metaplastic Mullerian epithelium. These findings suggested that the formation of Mullerian epithelium is a prerequisite step in the development of some types of epithelial ovarian cancer. Moreover, the expression was restricted to certain histologic subtypes: 93% of serous and 100% of endometrioid epithelial ovarian cancers expressed HE4, while only 50% and 0% of clear cell carcinomas and mucinous tumours, respectively, were positive. HE4 protein expression is restricted in normal tissue to the reproductive tracts and respiratory epithelium. In fact, tissue microarrays revealed that the majority of non-ovarian carcinomas do not express HE4 [100].

In 2008 the Food and Drug Administration (FDA) approved HE4 to monitor disease recurrence and this marker was recently incorporated into the clinical evaluation of ovarian cancer patients. Recently, Moore *et al.* published a series of papers that used a combination of CA125, HE4 and menopausal status to predict the presence of a malignant ovarian tumour and developed the Risk of Ovarian Malignancy Algorithm (ROMA), a simple biomarker based algorithm, which requires US [101, 102].

**4.5. Proteomic profiling of ovarian cancer for biomarker discovery**

development and validation.

An et al.,(2006)[111]

Petri et al., (2009) [112]

Li et al., (2009) [113]

Cortesi et al.,(2011) [114]

Unfortunately, current diagnostic tools have had very limited success in early detection. The search for an ovarian cancer screening method with improved specificity and sensitivity has led to the examination of serum biomarker patterns using new 'omic' technologies [107-110]. In recent years, the advancing techniques for proteomics have accelerated the research for ovarian cancer biomarkers. Numerous proteomics-based molecular biomarkers/panels have been identified and hold great potential for diagnostic applications, but they need further

Several studies have analysed the proteomic profiles of ovarian tumour tissue, cell lines, urine,

**AUTHORS IDENTIFIED BIOMARKER REGULATION IN CANCER**

NM23-H1 ↑ Annexin-1 ↑ Protein phosphatase-1 ↑ Ferritin light chain ↑ Proteasome alpha-6 ↑ NAGK (N-acetyl glucosamine kinase) ↑

Preventive Strategies for Ovarian Cancer http://dx.doi.org/10.5772/54686 61

fibrinogen alpha fragment ↑ collagen alpha 1 (III) fragment ↑ fibrinogen beta NT fragment ↑

mitochondrial short-chain enoyl-CoA hydratase ↑

Phosphatidylethanolamine-biding protein 1 (PEBP) ↓ glutathione S-transferase A2 (GSTA2) ↓

protein S100-A8-calgranulin A (S100A8) ↑ retinol binding protein (RET1) ↓

prx-II ↓ prx-III ↑ hsp27 ↑ hsp60 ↑

Prohibitin ↑

Annexin-5 (ANXA5) ↓

galectin-3 (LEG3) ↓

ascites fluid and blood samples from ovarian cancer patients (Table 10) [111- 114].

**Table 10.** Promising biomarkers discovered by proteomic technology for ovarian cancer diagnosis.

An *et al.* [111] identified that different histologic subtypes of ovarian malignant epithelial tumours showed distinctly different protein expression profiles. The potential candidate biomarkers screened in ovarian tumours and found to be significantly up-regulated in comparison to normal tissues were: NM23, annexin-1, protein phosphatase-1, ferritin light chain, proteasome R-6, and NAGK (N-acetylglucosamine kinase). More recently, Petri *et al.* [112] examined whether urine could be used to measure specific ovarian cancer proteomic

In the last few years, several multi-modal screenings of women at high risk, combining different approaches, were carried out to improve ovarian cancer diagnostic test performance [103,104]. In 2010, a prospective case-control study was designed to evaluate the independent contributions of HE4, CA125 and the Symptom Index (SI) to predict ovarian cancer status in a multivariate model [105]. The SI is a screening tool that evaluates specific symptoms in conjunction with their frequency and duration to identify women who are at risk of ovarian cancer [106]. The SI, HE4 and CA125 all made significant independent contributions to ovarian cancer prediction. A rule for the positive cut-off based on anyone of the three tests being positive had a sensitivity of 95% with specificity of 80%. A rule based on any two of the three tests being positive had a sensitivity of 84% with a specificity of 98.5%. The SI alone had sensitivity of 64% with specificity of 88%. If the SI index is used to select women for CA125 and HE4 testing, specificity is 98.5% and sensitivity is 58% using the 2-of-3-positive positive cut-off rule. A comparison between different markers in ovarian cancer early diagnosis is presented in Table 9.


**Table 9.** Description of screening tests and biomarker combinations.

#### **4.5. Proteomic profiling of ovarian cancer for biomarker discovery**

patients. Recently, Moore *et al.* published a series of papers that used a combination of CA125, HE4 and menopausal status to predict the presence of a malignant ovarian tumour and developed the Risk of Ovarian Malignancy Algorithm (ROMA), a simple biomarker based

In the last few years, several multi-modal screenings of women at high risk, combining different approaches, were carried out to improve ovarian cancer diagnostic test performance [103,104]. In 2010, a prospective case-control study was designed to evaluate the independent contributions of HE4, CA125 and the Symptom Index (SI) to predict ovarian cancer status in a multivariate model [105]. The SI is a screening tool that evaluates specific symptoms in conjunction with their frequency and duration to identify women who are at risk of ovarian cancer [106]. The SI, HE4 and CA125 all made significant independent contributions to ovarian cancer prediction. A rule for the positive cut-off based on anyone of the three tests being positive had a sensitivity of 95% with specificity of 80%. A rule based on any two of the three tests being positive had a sensitivity of 84% with a specificity of 98.5%. The SI alone had sensitivity of 64% with specificity of 88%. If the SI index is used to select women for CA125 and HE4 testing, specificity is 98.5% and sensitivity is 58% using the 2-of-3-positive positive cut-off rule. A comparison between different markers in ovarian cancer early diagnosis is

CA125 CA125 was dichotomized at 95th percentile in the control group. Subjects with a

SI or CA125 Screen considered positive if either the SI or CA125 was positive, or if both were

SI or HE4 Screen considered positive if either the SI or HE4 was positive, or if both were positive. Any 1 of 3 tests positive Screen considered positive if any one of the SI or CA125 or HE4 was positive, or if two

positive HE4, or if all three tests were positive.

quickly.

positive.

were positive.

**Table 9.** Description of screening tests and biomarker combinations.

CA125 or HE4 Screen considered positive if CA125, HE4 or both were positive.

or more tests were positive.

HE4 HE4 was dichotomized at 95th percentile in the control group. Subjects with a marker

marker value above that threshold were considered to be positive for CA125.

The SI was considered to be positive if the patient had at least one of the following symptoms for less than one year but more than 12 times per month: bloating or increased abdominal size, abdominal or pelvic pain, difficulty eating or feeling full

Screen considered positive if both the SI and CA125 were positive, or if both the SI and HE4 were positive, or if both CA125 and HE4 were positive, or if all three tests

Classified as positive if SI was positive in addition to either a positive CA125 or a

value above that threshold were considered to be positive for HE4.

algorithm, which requires US [101, 102].

60 Ovarian Cancer - A Clinical and Translational Update

**SCREENING TESTS DESCRIPTION**

presented in Table 9.

**Single Markers**

Symptom Index (SI)

**Marker Combinations**

Any 2 of 3 tests positive

positive

SI and at least 1 additional test

Unfortunately, current diagnostic tools have had very limited success in early detection. The search for an ovarian cancer screening method with improved specificity and sensitivity has led to the examination of serum biomarker patterns using new 'omic' technologies [107-110]. In recent years, the advancing techniques for proteomics have accelerated the research for ovarian cancer biomarkers. Numerous proteomics-based molecular biomarkers/panels have been identified and hold great potential for diagnostic applications, but they need further development and validation.

Several studies have analysed the proteomic profiles of ovarian tumour tissue, cell lines, urine, ascites fluid and blood samples from ovarian cancer patients (Table 10) [111- 114].


**Table 10.** Promising biomarkers discovered by proteomic technology for ovarian cancer diagnosis.

An *et al.* [111] identified that different histologic subtypes of ovarian malignant epithelial tumours showed distinctly different protein expression profiles. The potential candidate biomarkers screened in ovarian tumours and found to be significantly up-regulated in comparison to normal tissues were: NM23, annexin-1, protein phosphatase-1, ferritin light chain, proteasome R-6, and NAGK (N-acetylglucosamine kinase). More recently, Petri *et al.* [112] examined whether urine could be used to measure specific ovarian cancer proteomic profiles and whether one peak alone or in combination with CA125 or other peaks had the sensitivity and specificity to discriminate between ovarian cancer pelvic mass and benign pelvic mass. Twenty-one significantly different peaks (p<0.001) were examined and the three most significant peaks were identified as fibrinogen alpha fragment, collagen alpha 1 (III) fragment and fibrinogen beta NT fragment. These results supported the feasibility of using urine as a diagnostic tool and suggested the enhanced prediction performance of combined marker analysis. Li *et al.* [113] performed a comparative proteomic study of normal ovarian epithelial and ovarian epithelial serous cystadenocarcinoma tissue and identified six proteins significantly differentially expressed. In particular, Prx-II expression was found to be linearly decreased from normal ovarian tissue, to benign ovarian lesions, and ovarian malignancies. No statistical difference between carcinoma groups in different clinical stages, differentiation status, and histological type was seen, suggesting that the decreased level of Prx-II is a common marker for ovarian malignancies. This was the first report on the altered expression of Prx-II in ovarian cancer.

Five were found to be down-regulated and identified as galectin 3, glutathione S-transferase A-2, retinol binding protein 1, phosphatidylethanolamine-binding protein and annexin 5, while the calgranulin, was significantly up-regulated in all pathological samples, including the ascitic fluid. This is the first study to report an over-expression of calgranulin by 2-DE analysis combined with MS/MS on surgical biopsy. As previously reported, the reduced expression of galectin 3 and retinol binding protein 1 in cystic fluid and serum of patients with early stage disease is confirmed in this study. The results highlight alterations in proteins that control cell-cycle progression and apoptosis, as well as factors that modulate the activity of signal transduction pathways. Moreover, this study suggests that calgranulin expression may

Preventive Strategies for Ovarian Cancer http://dx.doi.org/10.5772/54686 63

However, critical assessment of the results has shown significant shortcomings and uncer‐ tainties with regard to the reproducibility of the findings and identity of the proteins behind the peak patterns, thus, the validation of the newly discovered biomarkers still remains the most challenging aspect of clinical proteomics. The advancing techniques for proteomics have shown promise in a variety of studies and have provided new insights into ovarian cancer diagnosis, but few have turned out to be useful in the clinic. At present, the development of an effective strategy for early detection of ovarian cancer is still a work in progress [110].

Primary and secondary prevention of ovarian cancer play a crucial role in the attempt to improve the overall survival from the disease. In particular, primary prevention is based on avoiding risk factors and increasing protective factors. Despite the identification of several risk and protective factors among the general population, most of the common factors described to date only slightly influence the risk of developing ovarian cancer, thus, the knowledge of these factors has still not been translated into practical strategies to prevent ovarian cancer. On the other hand, primary prevention could represent a good opportunity for high-risk women. Women who inherit a mutation in either the *BRCA1* or *BRCA2* gene have greatly elevated lifetime risks of ovarian cancer, fallopian tube cancer and breast cancer. Surveillance for ovarian and fallopian tube cancer has not been proven to be effective. For this reason, preventive surgical removal of the ovaries and fallopian tubes (salpingo-oophorectomy) is actively recommended to these women by the age of 35 or 40 years, often prior to natural menopause, to prevent cancer. Moreover, women at increased risk may join a cancer preven‐ tion clinical trial or a chemoprevention trial. In particular, oral contraceptive use can be considered as an alternative strategy in the chemoprevention of ovarian cancer in *BRCA1* mutation carriers who do not accept RRSO above the age of 30 years. Other chemopreventive agents such as retinoids, analgesic drugs, vitamin D, cyclo-oxygenase inhibitors and peroxi‐ some proliferator activated receptor-gamma ligands have shown promise in early investiga‐

Regarding radiological methods to investigate ovaries and their adnexes, new techniques besides TVUS need to be explored. Pelvic Magnetic Radiological Imaging could be of interest

be used as a diagnostic and/or prognostic biomarker [114].

**5. Discussion**

tions of disease prevention.

A recent comparative proteomic study investigated and defined protein expression patterns associated with advanced stage ovarian cancer, to define a panel of diagnostic and/or prog‐ nostic markers. The study also investigated proteins secreted by the cancer cell into the interstitial fluid, as cancer growth and progression also depends on stromal factors present in the tumour microenvironment. Moreover, many biomarkers present in biopsied cancer tissues can also be found in blood serum, representing potential biomarkers of the disease. Proteomic profiling of differentially expressed proteins in cancer ovarian tissue, tumoral interstitial fluid (TIF) and ascitic fluid, compared with healthy tissue samples and normal interstitial fluid (NIF), allowed the identification of protein spots consistently differentially expressed between normal and cancer samples. Protein expression/identification was evaluated by 2-DE (twodimensional gel electrophoresis) and MS (mass spectrometry) analysis and was confirmed by immunohistochemistry. Six proteins showed differential expression in tumoral interstitial fluid and tumour tissue compared to normal interstitial fluid and healthy tissue. Differential protein expression between tumoral and normal ovarian tissue is presented in Table 11.


The fold change indicates the direction and the magnitude of the change in expression level. Data are expressed as mean ± standard deviation.

**Table 11.** Modification in protein expression in tumoral tissue and interstitial fluid

Five were found to be down-regulated and identified as galectin 3, glutathione S-transferase A-2, retinol binding protein 1, phosphatidylethanolamine-binding protein and annexin 5, while the calgranulin, was significantly up-regulated in all pathological samples, including the ascitic fluid. This is the first study to report an over-expression of calgranulin by 2-DE analysis combined with MS/MS on surgical biopsy. As previously reported, the reduced expression of galectin 3 and retinol binding protein 1 in cystic fluid and serum of patients with early stage disease is confirmed in this study. The results highlight alterations in proteins that control cell-cycle progression and apoptosis, as well as factors that modulate the activity of signal transduction pathways. Moreover, this study suggests that calgranulin expression may be used as a diagnostic and/or prognostic biomarker [114].

However, critical assessment of the results has shown significant shortcomings and uncer‐ tainties with regard to the reproducibility of the findings and identity of the proteins behind the peak patterns, thus, the validation of the newly discovered biomarkers still remains the most challenging aspect of clinical proteomics. The advancing techniques for proteomics have shown promise in a variety of studies and have provided new insights into ovarian cancer diagnosis, but few have turned out to be useful in the clinic. At present, the development of an effective strategy for early detection of ovarian cancer is still a work in progress [110].

#### **5. Discussion**

profiles and whether one peak alone or in combination with CA125 or other peaks had the sensitivity and specificity to discriminate between ovarian cancer pelvic mass and benign pelvic mass. Twenty-one significantly different peaks (p<0.001) were examined and the three most significant peaks were identified as fibrinogen alpha fragment, collagen alpha 1 (III) fragment and fibrinogen beta NT fragment. These results supported the feasibility of using urine as a diagnostic tool and suggested the enhanced prediction performance of combined marker analysis. Li *et al.* [113] performed a comparative proteomic study of normal ovarian epithelial and ovarian epithelial serous cystadenocarcinoma tissue and identified six proteins significantly differentially expressed. In particular, Prx-II expression was found to be linearly decreased from normal ovarian tissue, to benign ovarian lesions, and ovarian malignancies. No statistical difference between carcinoma groups in different clinical stages, differentiation status, and histological type was seen, suggesting that the decreased level of Prx-II is a common marker for ovarian malignancies. This was the first report on the altered expression of Prx-II

A recent comparative proteomic study investigated and defined protein expression patterns associated with advanced stage ovarian cancer, to define a panel of diagnostic and/or prog‐ nostic markers. The study also investigated proteins secreted by the cancer cell into the interstitial fluid, as cancer growth and progression also depends on stromal factors present in the tumour microenvironment. Moreover, many biomarkers present in biopsied cancer tissues can also be found in blood serum, representing potential biomarkers of the disease. Proteomic profiling of differentially expressed proteins in cancer ovarian tissue, tumoral interstitial fluid (TIF) and ascitic fluid, compared with healthy tissue samples and normal interstitial fluid (NIF), allowed the identification of protein spots consistently differentially expressed between normal and cancer samples. Protein expression/identification was evaluated by 2-DE (twodimensional gel electrophoresis) and MS (mass spectrometry) analysis and was confirmed by immunohistochemistry. Six proteins showed differential expression in tumoral interstitial fluid and tumour tissue compared to normal interstitial fluid and healthy tissue. Differential protein expression between tumoral and normal ovarian tissue is presented in Table 11.

**P-value**

ANXA5 -1,88 ± -0,48 <0,0001 -5,605 ± -3,29 <0,01 PEBP -4,21 ± -2,90 <0,01 -2,82 ± -0,69 <0,0001 GSTA2 -4,67 ± -1,88 <0,0001 -27,39 ± -21,24 <0,01 LEG3 -2,19 ± -0,69 <0,0001 -5,10 ± -4,42 <0,05 S100A8 3,67 ± 1,50 <0,001 3,58 ± 1,11 <0,0001 RET1 -6,33 ± -3,30 <0,001 -5,01 ± -4,28 <0,05

The fold change indicates the direction and the magnitude of the change in expression level. Data are expressed as mean

**FOLD CHANGE TIF VERSUS NIF**

**P-value**

in ovarian cancer.

62 Ovarian Cancer - A Clinical and Translational Update

**PROTEIN NAME**

± standard deviation.

**FOLD CHANGE TUMORAL VERSUS NORMAL TISSUE**

**Table 11.** Modification in protein expression in tumoral tissue and interstitial fluid

Primary and secondary prevention of ovarian cancer play a crucial role in the attempt to improve the overall survival from the disease. In particular, primary prevention is based on avoiding risk factors and increasing protective factors. Despite the identification of several risk and protective factors among the general population, most of the common factors described to date only slightly influence the risk of developing ovarian cancer, thus, the knowledge of these factors has still not been translated into practical strategies to prevent ovarian cancer.

On the other hand, primary prevention could represent a good opportunity for high-risk women. Women who inherit a mutation in either the *BRCA1* or *BRCA2* gene have greatly elevated lifetime risks of ovarian cancer, fallopian tube cancer and breast cancer. Surveillance for ovarian and fallopian tube cancer has not been proven to be effective. For this reason, preventive surgical removal of the ovaries and fallopian tubes (salpingo-oophorectomy) is actively recommended to these women by the age of 35 or 40 years, often prior to natural menopause, to prevent cancer. Moreover, women at increased risk may join a cancer preven‐ tion clinical trial or a chemoprevention trial. In particular, oral contraceptive use can be considered as an alternative strategy in the chemoprevention of ovarian cancer in *BRCA1* mutation carriers who do not accept RRSO above the age of 30 years. Other chemopreventive agents such as retinoids, analgesic drugs, vitamin D, cyclo-oxygenase inhibitors and peroxi‐ some proliferator activated receptor-gamma ligands have shown promise in early investiga‐ tions of disease prevention.

Regarding radiological methods to investigate ovaries and their adnexes, new techniques besides TVUS need to be explored. Pelvic Magnetic Radiological Imaging could be of interest even if it is difficult to imagine such an expensive technique being employed in the screening of high-risk women. For high-risk women, recommended cancer screening strategies, which need to be adjusted depending on the earliest age of onset in a family, have not been assessed by randomized trials or case-control studies. Ovarian cancer screening relies on a combination of annual or semi-annual pelvic examination, annual or semi-annual transvaginal ultrasound examination with colour Doppler, and annual measurement of serum CA125 concentrations.

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[3] Marth C, Hiebl S, Oberaigner W, Winter R, Leodolter S, Sevelda P. Influence of department volume on survival for ovarian cancer: results from a prospective quality assurance program of the Austrian Association for Gynecologic Oncology. Int J

[4] Chan JK, Urban R, Cheung MK, Osann K, Shin JY, Husain A, Teng NN, Kapp DS, Berek JS, Leiserowitz GS. Ovarian cancer in younger vs older women: a population-based

[5] Nagle CM, Bain CJ, Green AC, Webb PM. The influence of reproductive and hormonal

[6] Schorge JO, Modesitt SC, Coleman RL, Cohn DE, Kauff ND, Duska LR, Herzog TJ. SGO White Paper on ovarian cancer: etiology, screening and surveillance. Gynecol Oncol.

[7] Schouten LJ, Rivera C, Hunter DJ, Spiegelman D, Adami HO, Arslan A, Beeson WL, van den Brandt PA, Buring JE, Folsom AR, Fraser GE, Freudenheim JL, Goldbohm RA, Hankinson SE, Lacey JV Jr, Leitzmann M, Lukanova A, Marshall JR, Miller AB, Patel AV, Rodriguez C, Rohan TE, Ross JA, Wolk A, Zhang SM, Smith-Warner SA. Height, body mass index, and ovarian cancer: a pooled analysis of 12 cohort studies. Cancer

[8] Lahmann PH, Cust AE, Friedenreich CM, Schulz M, Lukanova A, Kaaks R, Lundin E, Tjønneland A, Halkjaer J, Severinsen MT, Overvad K, Fournier A, Chabbert-Buffet N, Clavel-Chapelon F, Dossus L, Pischon T, Boeing H, Trichopoulou A, Lagiou P, Naska A, Palli D, Grioni S, Mattiello A, Tumino R, Sacerdote C, Redondo ML, Jakszyn P, Sánchez MJ, Tormo MJ, Ardanaz E, Arriola L, Manjer J, Jirström K, Bueno-de-Mesquita HB, May AM, Peeters PH, Onland-Moret NC, Bingham S, Khaw KT, Allen NE, Spencer E, Rinaldi S, Slimani N, Chajes V, Michaud D, Norat T, Riboli E. Anthropometric measures and epithelial ovarian cancer risk in the European Prospective Investigation

[9] Camargo MC, Stayner LT, Straif K, Reina M, Al-Alem U, Demers PA, Landrigan PJ. Occupational exposure to asbestos and ovarian cancer: a meta-analysis. Environ Health

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[11] Beral V; Million Women Study Collaborators, Bull D, Green J, Reeves G. Ovarian cancer and hormone replacement therapy in the Million Women Study. Lancet.

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Current approaches are a futile attempt to detect ovarian cancer in the early stages, but future research should be directed to better characterizing critical pathways in ovarian carcinogenesis and to identifying appropriate surveillance programs based on biomarker tests and/or radiological investigations, in order to improve overall survival, which dramatically decreases in the first 5 years. Due to the fact that an analysis of potentially thousands of proteins which could be simultaneously altered is necessary, comparative proteomics is a promising mode of potential biomarker discovery for cancer detection and monitoring. A better estimation of the biological importance of certain proteins with regard to the progression from pre-neoplastic tissue alterations to malignant tumours, as well as the prediction of the metastasis-forming potential by biomarkers, will be a necessary prerequisite to provide a more detailed insight and understanding of tumour progression.

#### **Acknowledgements**

The authors thank the "Angela Serra" Association for Cancer Research (Modena, Italy) and the "Fondazione Cassa di Risparmio "of Modena, Italy for financial support. A special thanks to Dr Adriano Benedetti and Dr Daniela Manzini (C.I.G.S., University of Modena and Reggio Emilia) for skilled assistance in protein analysis by ESI-Q-TOF MS. This work was supported by grants from the Italian Minister of University and Research, PRIN 2008 (20088RFCMH).

#### **Author details**

L. Cortesi, A. Toss and E. De Matteis

Department of Oncology and Haematology, University of Modena and Reggio Emilia, Modena, Italy

#### **References**

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even if it is difficult to imagine such an expensive technique being employed in the screening of high-risk women. For high-risk women, recommended cancer screening strategies, which need to be adjusted depending on the earliest age of onset in a family, have not been assessed by randomized trials or case-control studies. Ovarian cancer screening relies on a combination of annual or semi-annual pelvic examination, annual or semi-annual transvaginal ultrasound examination with colour Doppler, and annual measurement of serum CA125 concentrations.

Current approaches are a futile attempt to detect ovarian cancer in the early stages, but future research should be directed to better characterizing critical pathways in ovarian carcinogenesis and to identifying appropriate surveillance programs based on biomarker tests and/or radiological investigations, in order to improve overall survival, which dramatically decreases in the first 5 years. Due to the fact that an analysis of potentially thousands of proteins which could be simultaneously altered is necessary, comparative proteomics is a promising mode of potential biomarker discovery for cancer detection and monitoring. A better estimation of the biological importance of certain proteins with regard to the progression from pre-neoplastic tissue alterations to malignant tumours, as well as the prediction of the metastasis-forming potential by biomarkers, will be a necessary prerequisite to provide a more detailed insight

The authors thank the "Angela Serra" Association for Cancer Research (Modena, Italy) and the "Fondazione Cassa di Risparmio "of Modena, Italy for financial support. A special thanks to Dr Adriano Benedetti and Dr Daniela Manzini (C.I.G.S., University of Modena and Reggio Emilia) for skilled assistance in protein analysis by ESI-Q-TOF MS. This work was supported by grants from the Italian Minister of University and Research, PRIN 2008 (20088RFCMH).

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**Acknowledgements**

**Author details**

Modena, Italy

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[83] Buys SS, Partridge E, Black A, Johnson CC, Lamerato L, Isaacs C, Reding DJ, Greenlee RT, Yokochi LA, Kessel B, Crawford ED, Church TR, Andriole GL, Weissfeld JL, Fouad MN, Chia D, O'Brien B, Ragard LR, Clapp JD, Rathmell JM, Riley TL, Hartge P, Pinsky PF, Zhu CS, Izmirlian G, Kramer BS, Miller AB, Xu JL, Prorok PC, Gohagan JK, Berg CD; PLCO Project Team. Effect of screening on ovarian cancer mortality: the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Randomized Controlled Trial.

[84] Scheuer L, Kauff N, Robson M, Kelly B, Barakat R, Satagopan J, Ellis N, Hensley M, Boyd J, Borgen P, Norton L, Offit K. Outcome of preventive surgery and screening for breast and ovarian cancer in BRCA mutation carriers. J Clin Oncol 2002;20(5):1260-8.

[85] Hermsen BB, Olivier RI, Verheijen RH, van Beurden M, de Hullu JA, Massuger LF, Burger CW, Brekelmans CT, Mourits MJ, de Bock GH, Gaarenstroom KN, van Boven HH, Mooij TM, Rookus MA. No efficacy of annual gynaecological screening in BRCA1/2 mutation carriers; an observational follow-up study. Br J Cancer 2007;96(9):

[86] Olivier RI, Lubsen-Brandsma MA, Verhoef S, van Beurden M. CA125 and transvaginal ultrasound monitoring in high-risk women cannot prevent the diagnosis of advanced

[87] Van Der Velde NM, Mourits MJ, Arts HJ, De Vries J, Leegte BK, Dijkhuis G, Oosterwijk JC, De Bock GH. Time to stop ovarian cancer screening in BRCA1/2 mutation carriers?

[88] Stirling D, Evans DG, Pichert G, Shenton A, Kirk EN, Rimmer S, Steel CM, Lawson S, Busby-Earle RM, Walker J, Lalloo FI, Eccles DM, Lucassen AM, Porteous ME. Screening for familial ovarian cancer: failure of current protocols to detect ovarian cancer at an early stage according to the international Federation of gynecology and obstetrics

[89] Laframboise S, Nedelcu R, Murphy J, Cole DE, Rosen B. Use of CA-125 and ultrasound

[90] Evans DG, Gaarenstroom KN, Stirling D, Shenton A, Maehle L, Dørum A, Steel M, Lalloo F, Apold J, Porteous ME, Vasen HF, van Asperen CJ, Moller P. Screening for familial ovarian cancer: poor survival of BRCA1/2 related cancers. J Med Genet.

[91] NCCN Clinical Practice Guidelines in Oncology. Genetic/Familial High-Risk Assess‐

[92] Burke W, Daly M, Garber J, Botkin J, Kahn MJ, Lynch P, McTiernan A, Offit K, Perlman J, Petersen G, Thomson E, Varricchio C. Recommendations for follow-up care of

ment: Breast and Ovarian. http://nccn.org (accessed 19 September 2012)

ovarian cancer. Gynecol Oncol. 2006;100(1):20-6.

Int J Cancer. 2009;124(4):919-23.

2009;46(9):593-7.

system. J Clin Oncol. 2005;23(24):5588-96.

in high-risk women. Int J Gynecol Cancer. 2002;12(1):86-91.

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JAMA 2011;305(22):2295-303.

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[104] Macuks R, Baidekalna I, Donina S. An ovarian cancer malignancy risk index composed of HE4, CA125, ultrasonographic score, and menopausal status: use in differentiation of ovarian cancers and benign lesions. Tumour Biol. 2012 Jun 14. [Epub ahead of print] **Section 2**

**Pathology**


**Section 2**

### **Pathology**

[104] Macuks R, Baidekalna I, Donina S. An ovarian cancer malignancy risk index composed of HE4, CA125, ultrasonographic score, and menopausal status: use in differentiation of ovarian cancers and benign lesions. Tumour Biol. 2012 Jun 14. [Epub ahead of print]

[105] Andersen MR, Goff BA, Lowe KA, Scholler N, Bergan L, Drescher CW, Paley P, Urban N. Use of a Symptom Index, CA125, and HE4 to predict ovarian cancer. Gynecol Oncol.

[106] Goff BA, Mandel LS, Drescher CW, Urban N, Gough S, Schurman KM, Patras J, Mahony BS, Andersen MR. Development of an ovarian cancer symptom index: possibilities for

[107] Dann RB, Kelley JL, Zorn KK. Strategies for ovarian cancer prevention. Obstet Gynecol

[108] Petricoin EF, Ardekani AM, Hitt BA, Levine PJ, Fusaro VA, Steinberg SM, Mills GB, Simone C, Fishman DA, Kohn EC, Liotta LA. Use of proteomic patterns in serum to

[109] Nossov V, Amneus M, Su F, Lang J, Janco JM, Reddy ST, Farias-Eisner R. The early detection of ovarian cancer: from traditional methods to proteomics. Can we really do

[110] Zhang B, Barekati Z, Kohler C, Radpour R, Asadollahi R, Holzgreve W, Zhong XY. Proteomics and biomarkers for ovarian cancer diagnosis. Ann Clin Lab Sci. 2010;40(3):

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[112] Petri AL, Simonsen AH, Yip TT, Hogdall E, Fung ET, Lundvall L, Hogdall C. Three new potential ovarian cancer biomarkers detected in human urine with equalizer bead

[113] Li XQ, Zhang SL, Cai Z, Zhou Y, Ye TM, Chiu JF. Proteomic identification of tumorassociated protein in ovarian serous cystadenocarinoma. Cancer Lett. 2009; 275(1):

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ovarian cancer. Electrophoresis 2011;32(15):1992-2003.

Clin North Am. 2007;34(4):667-86.

**Chapter 4**

**Borderline Epithelial Tumors of the Ovary**

Borderline ovarian tumors (BOT) were first described in 1929 by Taylor, which, due to the characteristics of the tumor, called it "semi malignant" or "borderline" [1]; subsequently, this group of tumors of the ovary were classified in 1973 by the World Health Organization as "low malignant potential ovarian tumor" [2] and, finally, in 2003 WHO separates them from

Another term accepted to designate these independent ovarian neoplasms is "atypical

Borderline ovarian tumors represent 10-20% of epithelial ovarian neoplasm's [5] with an incidence of 1.8-4.8 out of 100.000 women per year [6] and typically have an excellent prognosis.

Unlike the invasive carcinomas, borderline ovarian tumors are characterized by cytoplasmic and nuclear atypia, (element of differential diagnosis with benign tumors), absence of stromal invasion, (element of differential diagnosis with malignant tumors), unusual degree of proliferation of the epithelial cells with cellular stratification including remarkable architec‐ tural atypia and the formation of papillary protuberances. The absence of obvious stromal invasion is a principal diagnostic criterion for BOTs. Histologically, most of them are serous or mucinous, but endometrioid, clear cell, Brenner (transitional cell) or mixed histotypes can

To date, there are still no prospective randomized trials to clinical management, although they have an excellent prognosis, with a 5-year overall survival rate of almost 100% in early-stage disease (stage I-II) and between 86% and 92% in more advanced disease (stage III-IV). [8]

and reproduction in any medium, provided the original work is properly cited.

© 2013 Cormio et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Gennaro Cormio, Vera Loizzi, Maddalena Falagario,

Doriana Scardigno, Donatella Latorre and

Additional information is available at the end of the chapter

carcinomas and call them borderline tumors. [3]

proliferating (or atypical proliferative) tumor". [4]

Luigi E. Selvaggi

**1. Introduction**

be also seen. [7]

http://dx.doi.org/10.5772/54828

### **Borderline Epithelial Tumors of the Ovary**

Gennaro Cormio, Vera Loizzi, Maddalena Falagario, Doriana Scardigno, Donatella Latorre and Luigi E. Selvaggi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54828

#### **1. Introduction**

Borderline ovarian tumors (BOT) were first described in 1929 by Taylor, which, due to the characteristics of the tumor, called it "semi malignant" or "borderline" [1]; subsequently, this group of tumors of the ovary were classified in 1973 by the World Health Organization as "low malignant potential ovarian tumor" [2] and, finally, in 2003 WHO separates them from carcinomas and call them borderline tumors. [3]

Another term accepted to designate these independent ovarian neoplasms is "atypical proliferating (or atypical proliferative) tumor". [4]

Borderline ovarian tumors represent 10-20% of epithelial ovarian neoplasm's [5] with an incidence of 1.8-4.8 out of 100.000 women per year [6] and typically have an excellent prognosis.

Unlike the invasive carcinomas, borderline ovarian tumors are characterized by cytoplasmic and nuclear atypia, (element of differential diagnosis with benign tumors), absence of stromal invasion, (element of differential diagnosis with malignant tumors), unusual degree of proliferation of the epithelial cells with cellular stratification including remarkable architec‐ tural atypia and the formation of papillary protuberances. The absence of obvious stromal invasion is a principal diagnostic criterion for BOTs. Histologically, most of them are serous or mucinous, but endometrioid, clear cell, Brenner (transitional cell) or mixed histotypes can be also seen. [7]

To date, there are still no prospective randomized trials to clinical management, although they have an excellent prognosis, with a 5-year overall survival rate of almost 100% in early-stage disease (stage I-II) and between 86% and 92% in more advanced disease (stage III-IV). [8]

© 2013 Cormio et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **2. Classification, pathology and clinical behavior**

Although they might occur in every age, most of the cases are diagnosed in pre-menopausal women between 34 and 40 years [9], while malignant ovarian cancer usually is diagnosed in patients between 50 and 70 years.

presence of foci resembling non invasive desmoplastic peritoneal implants with a welldelineated border on the ovarian surface, this phenomenon does not have any clear known pathogenesis nor clinical significance. [13, 19, 20, 16, 17] Signs of necrosis are very rare. [4]

Borderline Epithelial Tumors of the Ovary http://dx.doi.org/10.5772/54828 79

APTSs are usually positive for CK7, OC-125 and cytokeratin and express estrogen and

Non-invasive MPSCs, are serous borderline tumors characterized by the presence of micro‐ papillae arising from central papilla, when this specific pattern constitutes either a 5 mm or

In invasive MPSCs (synonymous of low-grade serous carcinoma) the stromal invasion must

Non-invasive MPSCs represent 14% of all BOTs. The mean age at diagnosis is about 42 years, in 70% of cases they are bilateral and 50% of patients are in stage I at the time of diagnosis,

On gross appearance, non-invasive MPSC s look like cysts with papillae without or with little necrosis just like APSTs but in contrast to them they present mostly with peritoneal implant

Histologically they are neoplasm with high degree of epithelial proliferation in a non hier‐

The differential diagnosis should be done with serous cystadenomas, serous mucinous or

Survival in serous BOTs differs significatively from serous invasive ovarian cancer and is

archical branching architecture, with micropapillary and cribriform patterns. [4]

endometrioid borderline tumors and with malignant neoplasm.

progesterone receptors. [4]

exceed 5 mm.

grater area or 10% or greater proportion.

while the other 50% are in stage II or III.

and bilaterality; the mean size is about 8 cm.

characterized by an excellent prognosis.

**Figure 1.** Serous borderline ovarian tumor

Risk factors for the development of BOTs are absolutely similar to those known for ovarian cancer, menarche, age at first pregnancy, age at first delivery, menstrual history, smoking history and family history of ovarian cancer, except that BOTs seem to have a lower frequency of BRCA mutations.

Borderline ovarian tumors are staged according to the FIGO classification of ovarian cancer. In 80% of cases patients with BOTs are in FIGO stage I at the time of diagnosis, about 30% of patients are in stage II-III in the same percentages each, while stage IV BOTs are very rare. [10]

#### **2.1. Serous borderline tumors**

They represent the 70% of BOTs, and 9-15% of all serous neoplasms [11,12] the mean age at presentation is 38 years old (range 17-77). [13] According to the FIGO staging system, [14] 68% are Stage I, 11% Stage II, 21% Stage III and less than 1% Stage IV. [15]

These neoplasms can be divided in two subtypes:


APTSs in 25-30% of cases they are bilateral, macroscopically they appear as cysts with serous contents with friable and exuberant papillary projections. (Figure 1) These papillae are mostly observed on the inner surface of the cyst, but in 70% of cases also in the external one. Rarely these serous BOTs show solid components.

Histologically APTSs show the presence of papillae with extensive epithelial stratification and budding, the epithelial cells have low or moderate atypias, in the fluid a detachment of single cells can be seen, there must not be any sign of invasion (Figure 2), but microinvasion (not more than 10 mm2 ) can be present in up to 15% of cases. F [13, 16, 17] Some patients with stage I microinvasive tumors have developed progressive disease and microinvasion for some authors can be considered as risk factor for patients with high-stage disease. [18]

The cells in APTS can show an epithelial and occasionally a mesothelial differentiation. The nuclei of these cells present more atypia than those seen in benign cystoadenomas, the nuclei are usually basally located and ovoid or rounded, the nucleoli are only occasionally prominent and the mitosis are not so common (usually less than four per ten high-power fields, HPF). [4]

APTSs are occasionally also associated to the presence of endosalpingiosis or non invasive peritoneal implants, referred as the phenomenon of autoimplantation can be observed as the presence of foci resembling non invasive desmoplastic peritoneal implants with a welldelineated border on the ovarian surface, this phenomenon does not have any clear known pathogenesis nor clinical significance. [13, 19, 20, 16, 17] Signs of necrosis are very rare. [4]

APTSs are usually positive for CK7, OC-125 and cytokeratin and express estrogen and progesterone receptors. [4]

Non-invasive MPSCs, are serous borderline tumors characterized by the presence of micro‐ papillae arising from central papilla, when this specific pattern constitutes either a 5 mm or grater area or 10% or greater proportion.

In invasive MPSCs (synonymous of low-grade serous carcinoma) the stromal invasion must exceed 5 mm.

Non-invasive MPSCs represent 14% of all BOTs. The mean age at diagnosis is about 42 years, in 70% of cases they are bilateral and 50% of patients are in stage I at the time of diagnosis, while the other 50% are in stage II or III.

On gross appearance, non-invasive MPSC s look like cysts with papillae without or with little necrosis just like APSTs but in contrast to them they present mostly with peritoneal implant and bilaterality; the mean size is about 8 cm.

Histologically they are neoplasm with high degree of epithelial proliferation in a non hier‐ archical branching architecture, with micropapillary and cribriform patterns. [4]

The differential diagnosis should be done with serous cystadenomas, serous mucinous or endometrioid borderline tumors and with malignant neoplasm.

Survival in serous BOTs differs significatively from serous invasive ovarian cancer and is characterized by an excellent prognosis.

**Figure 1.** Serous borderline ovarian tumor

**2. Classification, pathology and clinical behavior**

patients between 50 and 70 years.

78 Ovarian Cancer - A Clinical and Translational Update

**2.1. Serous borderline tumors**

of BRCA mutations.

Although they might occur in every age, most of the cases are diagnosed in pre-menopausal women between 34 and 40 years [9], while malignant ovarian cancer usually is diagnosed in

Risk factors for the development of BOTs are absolutely similar to those known for ovarian cancer, menarche, age at first pregnancy, age at first delivery, menstrual history, smoking history and family history of ovarian cancer, except that BOTs seem to have a lower frequency

Borderline ovarian tumors are staged according to the FIGO classification of ovarian cancer. In 80% of cases patients with BOTs are in FIGO stage I at the time of diagnosis, about 30% of patients are in stage II-III in the same percentages each, while stage IV BOTs are very rare. [10]

They represent the 70% of BOTs, and 9-15% of all serous neoplasms [11,12] the mean age at presentation is 38 years old (range 17-77). [13] According to the FIGO staging system, [14] 68%

**•** APTSs, Atypical proliferative serous tumors, behave in a benign way and show a papillary

**•** Non-invasive MPSCs, micopapillary serous borderline tumors, with a non hierarchical pattern, characterized by the presence of micropapilla, they are more associated with

APTSs in 25-30% of cases they are bilateral, macroscopically they appear as cysts with serous contents with friable and exuberant papillary projections. (Figure 1) These papillae are mostly observed on the inner surface of the cyst, but in 70% of cases also in the external one. Rarely

Histologically APTSs show the presence of papillae with extensive epithelial stratification and budding, the epithelial cells have low or moderate atypias, in the fluid a detachment of single cells can be seen, there must not be any sign of invasion (Figure 2), but microinvasion (not

I microinvasive tumors have developed progressive disease and microinvasion for some

The cells in APTS can show an epithelial and occasionally a mesothelial differentiation. The nuclei of these cells present more atypia than those seen in benign cystoadenomas, the nuclei are usually basally located and ovoid or rounded, the nucleoli are only occasionally prominent and the mitosis are not so common (usually less than four per ten high-power fields, HPF). [4] APTSs are occasionally also associated to the presence of endosalpingiosis or non invasive peritoneal implants, referred as the phenomenon of autoimplantation can be observed as the

authors can be considered as risk factor for patients with high-stage disease. [18]

) can be present in up to 15% of cases. F [13, 16, 17] Some patients with stage

are Stage I, 11% Stage II, 21% Stage III and less than 1% Stage IV. [15]

These neoplasms can be divided in two subtypes:

invasive implants and a worse prognosis than APTSs.

architecture with a hierarchical pattern

these serous BOTs show solid components.

more than 10 mm2

Atypical proliferative mucinous tumors of endocervical-like type are more frequently bilateral, smaller and are often associated with endometriosis. Macroscopically and microscopically they resemble APTS with a combination of endocervical mucinous and serous epithelium. These neoplasms very rarely present with peritoneal implants or signs of microinvasion (defined as the presence of single or small cluster of cells within the stroma) and have a benign

Borderline Epithelial Tumors of the Ovary http://dx.doi.org/10.5772/54828 81

In same cases these tumors can show a severe atypia and epithelial overgrowth still without any sign of stromal invasion, these cases are referred to "non invasive or intraepithelial

The immunohistochemistry pattern of mucinous BOTs is characterized by the expression of cytokeratin (CK) 7 and 20, but no positivity for estrogen and progesterone receptors and Ca125; in the differential diagnosis with intestinal tract tumors this can be very helpful (the neoplasms

The differential diagnosis should be done with metastatic mucinous carcinomas to the ovary

Endometrioid tumors of the ovary are usually carcinomas, while borderline forms are very rare; they can arise from endometriosis and can also be associated to endometrial hyperplasia.

Endometrioid BOTs, also called Atypical Proliferative Endometrioid Tumors (APET), account

Macroscopically they appear as cyst sometimes with solid compounds with hemorrhagic

brown fluid inside, in about 60% of cases endometriotic foci are also associated. [4]

carcinoma" and have still an excellent prognosis in stage I. [4]

and benign or invasive mucinous neoplasms of ovarian origin.

of intestinal tract origin express CK20 but not CK 7). [4]

**2.3. Endometrioid borderline tumors**

**Figure 3.** Endometrioid borderline ovarian tumor

for the 0,2% of ovarian epithelial neoplasms. [4]

behaviour.

**Figure 2.** Serous borderline ovarian tumors

#### **2.2. Mucinous borderline tumors**

Mucinous BOTs are less common than their serous counterparts. They are also called "atypical proliferative mucinous tumor" (APMT) or "mucinous tumor of low malignant potential". They are often associated to pseudomyxoma peritonei (PMP) a condition characterized by the presence of mucinous ascites and mucoid peritoneal implant

They can be divided into two subtypes:


The first type in 95% of cases is unilateral and appears macroscopically as a multicystic large neoplasm (mean size of about 20 cm) with a smooth capsule.

The cysts contain inside a mucinous material and their surfaces very rarely show the presence of papillary projections.

Histologically, the stromal invasion is absent, the epithelium is stratified, mucinous gastroin‐ testinal-type, with villoglandular of papillary intraglandular growth; the cells show moderate atypia in their nuclei. [4]

Their biological behaviour is very benign with a survival rate of nearly in early stages 100%. The tumors in advanced stage have a mortality of 50%, but mostly are associated with pseudomyxoma peritonei and probably all these case can be considered of primary gastroin‐ testinal and not ovarian origin (usually appendix but also pancreas and biliary tract). For this reason is generally accepted that the true primary APMT in advanced stage do not really exist and that those cases with mucin or benign mucinous epithelium implant on the peritoneum can be explained by the rupture of the cyst and should not be classified as PMP or as APMT with peritoneal implants. [4]

Atypical proliferative mucinous tumors of endocervical-like type are more frequently bilateral, smaller and are often associated with endometriosis. Macroscopically and microscopically they resemble APTS with a combination of endocervical mucinous and serous epithelium. These neoplasms very rarely present with peritoneal implants or signs of microinvasion (defined as the presence of single or small cluster of cells within the stroma) and have a benign behaviour.

In same cases these tumors can show a severe atypia and epithelial overgrowth still without any sign of stromal invasion, these cases are referred to "non invasive or intraepithelial carcinoma" and have still an excellent prognosis in stage I. [4]

The immunohistochemistry pattern of mucinous BOTs is characterized by the expression of cytokeratin (CK) 7 and 20, but no positivity for estrogen and progesterone receptors and Ca125; in the differential diagnosis with intestinal tract tumors this can be very helpful (the neoplasms of intestinal tract origin express CK20 but not CK 7). [4]

The differential diagnosis should be done with metastatic mucinous carcinomas to the ovary and benign or invasive mucinous neoplasms of ovarian origin.

#### **2.3. Endometrioid borderline tumors**

**Figure 2.** Serous borderline ovarian tumors

80 Ovarian Cancer - A Clinical and Translational Update

**2.2. Mucinous borderline tumors**

They can be divided into two subtypes:

**•** Gastrointestinal type

of papillary projections.

atypia in their nuclei. [4]

with peritoneal implants. [4]

presence of mucinous ascites and mucoid peritoneal implant

neoplasm (mean size of about 20 cm) with a smooth capsule.

**•** Endocervical-like type (müllerian or seromucinous)

Mucinous BOTs are less common than their serous counterparts. They are also called "atypical proliferative mucinous tumor" (APMT) or "mucinous tumor of low malignant potential". They are often associated to pseudomyxoma peritonei (PMP) a condition characterized by the

The first type in 95% of cases is unilateral and appears macroscopically as a multicystic large

The cysts contain inside a mucinous material and their surfaces very rarely show the presence

Histologically, the stromal invasion is absent, the epithelium is stratified, mucinous gastroin‐ testinal-type, with villoglandular of papillary intraglandular growth; the cells show moderate

Their biological behaviour is very benign with a survival rate of nearly in early stages 100%. The tumors in advanced stage have a mortality of 50%, but mostly are associated with pseudomyxoma peritonei and probably all these case can be considered of primary gastroin‐ testinal and not ovarian origin (usually appendix but also pancreas and biliary tract). For this reason is generally accepted that the true primary APMT in advanced stage do not really exist and that those cases with mucin or benign mucinous epithelium implant on the peritoneum can be explained by the rupture of the cyst and should not be classified as PMP or as APMT

Endometrioid tumors of the ovary are usually carcinomas, while borderline forms are very rare; they can arise from endometriosis and can also be associated to endometrial hyperplasia.

Endometrioid BOTs, also called Atypical Proliferative Endometrioid Tumors (APET), account for the 0,2% of ovarian epithelial neoplasms. [4]

Macroscopically they appear as cyst sometimes with solid compounds with hemorrhagic brown fluid inside, in about 60% of cases endometriotic foci are also associated. [4]

**Figure 3.** Endometrioid borderline ovarian tumor

Histologically they have glandular and papillary proliferation with different grade of com‐ plexity, with moderate or mild atypia in their cells, sometimes they show also squamous metaplasia and necrosis (Figure 3). A microinvasive APET can also be described if the glan‐ dular proliferation becomes confluent and the confluent area is less than 5 mm (otherwise it becomes a carcinoma) and this does not seem to be a negative prognostic factors.

In immunohistochemistry these neoplasms are usually positive for CEA, EGFR, Ras and

Borderline Epithelial Tumors of the Ovary http://dx.doi.org/10.5772/54828 83

The prognosis is very good, with only one lethal case of recurrence occurred 50 months after

The only certain diagnosis of BOT can be done by pathologists on the histological examina‐ tions, despite this, better understanding before surgery if an adnexal mass is benign, borderline or malignant is very important to decide if surgery is required and the surgical approach. The diagnosis of BOT can be suggested by the presence of certain symptoms, serum markers and

The range and type of symptoms claimed by BOT patients are similar to invasive cancer

Most commonly [80%) patients with Borderline tumors of the ovary complain of abdominal symptoms like abdominal pain or increased abdominal size, discomfort, tense abdomen; 10-35% of these patients complain of gastrointestinal symptomatology like changes in bowel habits, nausea or constipation; 15% complain of gynaecological symptoms like abnormal vaginal bleeding and dyspareunia (more patients when compared with invasive cancer); 5-26% complain of urinary symptoms especially urinary frequency or urgency; 5-7% present with weight loss and malaise and increased urinary urgency or frequency, very few patients (around3%) complain chest pain or breathing problems. Some studies demonstrated that patients with borderline ovarian tumour are more likely to have no symptoms than patient

Olsen et al compared symptoms of women with benign, borderline and invasive ovarian tumors, and demonstrated that patients with invasive cancer reported a greater number of symptoms (3.1 and 3.6 for Stages I-II and III-IV, respectively) than women with borderline or

Many studies tried to identify a serum marker that could distinguish BOT from invasive and

Ca-125 increases in BOT patients, less than in women with invasive cancer; anyway this marker is not so useful in the diagnosis especially because it can overlap between patients with stage I ovarian carcinoma or benign adnexal masses like endometriomas, abscesses or myomas and

negative for p16, p53 and cyclin d.

**3. Diagnosis**

**3.1. Symptoms**

**3.2. Serum markers**

benign ovarian tumors.

BOT patients. [27, 32, 33]

patients,

image techniques patterns.

the primary surgery reported in literature. [4, 23]

with invasive cancer. [24, 25, 26, 27, 28, 29, 30, 31]

benign tumors. (2.8 and 2.2 respectively; p < 0.0001). [31]

Also in case of APET with intraepithelial carcinoma (referred to the presence of severely atypical cells, but without any sign of invasion) the prognosis remains benign.

The immunohistochemistry pattern of endometrioid BOTs is characterized by the expression of CK7, CK20 and p16 only focally. [4, 21]

#### **2.4. Clear cell borderline tumors**

Clear cell tumors of the ovary are usually carcinomas, while borderline forms are very rare; they are usually associated to endometriosis and sometimes to endometrial disorders.

Clear cell BOTs, also called Atypical Proliferative Clear Cell Tumors (APCCT), represent the 0,2% of ovarian epithelial neoplasm, their incidence is higher in elder people than other BOTs (mean age 60-70 years).

Macroscopically, they usually appear as cyst with a smooth lobulated surface, clear fluid inside and the cut surface has minute cyst in a rubbery stroma (honeycomb appearance). Microscop‐ ically, they are characterized by the presence of tubular glands lined by more layers of hobnail cells, with a more crowded architecture, more epithelial proliferation and more atypia in their cells, when compared to their benign counterparts (clear cell adenofibromas). [4]

As for other BOTs microinvasive APCCT or APCCT with intraepithelial carcinoma can be described, but they are actually very rare, while peritoneal implants have not been described.

The prognosis of these forms of BOTs in early stage is also very benign. [4, 22]

#### **2.5. Borderline brenner (transitional cell) tumors**

Transitional cell tumors of the ovary account for 10% of all the epithelial ovarian neoplasm, they are usually benign while malignant and borderline forms have been described but are very uncommon.

Borderline Brenner (Transitional cell) tumors are also called Atypical Proliferative Brenner (Transitional cell) tumors, the mean age at presentation is about 69 years; they are usually unilateral and in stage I at the moment of diagnosis.

Macroscopically, they are cystic, quite large (mean diameter of about 20 cm) with papillary projections in the inner surface.

Histologically, they are characterized by a transitional urothelial like epithelium, with benign areas and parts with proliferation and atypia. No cases of intraepithelial carcinoma or microinvasion have been described in literature.

In immunohistochemistry these neoplasms are usually positive for CEA, EGFR, Ras and negative for p16, p53 and cyclin d.

The prognosis is very good, with only one lethal case of recurrence occurred 50 months after the primary surgery reported in literature. [4, 23]

#### **3. Diagnosis**

Histologically they have glandular and papillary proliferation with different grade of com‐ plexity, with moderate or mild atypia in their cells, sometimes they show also squamous metaplasia and necrosis (Figure 3). A microinvasive APET can also be described if the glan‐ dular proliferation becomes confluent and the confluent area is less than 5 mm (otherwise it

Also in case of APET with intraepithelial carcinoma (referred to the presence of severely

The immunohistochemistry pattern of endometrioid BOTs is characterized by the expression

Clear cell tumors of the ovary are usually carcinomas, while borderline forms are very rare; they are usually associated to endometriosis and sometimes to endometrial disorders.

Clear cell BOTs, also called Atypical Proliferative Clear Cell Tumors (APCCT), represent the 0,2% of ovarian epithelial neoplasm, their incidence is higher in elder people than other BOTs

Macroscopically, they usually appear as cyst with a smooth lobulated surface, clear fluid inside and the cut surface has minute cyst in a rubbery stroma (honeycomb appearance). Microscop‐ ically, they are characterized by the presence of tubular glands lined by more layers of hobnail cells, with a more crowded architecture, more epithelial proliferation and more atypia in their

As for other BOTs microinvasive APCCT or APCCT with intraepithelial carcinoma can be described, but they are actually very rare, while peritoneal implants have not been described.

Transitional cell tumors of the ovary account for 10% of all the epithelial ovarian neoplasm, they are usually benign while malignant and borderline forms have been described but are

Borderline Brenner (Transitional cell) tumors are also called Atypical Proliferative Brenner (Transitional cell) tumors, the mean age at presentation is about 69 years; they are usually

Macroscopically, they are cystic, quite large (mean diameter of about 20 cm) with papillary

Histologically, they are characterized by a transitional urothelial like epithelium, with benign areas and parts with proliferation and atypia. No cases of intraepithelial carcinoma or

cells, when compared to their benign counterparts (clear cell adenofibromas). [4]

The prognosis of these forms of BOTs in early stage is also very benign. [4, 22]

**2.5. Borderline brenner (transitional cell) tumors**

unilateral and in stage I at the moment of diagnosis.

microinvasion have been described in literature.

becomes a carcinoma) and this does not seem to be a negative prognostic factors.

atypical cells, but without any sign of invasion) the prognosis remains benign.

of CK7, CK20 and p16 only focally. [4, 21]

82 Ovarian Cancer - A Clinical and Translational Update

**2.4. Clear cell borderline tumors**

(mean age 60-70 years).

very uncommon.

projections in the inner surface.

The only certain diagnosis of BOT can be done by pathologists on the histological examina‐ tions, despite this, better understanding before surgery if an adnexal mass is benign, borderline or malignant is very important to decide if surgery is required and the surgical approach. The diagnosis of BOT can be suggested by the presence of certain symptoms, serum markers and image techniques patterns.

#### **3.1. Symptoms**

The range and type of symptoms claimed by BOT patients are similar to invasive cancer patients,

Most commonly [80%) patients with Borderline tumors of the ovary complain of abdominal symptoms like abdominal pain or increased abdominal size, discomfort, tense abdomen; 10-35% of these patients complain of gastrointestinal symptomatology like changes in bowel habits, nausea or constipation; 15% complain of gynaecological symptoms like abnormal vaginal bleeding and dyspareunia (more patients when compared with invasive cancer); 5-26% complain of urinary symptoms especially urinary frequency or urgency; 5-7% present with weight loss and malaise and increased urinary urgency or frequency, very few patients (around3%) complain chest pain or breathing problems. Some studies demonstrated that patients with borderline ovarian tumour are more likely to have no symptoms than patient with invasive cancer. [24, 25, 26, 27, 28, 29, 30, 31]

Olsen et al compared symptoms of women with benign, borderline and invasive ovarian tumors, and demonstrated that patients with invasive cancer reported a greater number of symptoms (3.1 and 3.6 for Stages I-II and III-IV, respectively) than women with borderline or benign tumors. (2.8 and 2.2 respectively; p < 0.0001). [31]

#### **3.2. Serum markers**

Many studies tried to identify a serum marker that could distinguish BOT from invasive and benign ovarian tumors.

Ca-125 increases in BOT patients, less than in women with invasive cancer; anyway this marker is not so useful in the diagnosis especially because it can overlap between patients with stage I ovarian carcinoma or benign adnexal masses like endometriomas, abscesses or myomas and BOT patients. [27, 32, 33]

Ca-125 can instead, be used in the follow up and to primarily assess the severity of the disease because several studies demonstrated that it increases more in advanced stage BOT than early ones. [34, 35, 36, 37, 38]

Ca-19.9 increases in 18,8 – 48,8% of patients, probably more in serous hystotype, [35, 39], while Carcinoembryonic antigen (CEA) levels increases in 17% of patients and more in mucinous tumor. [35, 39, 40]

Ca 72-4 increases in BOT with no differences within the hystotypes, anyway the levels of this serum marker are similar in patients with ovarian cancer. [35, 41]

#### **3.3. Ultrasound**

Transvaginal ultrasound is well known to be an effective primary screening imaging technique in patients with adnexal masses to distinguish benign from malignant conditions.

Up to 63% of patients with BOT present on the ultrasound a cyst with papillae inside, but without solid patterns, septa or any other sign of complexity. [42, 43]

BOTs appears on ultrasound images usually as:

**•** unilocular cyst with solid papillary projections (defined as any projections with a height greater than or equal to 3 mm) arising from the inner wall and with a positive ovarian crescent sign (Figure 4, 5)

**Figure 5.** Papillary projections in BOTs

**Figure 6.** Honeycomb nodule in BOTs

11% of these tumors can appear as simple anechoic cysts without any papillae, and up to 30% as cysts with septa. (Figure 7) [42, 43, 45]. For these reasons, neither the presence of papillae nor septa can be considered as sensitive sonographic markers of borderline tumors, in fact it

Borderline Epithelial Tumors of the Ovary http://dx.doi.org/10.5772/54828 85

has been shown that also benign tumors can contain papillae or septa. [42, 46]

**•** cyst with a "honeycomb nodule", defined as a multilocular nodule mostly with a solid pattern with cystic areas arising from the inner cyst wall. (Figure 6) [42, 43, 44, 45]

**Figure 4.** Unilocular cyst with solid papillary projections in BOTs

**Figure 5.** Papillary projections in BOTs

Ca-125 can instead, be used in the follow up and to primarily assess the severity of the disease because several studies demonstrated that it increases more in advanced stage BOT than early

Ca-19.9 increases in 18,8 – 48,8% of patients, probably more in serous hystotype, [35, 39], while Carcinoembryonic antigen (CEA) levels increases in 17% of patients and more in mucinous

Ca 72-4 increases in BOT with no differences within the hystotypes, anyway the levels of this

Transvaginal ultrasound is well known to be an effective primary screening imaging technique

Up to 63% of patients with BOT present on the ultrasound a cyst with papillae inside, but

**•** unilocular cyst with solid papillary projections (defined as any projections with a height greater than or equal to 3 mm) arising from the inner wall and with a positive ovarian

**•** cyst with a "honeycomb nodule", defined as a multilocular nodule mostly with a solid pattern with cystic areas arising from the inner cyst wall. (Figure 6) [42, 43, 44, 45]

in patients with adnexal masses to distinguish benign from malignant conditions.

serum marker are similar in patients with ovarian cancer. [35, 41]

without solid patterns, septa or any other sign of complexity. [42, 43]

BOTs appears on ultrasound images usually as:

**Figure 4.** Unilocular cyst with solid papillary projections in BOTs

crescent sign (Figure 4, 5)

ones. [34, 35, 36, 37, 38]

84 Ovarian Cancer - A Clinical and Translational Update

tumor. [35, 39, 40]

**3.3. Ultrasound**

**Figure 6.** Honeycomb nodule in BOTs

11% of these tumors can appear as simple anechoic cysts without any papillae, and up to 30% as cysts with septa. (Figure 7) [42, 43, 45]. For these reasons, neither the presence of papillae nor septa can be considered as sensitive sonographic markers of borderline tumors, in fact it has been shown that also benign tumors can contain papillae or septa. [42, 46]

present mostly in benign masses, while intramural or intrapapillae flow is present mostly in

Borderline Epithelial Tumors of the Ovary http://dx.doi.org/10.5772/54828 87

The study of the distribution of the flow or the resistance and pulsatility indexes cannot be considered effective neither in the differential diagnosis between the different histotypes. [53]

The use of contrast medium injected intravenously has been also suggested as a technique able to discriminate benign from borderline and malignant condition, a multicentre study including 10 cases of BOT and totally 89 patients with ovarian masses concluded that the use of second generation contrast agent like Sono Vue, Bracco, Netherland can be useful in the differential diagnosis between benign and malignant condition but not between borderline and benign

**3.4. Computerized tomography, magnetic resonance and positron emission tomography**

Computerized tomography seems not to discriminate BOT from malignant ovarian tumors, it can recognize the complex architecture of BOT, but the tissue contrast is limited so that it is not so clear the contrast between the solid and cystic components of these tumors; the role of CT scan is then mostly limited to detect the presence of metastases and to estimate the FIGO

Magnetic Resonance is the best image technique to characterize borderline ovarian tumors. Bent et al described the appearance of BOT on MR images, and identified four morphological categories: unilocular cysts (19%), minimally septate cysts with papillae (19%), markedly septate lesions with plaque-like excrescences (45%) and predominantly solid with exophytic

borderline or malignant conditions. (Figure 8, 9) [43]

**Figure 8.** Flow distribution in BOTs

ovarian masses. [54]

stage. [55, 56]

**Figure 7.** Cyst with septa in BOTs

The ovarian crescent sign is defined as the presence of healthy ovarian tissue adjacent to the cyst wall seen on the ultrasound images as an hypoechogenic area with or without ovarian follicles that cannot be separated from the mass when applying pressure with the transvaginal probe; it has been shown that the presence of this sign can be used to exclude the diagnosis of invasive ovarian cancer. [47]

Yazbek et al demonstrated that the presence of papillae and crescent signs are suggestive of a serous or endocervical hystotype while the presence of thick echogenic fluid and honeycomb nodules are suggestive of gastrointestinal hystotype, moreover Exacoustos et al found that the serous types seem to be smaller than mucinous. [43, 45]

Yazbek et al conclude that the diagnosis of BOT with ultrasound can be achieved in 68.6%, more in serous or endocervical (75%) than in gastrointestinal hystotype (60%). [45]

The role of Doppler ultrasound is still not clear in the diagnosis of BOTs, some authors found a difference in the resistance index and pulsatility index between BOT, benign tumors and invasive cancers, these indexes seem to gradually decrease with the grade of malignancy of the condition. [48, 49, 50]

Otherwise, Tekay et al found no statistically significative differences in the resistance and pulsatility indexes values between invasive, borderline and benign ovarian tumors. [51]

The vessel distribution within the tumor tissue has also been studied deeply but there are not still any clear conclusions, some authors demonstrated that BOT show similar vascular patterns to benign or malignant conditions. [42, 43, 49, 52]

Exacoustos et al found that the flow was present respectively in benign, borderline and malignant tumors in 80, 97 and 100% of cases and that usually a peripheral vascularization is present mostly in benign masses, while intramural or intrapapillae flow is present mostly in borderline or malignant conditions. (Figure 8, 9) [43]

The study of the distribution of the flow or the resistance and pulsatility indexes cannot be considered effective neither in the differential diagnosis between the different histotypes. [53]

**Figure 8.** Flow distribution in BOTs

**Figure 7.** Cyst with septa in BOTs

86 Ovarian Cancer - A Clinical and Translational Update

invasive ovarian cancer. [47]

the condition. [48, 49, 50]

serous types seem to be smaller than mucinous. [43, 45]

patterns to benign or malignant conditions. [42, 43, 49, 52]

The ovarian crescent sign is defined as the presence of healthy ovarian tissue adjacent to the cyst wall seen on the ultrasound images as an hypoechogenic area with or without ovarian follicles that cannot be separated from the mass when applying pressure with the transvaginal probe; it has been shown that the presence of this sign can be used to exclude the diagnosis of

Yazbek et al demonstrated that the presence of papillae and crescent signs are suggestive of a serous or endocervical hystotype while the presence of thick echogenic fluid and honeycomb nodules are suggestive of gastrointestinal hystotype, moreover Exacoustos et al found that the

Yazbek et al conclude that the diagnosis of BOT with ultrasound can be achieved in 68.6%,

The role of Doppler ultrasound is still not clear in the diagnosis of BOTs, some authors found a difference in the resistance index and pulsatility index between BOT, benign tumors and invasive cancers, these indexes seem to gradually decrease with the grade of malignancy of

Otherwise, Tekay et al found no statistically significative differences in the resistance and pulsatility indexes values between invasive, borderline and benign ovarian tumors. [51]

The vessel distribution within the tumor tissue has also been studied deeply but there are not still any clear conclusions, some authors demonstrated that BOT show similar vascular

Exacoustos et al found that the flow was present respectively in benign, borderline and malignant tumors in 80, 97 and 100% of cases and that usually a peripheral vascularization is

more in serous or endocervical (75%) than in gastrointestinal hystotype (60%). [45]

The use of contrast medium injected intravenously has been also suggested as a technique able to discriminate benign from borderline and malignant condition, a multicentre study including 10 cases of BOT and totally 89 patients with ovarian masses concluded that the use of second generation contrast agent like Sono Vue, Bracco, Netherland can be useful in the differential diagnosis between benign and malignant condition but not between borderline and benign ovarian masses. [54]

#### **3.4. Computerized tomography, magnetic resonance and positron emission tomography**

Computerized tomography seems not to discriminate BOT from malignant ovarian tumors, it can recognize the complex architecture of BOT, but the tissue contrast is limited so that it is not so clear the contrast between the solid and cystic components of these tumors; the role of CT scan is then mostly limited to detect the presence of metastases and to estimate the FIGO stage. [55, 56]

Magnetic Resonance is the best image technique to characterize borderline ovarian tumors. Bent et al described the appearance of BOT on MR images, and identified four morphological categories: unilocular cysts (19%), minimally septate cysts with papillae (19%), markedly septate lesions with plaque-like excrescences (45%) and predominantly solid with exophytic papillary projections (16%); they also concluded that MRI can be helpful in the differential diagnosis of BOT and in surgical planning. [57]

be performed in case of fertility sparing surgery. Several reports suggest that the overall disease-specific survival rates between the radical e the fertility sparing surgical approaches are not different. [70, 72] Thus, it appears that young women who desire future fertility can be safety treated with fertility-sparing surgery without compromising their overall survival.

Borderline Epithelial Tumors of the Ovary http://dx.doi.org/10.5772/54828 89

Barnhill DA et al suggested that a simple cystectomy should be performed only in selected cases if the tumor is in stage I, can be removed completely and is loosely attached. [73]

The contralateral ovary must be carefully macroscopically inspected, but performing a biopsy is not recommended in order to avoid the occurrence of adhesions that can affect the future fertility capacity of the patient. Some surgeon suggest to women with BOTs who had under‐ gone fertility sparing surgery to complete the radical surgery after the completion of child‐

The role of adjuvant therapy is still not clear. At this time, there is no proven benefit from adju‐ vant therapy, even in advanced-stage disease and with the presence of invasive implants. [74] Generally, in absence of invasive implants, watchful expectancy should be considered, while adjuvant chemotherapy (usually platin based chemotherapy) should be offered to patients with invasive implants, with the persistence of residual tumor after surgery and in clinically

The follow up of these patients must be performed for more than 10 years after the primary treatment since long term recurrence (even after 20 years) have been observed especially in women who underwent a conservative surgical approach. The follow up should include pelvic

Fifty-five women with borderline ovarian tumors were identified a tour institution from 1991 to 2011, median age at diagnosis was 40 years (range 13-79). The most common symptoms complained by patients at the moment of diagnosis were abdominal-or pelvic pain and discomfort. The tumor diameter ranged between 0.5 and 10 cm and 5.4% of patients presented

Only in the 47% patients, [26] tumor markers were evaluated before primary surgery, specif‐ ically CA125 was higher in 13 (23.6%), CA19.9 in 2 (3.6%) and 4 patients (7.3%) presented with

Our expert pathologist in Gynecological oncology pathology found 33 serous, 18 mucinous, 1

All women underwent surgery as primary treatment, 72,8% with laparotomic approach, whereas 13 women (23.6%) underwent a laparoscopic one; in particular 20 patients (36.4%) had a total abdominal hysterectomy with bilateral salpingo-oophorectomy, 2 patients (3.6%)

and gynecological examinations, ultrasound and measurement of serum markers.

BOTs in advanced stage should be treated with debulking surgery.

bearing.

progressive disease.

**5. Our experience: 55 cases**

ascites at the time of diagnosis.

both of these markers increased.

endometrioid and 3 mixed borderline ovarian tumors.

In T1- and T2-weighted images BOTs solid tissues are usually intermediate in signal intensity and they demonstrate also enhancement after the administration of gadolinium-based contrast media. [57, 58, 59, 60] The enhancement pattern seems to be useful in the differential diagnosis between benign, borderline and invasive ovarian tumors. [61]

In a series of 168 ovarian masses (23 BOT) Bazot et al estimated that the sensitivity and specificity of MRI for the diagnosis of BOT are 45.5% and 96.1%, respectively. [62]

Positron emission tomography can increase the accuracy of other imaging techniques in the diagnosis of BOT.

Malignant cells use glucose to survive, for this reason invasive cancer are characterized on PET images by a higher uptake of 18F-fluorodeoxyglucose than both BOT and benign tumors that do not have a high glycolitic rate. [64, 65, 66, 67]

Nam et al investigated the role of combined 18F-fluorodeoxyglucose positron emission tomography/computerized tomography (FDG-PET/CT) and found that it can be more accurate than ultrasound, CT and MRI in the differential diagnosis between BOT, benign and malignant ovarian cancer. [68]

#### **4. Treatment and follow up**

Surgery represents the gold standard treatment and a complete surgical staging is mandatory and very important. Lin et al found out that only in 12% of cases the primary surgical stadiation is actually right. Moreover, BOTs are also difficult to diagnosed in frozen section, many apparent BOTs on frozen section are found to be frankly malignant in permanent sections (the correct diagnosis is achieved in 58-86%) of patients and it depends especially on the experience of the pathologists.

The surgical approach in the management of BOT is similar to the one used in the malignant forms and includes: total abdominal hysterectomy, bilateral salpinogo-oophorectomy, omentectomy, peritoneal washings, and multiple biopsies, including pelvic and pariaortic lymph nodes sampling for the stadiation.

In mucinous BOTs is strongly recommended to perform appendectomy and to carefully analyze the entire intestinal tract to exclude a gastrointestinal tumor.

Because of the excellent prognosis and the young age of these women the treatment is becoming always more conservative and fertility sparing surgery can be considered [69, 70], it consists in ovariectomy or simple cystectomy.

Because 15% of patients who undergone unilateral salpingo oophorectomy develop a primary tumor in the preserved ovary [13, 71] the conservative approach should be considered carefully. In all cases a carefully inspection of the capsule to find any sign of rupture should be performed in case of fertility sparing surgery. Several reports suggest that the overall disease-specific survival rates between the radical e the fertility sparing surgical approaches are not different. [70, 72] Thus, it appears that young women who desire future fertility can be safety treated with fertility-sparing surgery without compromising their overall survival.

Barnhill DA et al suggested that a simple cystectomy should be performed only in selected cases if the tumor is in stage I, can be removed completely and is loosely attached. [73]

The contralateral ovary must be carefully macroscopically inspected, but performing a biopsy is not recommended in order to avoid the occurrence of adhesions that can affect the future fertility capacity of the patient. Some surgeon suggest to women with BOTs who had under‐ gone fertility sparing surgery to complete the radical surgery after the completion of child‐ bearing.

BOTs in advanced stage should be treated with debulking surgery.

The role of adjuvant therapy is still not clear. At this time, there is no proven benefit from adju‐ vant therapy, even in advanced-stage disease and with the presence of invasive implants. [74]

Generally, in absence of invasive implants, watchful expectancy should be considered, while adjuvant chemotherapy (usually platin based chemotherapy) should be offered to patients with invasive implants, with the persistence of residual tumor after surgery and in clinically progressive disease.

The follow up of these patients must be performed for more than 10 years after the primary treatment since long term recurrence (even after 20 years) have been observed especially in women who underwent a conservative surgical approach. The follow up should include pelvic and gynecological examinations, ultrasound and measurement of serum markers.

#### **5. Our experience: 55 cases**

papillary projections (16%); they also concluded that MRI can be helpful in the differential

In T1- and T2-weighted images BOTs solid tissues are usually intermediate in signal intensity and they demonstrate also enhancement after the administration of gadolinium-based contrast media. [57, 58, 59, 60] The enhancement pattern seems to be useful in the differential diagnosis

In a series of 168 ovarian masses (23 BOT) Bazot et al estimated that the sensitivity and

Positron emission tomography can increase the accuracy of other imaging techniques in the

Malignant cells use glucose to survive, for this reason invasive cancer are characterized on PET images by a higher uptake of 18F-fluorodeoxyglucose than both BOT and benign tumors that

Nam et al investigated the role of combined 18F-fluorodeoxyglucose positron emission tomography/computerized tomography (FDG-PET/CT) and found that it can be more accurate than ultrasound, CT and MRI in the differential diagnosis between BOT, benign and malignant

Surgery represents the gold standard treatment and a complete surgical staging is mandatory and very important. Lin et al found out that only in 12% of cases the primary surgical stadiation is actually right. Moreover, BOTs are also difficult to diagnosed in frozen section, many apparent BOTs on frozen section are found to be frankly malignant in permanent sections (the correct diagnosis is achieved in 58-86%) of patients and it depends especially on the experience

The surgical approach in the management of BOT is similar to the one used in the malignant forms and includes: total abdominal hysterectomy, bilateral salpinogo-oophorectomy, omentectomy, peritoneal washings, and multiple biopsies, including pelvic and pariaortic

In mucinous BOTs is strongly recommended to perform appendectomy and to carefully

Because of the excellent prognosis and the young age of these women the treatment is becoming always more conservative and fertility sparing surgery can be considered [69, 70],

Because 15% of patients who undergone unilateral salpingo oophorectomy develop a primary tumor in the preserved ovary [13, 71] the conservative approach should be considered carefully. In all cases a carefully inspection of the capsule to find any sign of rupture should

analyze the entire intestinal tract to exclude a gastrointestinal tumor.

specificity of MRI for the diagnosis of BOT are 45.5% and 96.1%, respectively. [62]

diagnosis of BOT and in surgical planning. [57]

88 Ovarian Cancer - A Clinical and Translational Update

do not have a high glycolitic rate. [64, 65, 66, 67]

diagnosis of BOT.

ovarian cancer. [68]

of the pathologists.

**4. Treatment and follow up**

lymph nodes sampling for the stadiation.

it consists in ovariectomy or simple cystectomy.

between benign, borderline and invasive ovarian tumors. [61]

Fifty-five women with borderline ovarian tumors were identified a tour institution from 1991 to 2011, median age at diagnosis was 40 years (range 13-79). The most common symptoms complained by patients at the moment of diagnosis were abdominal-or pelvic pain and discomfort. The tumor diameter ranged between 0.5 and 10 cm and 5.4% of patients presented ascites at the time of diagnosis.

Only in the 47% patients, [26] tumor markers were evaluated before primary surgery, specif‐ ically CA125 was higher in 13 (23.6%), CA19.9 in 2 (3.6%) and 4 patients (7.3%) presented with both of these markers increased.

Our expert pathologist in Gynecological oncology pathology found 33 serous, 18 mucinous, 1 endometrioid and 3 mixed borderline ovarian tumors.

All women underwent surgery as primary treatment, 72,8% with laparotomic approach, whereas 13 women (23.6%) underwent a laparoscopic one; in particular 20 patients (36.4%) had a total abdominal hysterectomy with bilateral salpingo-oophorectomy, 2 patients (3.6%) had a bilateral salpingo-oophorectomy with uterus sparing and the remaining 33 women [60%) performed a procedure strictly interested the ovary. Omentectomy was performed in 32 patients [58%) whereas para aortic lymph node dissection in only 1 patient and appendectomy in 17 patients (31%). Peritoneal biopsies were performed in 27 women (49%), peritoneal cytology in 29 cases (53%) and positive in only 2 (7%).

In any case, a long-term follow-up is highly recommended for these tumors because recur‐

, Doriana Scardigno,

Borderline Epithelial Tumors of the Ovary http://dx.doi.org/10.5772/54828 91

rences can occur several years after primary treatment.

Gennaro Cormio, Vera Loizzi, Maddalena Falagario\*

\*Address all correspondence to: mad.falagario@gmail.com

Department of Gynaecology, Obstetric and Neonatology, University of Bari, Italy

[1] Taylor, H. Malignant and semi-malignant tumors of the ovary. Surg Gynecol Obstet,

[2] FIGOInternational Federation of Gynecology and Obstetrics. Classification and staging of malignant tumours in the female pelvis. Acta Obstet Gynecol Scand (1971). , 50, 1-7.

[3] Tavassoli, F. A. Devilee P (eds). World Health Organization Classification of Tumours. Pathology and Genetics. Tumours of the Breast and Female Genital Organs. IARC

[4] Russell, P. Surface epithelial-stromal tumors of the ovary. In: Kurman RJ (ed). Blaus‐ tein's Pathology of the Female Genital Tract, 4th edn. Springer-Verlag: New York,

[5] Crispens, M. A. Borderline ovarian tumours: a review of the recent literature. Curr Opin

[6] Skirnisdottir, I, Garmo, H, Wilander, E, & Holmberg, L. Borderline ovarian tumors in Sweden 1960-2005: trends in incidence and age at diagnosis compared to ovarian

[7] Bostwick, D. G, Tazelaar, H. D, Ballon, S. C, Hendrickson, M. R, & Kempson, R. L. Ovarian epithelial tumors of borderline malignancy. A clinical and pathologic study of

[8] Benedet, J. L, Bender, H, & Jones, H. rd, Ngan, H. Y., and Pecorelli, S. FIGO staging classifications and clinical practice guidelines in the management of gynecologic cancers. FIGO Committee on Gynecologic Oncology. Int J Gynaecol Obstet, (2000). , 70,

Donatella Latorre and Luigi E. Selvaggi

(1929). , 48, 204-230.

Press: Lyon, (2003).

(1994). , 705-782.

209-262.

Obstet Gynecol, (2003). , 15, 39-43.

cancer. Int J Cancer, (2008). , 123, 1897-1901.

109 cases. Cancer, (1986). , 58, 2052-2065.

**Author details**

**References**

Forty-seven patients were in FIGO stage I (85.4%), most of these in IA stage (41 patients, 74.5%), 4 patients were in stage II (7.3%) and the last 4 patients in stage III (7.3%).

Fifty-four patients (98.2%) had no residual tumor after surgical procedure, while 1 patient (1.8%) had macroscopic residual tumor ≤2 cm in the ovary and peritoneal carcinomatosis.

After surgery only 2 patients (3.7%) were treated with adjuvant platinum-based combination chemotherapy for their stage IIC and IIIC; both patients achieved a complete response after treatment.

The other patients did not received any other treatment.

The median disease free survival and the 5-year survival rate of our patient population were 42 months (range 16-84) and 97%, respectively.

The statistical analysis performed with Kaplan Meier method and log rank test showed that the survival in patients who underwent fertility-sparing surgery did not differ from those who had a complete surgical staging (p=0.08). No significative differences were observed when comparing the different stages (Stage I-II vs Stage III; p=0.7), histological type (serous versus mucinous, endometrioid and mixed tumor; p=0.15), tumor size (> 10 cm vs < 10 cm; p=0.39), surgical approach (laparotomy vs laparoscopy; p=0.56), elevation of CA125 at diagnosis (positive vs negative marker; p=0.55).

Six patients developed a recurrence of the disease. All of them underwent a secondary laparotomy, four with a conservative approach and two with a complete surgical staging because of the presence of invasive implants. These two patients received then also chemo‐ therapy. All the six women were alive with no evidence of disease with a median survival of 39 months.

We were able to obtain the fertility status of 16 patients who underwent a fertility-sparing surgery. Four of these women became pregnant and the rest of them had not a desire of childbearing at the time of their last follow up. One of these pregnancies was obtained by in vitro fertilization techniques, while the rest of them were spontaneous.

#### **6. Conclusions**

In conclusion, BOTs have an excellent prognosis of nearly 100% of survival rate.

Conservative fertility sparing surgery should be considered for women in the reproductive age group who desire preservation of fertility.

In any case, a long-term follow-up is highly recommended for these tumors because recur‐ rences can occur several years after primary treatment.

#### **Author details**

had a bilateral salpingo-oophorectomy with uterus sparing and the remaining 33 women [60%) performed a procedure strictly interested the ovary. Omentectomy was performed in 32 patients [58%) whereas para aortic lymph node dissection in only 1 patient and appendectomy in 17 patients (31%). Peritoneal biopsies were performed in 27 women (49%), peritoneal

Forty-seven patients were in FIGO stage I (85.4%), most of these in IA stage (41 patients, 74.5%),

Fifty-four patients (98.2%) had no residual tumor after surgical procedure, while 1 patient (1.8%) had macroscopic residual tumor ≤2 cm in the ovary and peritoneal carcinomatosis.

After surgery only 2 patients (3.7%) were treated with adjuvant platinum-based combination chemotherapy for their stage IIC and IIIC; both patients achieved a complete response after

The median disease free survival and the 5-year survival rate of our patient population were

The statistical analysis performed with Kaplan Meier method and log rank test showed that the survival in patients who underwent fertility-sparing surgery did not differ from those who had a complete surgical staging (p=0.08). No significative differences were observed when comparing the different stages (Stage I-II vs Stage III; p=0.7), histological type (serous versus mucinous, endometrioid and mixed tumor; p=0.15), tumor size (> 10 cm vs < 10 cm; p=0.39), surgical approach (laparotomy vs laparoscopy; p=0.56), elevation of CA125 at diagnosis

Six patients developed a recurrence of the disease. All of them underwent a secondary laparotomy, four with a conservative approach and two with a complete surgical staging because of the presence of invasive implants. These two patients received then also chemo‐ therapy. All the six women were alive with no evidence of disease with a median survival of

We were able to obtain the fertility status of 16 patients who underwent a fertility-sparing surgery. Four of these women became pregnant and the rest of them had not a desire of childbearing at the time of their last follow up. One of these pregnancies was obtained by in

Conservative fertility sparing surgery should be considered for women in the reproductive

vitro fertilization techniques, while the rest of them were spontaneous.

In conclusion, BOTs have an excellent prognosis of nearly 100% of survival rate.

4 patients were in stage II (7.3%) and the last 4 patients in stage III (7.3%).

cytology in 29 cases (53%) and positive in only 2 (7%).

90 Ovarian Cancer - A Clinical and Translational Update

The other patients did not received any other treatment.

42 months (range 16-84) and 97%, respectively.

(positive vs negative marker; p=0.55).

age group who desire preservation of fertility.

treatment.

39 months.

**6. Conclusions**

Gennaro Cormio, Vera Loizzi, Maddalena Falagario\* , Doriana Scardigno, Donatella Latorre and Luigi E. Selvaggi

\*Address all correspondence to: mad.falagario@gmail.com

Department of Gynaecology, Obstetric and Neonatology, University of Bari, Italy

#### **References**


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**Chapter 5**

**Implication of Clear Cell and Mucinous Histology**

The incidence of ovarian cancer in 2008 was projected to be 225,500 new cases and 140,200 deaths worldwide, representing 3.7% of all female cancers and 4.2% of all cancer deaths in women [1]. Ovarian cancer, one of the major causes of death from cancer in women, is commonly diagnosed at an advanced stage. Cytoreductive surgery followed by chemotherapy combining platinum and taxane is currently the standard treatment for ovarian cancer [2]. Ovarian cancer is one of the most sensitive solid tumors, with objective responses ranging from 60 to 80% even in patients with advanced stage. However, most patients ultimately recur and

Resistance to chemotherapy presents a major obstacle in attempting to improve the prognosis of patients with ovarian cancer. Accordingly, it is important to elucidate the mechanisms of chemoresistance to manage ovarian cancer. Recently, the biological characteristics of ovarian cancer have been clarified. It has long been known that ovarian cancers of serous histology appear to be more sensitive to chemotherapy than other histological subtypes. Patients with clear cell carcinoma or mucinous adenocarcinoma of the ovary showed a significantly worse prognosis in a retrospective review of several Gynecologic Oncology Group (GOG) trials [3]. Therefore, it is important to determine optimal regimens based on histological subtype. In this chapter, clear cell carcinoma and mucinous adenocarcinoma of the ovary are discussed.

Clear cell carcinoma (CCC)hasunique clinical andbiologicalfeatures [4].InNorthAmerica and Europe, CCC is the third most common histological subtype of epithelial ovarian cancer (EOC), with an estimated prevalence of 1-12% [5, 6]. For unknown reason, CCC comprises more than

and reproduction in any medium, provided the original work is properly cited.

© 2013 Naniwa et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Jun Naniwa, Hiroaki Itamochi and Junzo Kigawa

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54931

develop resistance to platinum and taxane.

**2. Clear cell carcinoma**

**1. Introduction**


### **Implication of Clear Cell and Mucinous Histology**

Jun Naniwa, Hiroaki Itamochi and Junzo Kigawa

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54931

#### **1. Introduction**

[67] Yamamoto, Y, Oguri, H, Yamada, R, et al. (2008). Preoperative evaluation of pelvic masses with combined 18F-fluorodeoxyglucose positron emission tomography and

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[73] Barnhill, D. A, Kurman, R. J, Brady, M. F, et al. Preliminary analysis of the behavior of stage I ovarian serous tumors of low malignant potential: a Gynecologic Oncology

[74] Trope, C, Kaern, J, Vergote, I. B, Kristensen, G, & Abeler, V. Are borderline tumors of the ovary overtreated both surgically and systemically? A review of four prospective randomized trials including 253 patients with borderline tumors. Gynecol Oncol,

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96 Ovarian Cancer - A Clinical and Translational Update

tumors. Obstet Gynecol, (2000). , 95, 541-547.

Group study. J Clin Oncol (1995). , 13, 2752-2756.

The incidence of ovarian cancer in 2008 was projected to be 225,500 new cases and 140,200 deaths worldwide, representing 3.7% of all female cancers and 4.2% of all cancer deaths in women [1]. Ovarian cancer, one of the major causes of death from cancer in women, is commonly diagnosed at an advanced stage. Cytoreductive surgery followed by chemotherapy combining platinum and taxane is currently the standard treatment for ovarian cancer [2]. Ovarian cancer is one of the most sensitive solid tumors, with objective responses ranging from 60 to 80% even in patients with advanced stage. However, most patients ultimately recur and develop resistance to platinum and taxane.

Resistance to chemotherapy presents a major obstacle in attempting to improve the prognosis of patients with ovarian cancer. Accordingly, it is important to elucidate the mechanisms of chemoresistance to manage ovarian cancer. Recently, the biological characteristics of ovarian cancer have been clarified. It has long been known that ovarian cancers of serous histology appear to be more sensitive to chemotherapy than other histological subtypes. Patients with clear cell carcinoma or mucinous adenocarcinoma of the ovary showed a significantly worse prognosis in a retrospective review of several Gynecologic Oncology Group (GOG) trials [3]. Therefore, it is important to determine optimal regimens based on histological subtype. In this chapter, clear cell carcinoma and mucinous adenocarcinoma of the ovary are discussed.

#### **2. Clear cell carcinoma**

Clear cell carcinoma (CCC)hasunique clinical andbiologicalfeatures [4].InNorthAmerica and Europe, CCC is the third most common histological subtype of epithelial ovarian cancer (EOC), with an estimated prevalence of 1-12% [5, 6]. For unknown reason, CCC comprises more than

© 2013 Naniwa et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

20%of suchcancers inJapan[7-9].Interestingly,amongAsianwomenlivingintheUnitedStates, CCC was diagnosed twice as frequently (11.1%) compared to Caucasians (4.8%) [10].

The median age did not differ between patients with CCC and those with SAC. The percentage of patients at stage I was significantly higher in CCC than SAC (16.6%), while significantly fewer patients were at stage III had CCC than SAC (61.7%). By contrast, the incidence of stage III was significantly lower in CCC than in SAC. Recurrence in patients with CCC occurred in 29% of stage I patients, 30% of stage II, 62% of stage III, and 73% at stage IV. Although none of the patients with stage Ia CCC relapsed, 14 of 38 patients (37%) with stage Ic did relapse.

Implication of Clear Cell and Mucinous Histology

http://dx.doi.org/10.5772/54931

99

In stage III disease the median survival time was significantly shorter for patients with CCC than those with SAC. The survival rate for patients at stage III was significantly lower in the CCC group than in the SAC group. Although estimated survival rates at 3 and 5 years in patients with no gross tumor did not differ significantly between CCC and SAC, survival rates in both patients with <2 cm and >2 cm residual disease were significantly lower in CCC than in SAC. Jenison, et al. [23] showed that the survival rates for CCC were consistently lower in each of the FIGO stages compared with SAC, although there was no statistical significance. In their study, median survival time for stage I patients with CCC was significantly shorter than that for those with SAC. Similarly, the survival rate for patients with stage Ic CCC was lower compared with patients with stage Ic SAC. Additionally, the median survival time for stage I patients with CCC was worse than those with SAC (31.8 months vs 42.3 months) and the time to recurrence in patients with stage I/II CCC was definitely shorter (12.2 months) [9]. Twentyseven patients with stage III/IV CCC had measurable disease after initial surgery. The overall clinical response rate for SAC was 72.5%. In contrast, only three (11.1%) of 27 patients re‐ sponded to platinum-based chemotherapy in CCC. Patients with CCC showed a very low rate of response and a high incidence of progressive disease. Another authors demonstrated that platinum-based chemotherapy did not appear to improve the survival of patients with CCC, compared with survival after non-platinum-based chemotherapy [24]. Additionally, CCC patients with residual tumor showed a high recurrence rate (Fig 1). CCC has a more aggressive course and a more malignant behavior than SAC. Therefore, new treatment strategies for CCC,

including alternative regimens of chemotherapy, should be established.

**4. Mechanisms of platinum resistance in CCC of the ovary**

its downstream signaling pathways [25-29].

There is general acceptance of CCC that is insensitive to conventional platinum-based chemotherapy lead to a poor prognosis. Resistance to cisplatin (CDDP) is an important factor in the poor prognosis of patients with CCC. Several mechanisms involved in drug resistance have been proposed as explanations, including decreased drug accumulation, increased drug detoxification, increased DNA repair activity, and activation of receptor tyrosine kinases and

Adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporters, such as ABCB1 (also known as P glycoprotein), ABCC1 (multidrug resistance associated protein-1) and ABCC3, are known to lower intracellular drug concentrations and are important multidrug resistance factors [30]. An immunohistochemical study of ABCB1 and ABCC1 in CCC and SAC tumors revealed that their expression of these transporters did not differ between CCC and SAC [31].

Several studies have analyzed the risk factors for ovarian cancer by histologic subtype. CCC was associated (odds ratio: 2.2-2.3) with an increased body mass index (BMI 30) [11, 12]. However, in the NIH-AARP Diet and Health Study BMI was correlated only with endome‐ trioid histology [13].

It has long been recognized that CCC often is associated with endometriosis (22-70%), where‐ as hobnail cells bear a very strong morphological resemblance to endometrial Aria-Stella cells [14, 15]. Several studies have reported that endometriosis frequently shows a sequential change to EOC, including CCC. Therefore, atypical endometriosis is considered to be a precancerous change. Ovarian cancers associated with endometriosis tend to occur in younger women, and present5-6yearsearlier,onaverage,thanhigh-gradeserous carcinoma(HGSC)[16].InNational Cancer Institute Surveillance, Epidemiology and End Results (SEER) data, women with CCC wereyoungerthanpatientswithserousadenocarcinoma(SAC)(55vs.64years;medianage)[10].

An increased incidence of vascular thromboembolic complications is seen in patients with CCC [17, 18]. Up to 40% of patients with CCC may develop thromboembolic disease and this rate is double that in matched non-CCC controls with ovarian cancers [19].

#### **3. Clinicopathological features**

Ovarian CCC usually presents as a large pelvic mass [20, 21]. The size of masses range from 3 to 20 cm, with most tumors detected preoperatively either by clinical examination or imaging. Recent reports involving large institutional cohorts compared early-stage (I/II) to advancedstage ovarian cancers (III/IV) and showed that 57-81% of CCC were diagnosed at an early stage [9, 22]. In SEER data, 56% of CCC were stage I, compared to 19% for SAC [10]. Combining the low overall incidence of CCC and their early stage propensity, CCC may make up only 1-5% of advanced stage patients in chemotherapy trials, largely due to their overall low incidence and tendency for early stage distribution at the time for initial diagnosis [6].

Sugiyama, et al. [9] retrospectively reviewed 101 patients with CCC in Japan who underwent complete surgical staging to determine clinicopathological features of CCC. Histologic evaluation was performed under central pathological review. Tumors were diagnosed as CCC if the following appeared in 90% or more of all specimens: a small to large sheet of polyhedral clear cells with delicate fibrovascular septa, tubules and papillae, clear or hobnail, or eosino‐ philic cells of organoid appearance, or clear cells with coalescent vacuoles containing "targe‐ toid" eosinophilic PAS-positive globules. Of the 662 patients with EOC, 101 (15.3%) had CCC and 235 (35.5%) SAC. All patients underwent complete surgical staging, including intraperi‐ toneal cytology, bilateral salpingo-oophorectomy, hysterectomy, omentectomy, pelvic-/ paraaortic lymphadenectomy, and aggressive cytoreductive surgery for advanced cases. Ninety-seven (96%) of 101 patients with CCC and 229 (97%) of 235 with SAC underwent platinum-based chemotherapy after initial surgery.

The median age did not differ between patients with CCC and those with SAC. The percentage of patients at stage I was significantly higher in CCC than SAC (16.6%), while significantly fewer patients were at stage III had CCC than SAC (61.7%). By contrast, the incidence of stage III was significantly lower in CCC than in SAC. Recurrence in patients with CCC occurred in 29% of stage I patients, 30% of stage II, 62% of stage III, and 73% at stage IV. Although none of the patients with stage Ia CCC relapsed, 14 of 38 patients (37%) with stage Ic did relapse.

20%of suchcancers inJapan[7-9].Interestingly,amongAsianwomenlivingintheUnitedStates,

Several studies have analyzed the risk factors for ovarian cancer by histologic subtype. CCC was associated (odds ratio: 2.2-2.3) with an increased body mass index (BMI 30) [11, 12]. However, in the NIH-AARP Diet and Health Study BMI was correlated only with endome‐

It has long been recognized that CCC often is associated with endometriosis (22-70%), where‐ as hobnail cells bear a very strong morphological resemblance to endometrial Aria-Stella cells [14, 15]. Several studies have reported that endometriosis frequently shows a sequential change to EOC, including CCC. Therefore, atypical endometriosis is considered to be a precancerous change. Ovarian cancers associated with endometriosis tend to occur in younger women, and present5-6yearsearlier,onaverage,thanhigh-gradeserous carcinoma(HGSC)[16].InNational Cancer Institute Surveillance, Epidemiology and End Results (SEER) data, women with CCC wereyoungerthanpatientswithserousadenocarcinoma(SAC)(55vs.64years;medianage)[10].

An increased incidence of vascular thromboembolic complications is seen in patients with CCC [17, 18]. Up to 40% of patients with CCC may develop thromboembolic disease and this rate

Ovarian CCC usually presents as a large pelvic mass [20, 21]. The size of masses range from 3 to 20 cm, with most tumors detected preoperatively either by clinical examination or imaging. Recent reports involving large institutional cohorts compared early-stage (I/II) to advancedstage ovarian cancers (III/IV) and showed that 57-81% of CCC were diagnosed at an early stage [9, 22]. In SEER data, 56% of CCC were stage I, compared to 19% for SAC [10]. Combining the low overall incidence of CCC and their early stage propensity, CCC may make up only 1-5% of advanced stage patients in chemotherapy trials, largely due to their overall low incidence

Sugiyama, et al. [9] retrospectively reviewed 101 patients with CCC in Japan who underwent complete surgical staging to determine clinicopathological features of CCC. Histologic evaluation was performed under central pathological review. Tumors were diagnosed as CCC if the following appeared in 90% or more of all specimens: a small to large sheet of polyhedral clear cells with delicate fibrovascular septa, tubules and papillae, clear or hobnail, or eosino‐ philic cells of organoid appearance, or clear cells with coalescent vacuoles containing "targe‐ toid" eosinophilic PAS-positive globules. Of the 662 patients with EOC, 101 (15.3%) had CCC and 235 (35.5%) SAC. All patients underwent complete surgical staging, including intraperi‐ toneal cytology, bilateral salpingo-oophorectomy, hysterectomy, omentectomy, pelvic-/ paraaortic lymphadenectomy, and aggressive cytoreductive surgery for advanced cases. Ninety-seven (96%) of 101 patients with CCC and 229 (97%) of 235 with SAC underwent

is double that in matched non-CCC controls with ovarian cancers [19].

and tendency for early stage distribution at the time for initial diagnosis [6].

**3. Clinicopathological features**

platinum-based chemotherapy after initial surgery.

CCC was diagnosed twice as frequently (11.1%) compared to Caucasians (4.8%) [10].

trioid histology [13].

98 Ovarian Cancer - A Clinical and Translational Update

In stage III disease the median survival time was significantly shorter for patients with CCC than those with SAC. The survival rate for patients at stage III was significantly lower in the CCC group than in the SAC group. Although estimated survival rates at 3 and 5 years in patients with no gross tumor did not differ significantly between CCC and SAC, survival rates in both patients with <2 cm and >2 cm residual disease were significantly lower in CCC than in SAC. Jenison, et al. [23] showed that the survival rates for CCC were consistently lower in each of the FIGO stages compared with SAC, although there was no statistical significance. In their study, median survival time for stage I patients with CCC was significantly shorter than that for those with SAC. Similarly, the survival rate for patients with stage Ic CCC was lower compared with patients with stage Ic SAC. Additionally, the median survival time for stage I patients with CCC was worse than those with SAC (31.8 months vs 42.3 months) and the time to recurrence in patients with stage I/II CCC was definitely shorter (12.2 months) [9]. Twentyseven patients with stage III/IV CCC had measurable disease after initial surgery. The overall clinical response rate for SAC was 72.5%. In contrast, only three (11.1%) of 27 patients re‐ sponded to platinum-based chemotherapy in CCC. Patients with CCC showed a very low rate of response and a high incidence of progressive disease. Another authors demonstrated that platinum-based chemotherapy did not appear to improve the survival of patients with CCC, compared with survival after non-platinum-based chemotherapy [24]. Additionally, CCC patients with residual tumor showed a high recurrence rate (Fig 1). CCC has a more aggressive course and a more malignant behavior than SAC. Therefore, new treatment strategies for CCC, including alternative regimens of chemotherapy, should be established.

#### **4. Mechanisms of platinum resistance in CCC of the ovary**

There is general acceptance of CCC that is insensitive to conventional platinum-based chemotherapy lead to a poor prognosis. Resistance to cisplatin (CDDP) is an important factor in the poor prognosis of patients with CCC. Several mechanisms involved in drug resistance have been proposed as explanations, including decreased drug accumulation, increased drug detoxification, increased DNA repair activity, and activation of receptor tyrosine kinases and its downstream signaling pathways [25-29].

Adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporters, such as ABCB1 (also known as P glycoprotein), ABCC1 (multidrug resistance associated protein-1) and ABCC3, are known to lower intracellular drug concentrations and are important multidrug resistance factors [30]. An immunohistochemical study of ABCB1 and ABCC1 in CCC and SAC tumors revealed that their expression of these transporters did not differ between CCC and SAC [31].

MMR are highly tolerant to the methylating chemotherapeutic drugs streptozocin and temozolomide and, to a lesser extent, CDDP and doxorubicin [39]. Loss of MMR may be caused either by a germline mutation of two major MMR genes, hMLH1 or hMSH2, or by somatic MMR gene inactivation through epigenetic silencing via methylation of the hMLH1 promoter. Cai, et al. [40] reported that elevated expression of hMLH1 and hMSH2 proteins are involved in the development of a subset of CCC, and that there is a strong correlation between alterations

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Epidermal growth-factor receptor (EGFR) and v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2; HER2) are cell-surface-receptor tyrosine kinases and can activate both the signaling pathways of mitogen-activated protein kinase and phosphatidylinositol 3'-kinase (PI3K)-Akt [41]. Activating these pathways leads to phosphorylated Bc1-2 antagonist cell death (BAD) and B-cell leukemia/lymphoma (Bcl)-2, thereby inhibiting chemotherapy-induced apoptosis [42]. An immunohistochemical study showed found EGFR in 61% of CCC tumors [43]. Molecular analyzes of various types of ovarian tumors showed HER2 to be overexpressed in CCC relative to other major histological types of EOC [44]. In ovarian cancer, the HER2 protein is overexpressed as a consequence of HER2 gene amplification in 20 to 25% of cases

Cell proliferation is controlled by cyclin-dependent kinases (CDK), which are regulated by cyclin binding, phosphorylation, and CDK inhibitors (e.g. p16, p21, and p27) [47]. p53, known as a tumor suppressor protein, also up-regulates expression of p21 and causes cell the cycle to arrest at G1. Changes in the p53 gene are seen in 50 to 70% of cases of advanced serous adenocarcinoma [48, 49]. In contrast, the p53 mutation is rare in CCC, and immunohistochem‐ ical staining shows that CCC tends to express little or no p53 protein [50]. Cytotoxic drugs are primarily effective against proliferating cells; therefore, quiescent cells show a degree of resistance relative to cycling cells [51]. Dimanche-Boitrel, et al. [52] reported that less intracel‐ lular drug accumulates in resting cells. Itamochi et al. examined the proliferation activity and CDDP sensitivity of 11 CCC and 5 SAC cell lines, and this found that the doubling time for CCC cells was significantly longer than for SAC (61.4 vs 29.8 h) [7]. There was a significant reverse correlation between the S-phase fraction and the response to CDDP. These findings may relate to the high incidence of stage I patients with CCC, and also suggests that the resistance of CCC to CDDP may be caused by low cell proliferation. In addition, Ki-67, a nuclear antigen expressed in all states of the cell cycle except in resting cells in G0, has a significantly lower labeling index in CCC than in SAC [31]. Furthermore, a significantly higher Ki-67 labeling index (LI) is observed in responders than in non-responders in both CCC and

The 5-year survival rate for high LI patients (over 18.4%; mean value for CCC) was significantly greater than that for low LI (less than 18.4%) ( 46.3% vs. 9.2%, p<0.05) (Fig. 2). A multivariable analysis revealed that the Ki-67 labeling index and residual tumor size were independent prognostic factors. Other authors have reported that immunohistochemical staining of CCC reveals a low expression of Ki-67, p53, and cyclin A, and significantly increased expression of both p21 and cyclin E, which are other histological subtypes [53]. These results suggest that

in the expression of hMLH1 and hMSH2 and the presence of MSI in CCC tumors.

and predicts a poor prognosis [45, 46].

SAC tumors.

**Figure 1.** Progression free survival rate for stage III and IV patients, and residual tumor diameter. Progression free sur‐ vival rates for the patients without residual disease were significantly greater, but there was no significant difference between those with <1 cm residual disease and with >1 cm. RT: residual tumor diameter

In addition, no significant differences were observed in the expression of ABCB1 and ABCC1 between responders and non-responders to platinum-based chemotherapy in both tumor types. These results suggest that multidrug resistance proteins do not contribute to chemore‐ sistance in CCC. Ohishi, et al. [32] examined the expression of mRNA by ABCC superfamily members, ABCC1, ABCC2, and ABCC3, in CCC and SAC tumors. They found that only ABCC3 genes were expressed significantly more in CCC than SAC. Therefore, they concluded that increased expression of ABCC3 may, at least in part, be associated with the chemoresistant phenotype of CCC.

Several drug detoxification systems also can diminish intracellular drug activity. Cellular detoxification via the glutathione system is known to be involved in the metabolism of various cytotoxic agents, including the platinum agents, etoposide (VP-16), and mitomycin C (MMC) [27, 28, 33, 34]. Indeed, the glutathione concentrations in CCC cell lines increased significantly after exposure to CDDP or MMC [35]. A gene expression study showed that glutathione peroxidase 3 (GPx3), glutaredoxin (GLRX), and superoxide dismutase (SOD2) were expressed highly in CCC tumors and that the elevated levels of these, and perhaps other, antioxidant proteins may render the tumors more resistant to chemotherapy [36].

Nucleotide excision repair (NER) is a multienzyme DNA repair pathway in eukaryotes that has been implicated in drug resistance in human tumor cells [37]. Reed, et al. [38] examined the mRNA expression of two key genes, excision repaircross-complementing rodent repair deficiency, complementation group 1 (ERCC1) and xeroderma pigmentosum group B (XPB), that are involved in the NER pathway of EOC tumors. Expression of ERCC1 and XPB were higher in CCC tumors than in other histological tumor types. This phenomenon may be related to de novo drug resistance against chemotherapeutic agents in CCC. DNA mismatch repair systems (MMR), which correct errors that occur during DNA replication, also play a critical role in the sensitivity of DNA damaging agents. In experimental systems, cells deficient in MMR are highly tolerant to the methylating chemotherapeutic drugs streptozocin and temozolomide and, to a lesser extent, CDDP and doxorubicin [39]. Loss of MMR may be caused either by a germline mutation of two major MMR genes, hMLH1 or hMSH2, or by somatic MMR gene inactivation through epigenetic silencing via methylation of the hMLH1 promoter. Cai, et al. [40] reported that elevated expression of hMLH1 and hMSH2 proteins are involved in the development of a subset of CCC, and that there is a strong correlation between alterations in the expression of hMLH1 and hMSH2 and the presence of MSI in CCC tumors.

Epidermal growth-factor receptor (EGFR) and v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2; HER2) are cell-surface-receptor tyrosine kinases and can activate both the signaling pathways of mitogen-activated protein kinase and phosphatidylinositol 3'-kinase (PI3K)-Akt [41]. Activating these pathways leads to phosphorylated Bc1-2 antagonist cell death (BAD) and B-cell leukemia/lymphoma (Bcl)-2, thereby inhibiting chemotherapy-induced apoptosis [42]. An immunohistochemical study showed found EGFR in 61% of CCC tumors [43]. Molecular analyzes of various types of ovarian tumors showed HER2 to be overexpressed in CCC relative to other major histological types of EOC [44]. In ovarian cancer, the HER2 protein is overexpressed as a consequence of HER2 gene amplification in 20 to 25% of cases and predicts a poor prognosis [45, 46].

In addition, no significant differences were observed in the expression of ABCB1 and ABCC1 between responders and non-responders to platinum-based chemotherapy in both tumor types. These results suggest that multidrug resistance proteins do not contribute to chemore‐ sistance in CCC. Ohishi, et al. [32] examined the expression of mRNA by ABCC superfamily members, ABCC1, ABCC2, and ABCC3, in CCC and SAC tumors. They found that only ABCC3 genes were expressed significantly more in CCC than SAC. Therefore, they concluded that increased expression of ABCC3 may, at least in part, be associated with the chemoresistant

**Figure 1.** Progression free survival rate for stage III and IV patients, and residual tumor diameter. Progression free sur‐ vival rates for the patients without residual disease were significantly greater, but there was no significant difference

**0 20 40 60 80 100 120 140 Survival time (months)**

*p***=0.04**

**Median progression-free survival duration (months)**

**39**

**7**

**5**

**RT=0cm**

**RT<1cm**

**RT>1cm**

between those with <1 cm residual disease and with >1 cm. RT: residual tumor diameter

Several drug detoxification systems also can diminish intracellular drug activity. Cellular detoxification via the glutathione system is known to be involved in the metabolism of various cytotoxic agents, including the platinum agents, etoposide (VP-16), and mitomycin C (MMC) [27, 28, 33, 34]. Indeed, the glutathione concentrations in CCC cell lines increased significantly after exposure to CDDP or MMC [35]. A gene expression study showed that glutathione peroxidase 3 (GPx3), glutaredoxin (GLRX), and superoxide dismutase (SOD2) were expressed highly in CCC tumors and that the elevated levels of these, and perhaps other, antioxidant

Nucleotide excision repair (NER) is a multienzyme DNA repair pathway in eukaryotes that has been implicated in drug resistance in human tumor cells [37]. Reed, et al. [38] examined the mRNA expression of two key genes, excision repaircross-complementing rodent repair deficiency, complementation group 1 (ERCC1) and xeroderma pigmentosum group B (XPB), that are involved in the NER pathway of EOC tumors. Expression of ERCC1 and XPB were higher in CCC tumors than in other histological tumor types. This phenomenon may be related to de novo drug resistance against chemotherapeutic agents in CCC. DNA mismatch repair systems (MMR), which correct errors that occur during DNA replication, also play a critical role in the sensitivity of DNA damaging agents. In experimental systems, cells deficient in

proteins may render the tumors more resistant to chemotherapy [36].

phenotype of CCC.

**0**

**20**

**40**

**Cumulative percent (%)**

**60**

**80**

**100**

100 Ovarian Cancer - A Clinical and Translational Update

Cell proliferation is controlled by cyclin-dependent kinases (CDK), which are regulated by cyclin binding, phosphorylation, and CDK inhibitors (e.g. p16, p21, and p27) [47]. p53, known as a tumor suppressor protein, also up-regulates expression of p21 and causes cell the cycle to arrest at G1. Changes in the p53 gene are seen in 50 to 70% of cases of advanced serous adenocarcinoma [48, 49]. In contrast, the p53 mutation is rare in CCC, and immunohistochem‐ ical staining shows that CCC tends to express little or no p53 protein [50]. Cytotoxic drugs are primarily effective against proliferating cells; therefore, quiescent cells show a degree of resistance relative to cycling cells [51]. Dimanche-Boitrel, et al. [52] reported that less intracel‐ lular drug accumulates in resting cells. Itamochi et al. examined the proliferation activity and CDDP sensitivity of 11 CCC and 5 SAC cell lines, and this found that the doubling time for CCC cells was significantly longer than for SAC (61.4 vs 29.8 h) [7]. There was a significant reverse correlation between the S-phase fraction and the response to CDDP. These findings may relate to the high incidence of stage I patients with CCC, and also suggests that the resistance of CCC to CDDP may be caused by low cell proliferation. In addition, Ki-67, a nuclear antigen expressed in all states of the cell cycle except in resting cells in G0, has a significantly lower labeling index in CCC than in SAC [31]. Furthermore, a significantly higher Ki-67 labeling index (LI) is observed in responders than in non-responders in both CCC and SAC tumors.

The 5-year survival rate for high LI patients (over 18.4%; mean value for CCC) was significantly greater than that for low LI (less than 18.4%) ( 46.3% vs. 9.2%, p<0.05) (Fig. 2). A multivariable analysis revealed that the Ki-67 labeling index and residual tumor size were independent prognostic factors. Other authors have reported that immunohistochemical staining of CCC reveals a low expression of Ki-67, p53, and cyclin A, and significantly increased expression of both p21 and cyclin E, which are other histological subtypes [53]. These results suggest that CCC has low tumor proliferation activity and that this low proliferation activity in CCC could be associated with chemoresistance.

**6. Mucinous adenocarcinoma**

**Figure 3.** Criteria for central pathological review.

precursors to invasive tumors [57].

the stroma.

Ovarian mucinous adenocarcinoma (MAC) is divided into intraepithelial and invasive carcinomas (Fig. 3). Intraepithelial mucinous carcinoma is characterized by marled epithelial atypia in the absence of stromal invasion. Invasive mucinous carcinoma is diagnosed once stromal invasion measuring more than 5 mm or more than 10 mm2 is detected. Two types of invasive mucinous caricinoma are recognized: expansile and infiltrative. The former is characterized by conflicted glandular growth uninterrupted by normal ovarian parenchyma, while the latter demonstrates a presence of small glands, nests or individual cells infiltrating

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Intraepithelial mucinous carcinoma, FIGO stage I, has a recurrence rate of 5.8% [55]. Invasive mucinous carcinoma, FIGO stage I, has a 5-year survival rate of 91%. Patients with advanced tumors usually die of the disease [56]. Invasive mucinous carcinoma with an infiltrative pattern has a more aggressive course than mucinous carcinoma with an expansile pattern. Interest‐ ingly, invasive mucinous caricinoma of the ovary often coexistes alongside areas of mucinous borderline lesions and benign mucinous cystadenomas, suggesting that these lesions may be

Winter, et al. [3] reviewed the data from 6 GOG phase III trials of adjuvant chemotherapy with CDDP and PTX in women with stage III EOC after primary debulking surgery, both optimal and suboptimal. Of the 1,895 patients included in these 6 studies, 74% had SAC, while only 2% had MAC. The authors found that women with mucinous tumors had a progression-free survival of 10.5 months, compared to 16.9 months for women with serous tumors. Women with MAC had a relative risk of progression of 2.18 compared their serous counterparts

**Figure 2.** Estimated survival rates for patient with clear cell carcinoma. When the cut-off value of Ki-67 labeling index (LI) was set at 18.4% (the mean value of clear cell carcinoma), the estimated 5-year survival rate for elevated Ki-67 LI patients was significantly greater than that for low Ki-67 LI (46.3% vs. 9.2%).

#### **5. Future directions**

The clinicopathological features of CCC suggest that a new strategy for chemotherapy in CCC should be adopted, focusing on new agents without cross-resistance to platinum agents. Several anticancer agents with no cross-resistance to platinum analogues, such as paclitaxel (PTX), VP-16, and camptothecin (CPT-11) have been developed.

Activating the PI3K/Akt pathway and its downstream signaling mammalian target of rapa‐ mycin (mTOR) seems to indicate drug resistance and poor prognosis in many cancers. It has been reported that CCC has a high frequency of activating mutations of PIK3CA [54]. Because it is known well that activation of Akt signaling results in hypersensitivity to mTOR inhibition, CCC may be a good candidate for therapy with an mTOR inhibitor. Several clinical trials have shown potential antitumor activities for mTOR inhibitors (everolimus, deforolimus, and temsirolimus) in solid tumors. Temsirolimus (CCI779, a synthetic, ester analog of rapamycin) is indicated to treat advanced renal cell carcinoma. A phase II study is ongoing to evaluate the safety and efficacy of temsirolimus in combination with carboplatin and PTX followed by temsirolimus consolidation as first-line therapy for patients with stage III-IV CCC in the ovary (NCT01196429, ClinicalTrials.gov). We hope this combination therapy will improve the survival of patients with ovarian CCC.

#### **6. Mucinous adenocarcinoma**

CCC has low tumor proliferation activity and that this low proliferation activity in CCC could

**Figure 2.** Estimated survival rates for patient with clear cell carcinoma. When the cut-off value of Ki-67 labeling index (LI) was set at 18.4% (the mean value of clear cell carcinoma), the estimated 5-year survival rate for elevated Ki-67 LI

The clinicopathological features of CCC suggest that a new strategy for chemotherapy in CCC should be adopted, focusing on new agents without cross-resistance to platinum agents. Several anticancer agents with no cross-resistance to platinum analogues, such as paclitaxel

Activating the PI3K/Akt pathway and its downstream signaling mammalian target of rapa‐ mycin (mTOR) seems to indicate drug resistance and poor prognosis in many cancers. It has been reported that CCC has a high frequency of activating mutations of PIK3CA [54]. Because it is known well that activation of Akt signaling results in hypersensitivity to mTOR inhibition, CCC may be a good candidate for therapy with an mTOR inhibitor. Several clinical trials have shown potential antitumor activities for mTOR inhibitors (everolimus, deforolimus, and temsirolimus) in solid tumors. Temsirolimus (CCI779, a synthetic, ester analog of rapamycin) is indicated to treat advanced renal cell carcinoma. A phase II study is ongoing to evaluate the safety and efficacy of temsirolimus in combination with carboplatin and PTX followed by temsirolimus consolidation as first-line therapy for patients with stage III-IV CCC in the ovary (NCT01196429, ClinicalTrials.gov). We hope this combination therapy will improve the

patients was significantly greater than that for low Ki-67 LI (46.3% vs. 9.2%).

(PTX), VP-16, and camptothecin (CPT-11) have been developed.

**5. Future directions**

survival of patients with ovarian CCC.

be associated with chemoresistance.

102 Ovarian Cancer - A Clinical and Translational Update

Ovarian mucinous adenocarcinoma (MAC) is divided into intraepithelial and invasive carcinomas (Fig. 3). Intraepithelial mucinous carcinoma is characterized by marled epithelial atypia in the absence of stromal invasion. Invasive mucinous carcinoma is diagnosed once stromal invasion measuring more than 5 mm or more than 10 mm2 is detected. Two types of invasive mucinous caricinoma are recognized: expansile and infiltrative. The former is characterized by conflicted glandular growth uninterrupted by normal ovarian parenchyma, while the latter demonstrates a presence of small glands, nests or individual cells infiltrating the stroma.

**Figure 3.** Criteria for central pathological review.

Intraepithelial mucinous carcinoma, FIGO stage I, has a recurrence rate of 5.8% [55]. Invasive mucinous carcinoma, FIGO stage I, has a 5-year survival rate of 91%. Patients with advanced tumors usually die of the disease [56]. Invasive mucinous carcinoma with an infiltrative pattern has a more aggressive course than mucinous carcinoma with an expansile pattern. Interest‐ ingly, invasive mucinous caricinoma of the ovary often coexistes alongside areas of mucinous borderline lesions and benign mucinous cystadenomas, suggesting that these lesions may be precursors to invasive tumors [57].

Winter, et al. [3] reviewed the data from 6 GOG phase III trials of adjuvant chemotherapy with CDDP and PTX in women with stage III EOC after primary debulking surgery, both optimal and suboptimal. Of the 1,895 patients included in these 6 studies, 74% had SAC, while only 2% had MAC. The authors found that women with mucinous tumors had a progression-free survival of 10.5 months, compared to 16.9 months for women with serous tumors. Women with MAC had a relative risk of progression of 2.18 compared their serous counterparts (p<0.001) [69]. The relative risk of death from MAC for compared to SAC was 4.14 (p<0.001). Shimada, et al. [58] compared 24 women with primary MAC to 189 women with SAC and found response rates to platinum-based regimens of 12.5 % and 37.7% respectively. Pectasides, et al. [59] compared 47 women with advanced stage primary MAC to 94 with advanced-stage SAC, all of whom had received a platinum-based regimen in 1 of 9 Hellenic Cooperative Oncology Group studies. The authors found a better response rate in women with SAC (70% for serous vs 38.5 % for mucinous), although this did not translate into survival differences between the 2 groups.

and are commonly mutated not only in inherited SAC but also in many cases of sporadic SAC. Tonin, et al. [66] reviewed the histopathologic subtypes of ovarian carcinomas in 58 families with hereditary breast and ovarian carcinomas. In those patients with known BRCA mutations, 64% had SAC, and only 2% had MAC. In contrast, among women with ovarian cancer negative for BRCA mutation, 29% had MAC, and this proportion was significantly higher than among women with BRCA mutation. Similarly, in a review of the literature that included 636 BRCA mutation-positive women with ovarian cancer, only 2% were found to have mucinous subtypes. p53 also seems to play a prominent role in carcinogenesis of serous ovarian tumors but not mucinous ovarian tumors [70]. Mutations in p53 have been found in almost 60% of

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The expression of multiple individual proteins has been examined in serous and mucinous tumor specimens using immunohistochemical stains. Compared to serous tumors, mucinous tumors are more likely to express E-cadherin (62% vs. 4%, p<0.001) and less likely to stain positive for N-cadherin (8% vs. 68%, p<0.001). The cadherin family of glycoproteins helps cells establish contact with other cells and stabilize tissue architecture. The matrix metalloprotei‐ nases, which also play a role on cell migration and adhesion, have also been found to be expressed differently between serous and mucinous tumors. Kobel, et al. evaluated 21 proteins with immunohistochemistry in 500 ovarian cancer specimens. They found different expression between serous and mucinous subtypes in 20 of the 21 biomarkers they examined, including p53, cadherin, metalloproteinase, CA125, and WT-1. Collectively, these and other molecular studies point toward a distinct pathogenesis of MAC compared to other histological subtypes

The majority of MAC are either well or moderately differentiated and this contributes to the low risk of relapse for FIGO stage I tumors. It also is known that patients at an early stage show good outcomes. On the other hand, patients with advanced mucinous adenocarcinoma are recognized to have poorer outcomes. There was no significant difference in survival between mucinous invasive adenocarcinoma and SAC in patients with optimal surgical management [58]. In contrast, patients with suboptimal therapy showed a significantly worse prognosis

Whenamucinous tumorisgrosslylimitedtotheovary,thereis littlechanceofoccultlymphnode metastasis. Cho, et al. [71] reviewed 26 cases of MAC noted to be grossly stage I intraoperative‐ ly. All of these patients underwent lymphadenectomy as part of their staging procedures, and none were found to have lymph node disease. In contrast, 10% of patients with apparent stage I SAC of the ovary have been reported to have occult nodal metastasis at the time of diagnosis. Using the Swedish Family Canter Database of over 6,000 women with a diagnosis of ovarian cancer showed that the average overall survival was 34 months in women with serous sub‐ types, compared to 70 months for women with mucinous subtypes [72]. In addition, the hazard ratio for cause-specific survival from MAC compared to SAC was 0.49 (95% confidence inter‐ val, 0.41-0.57); the corresponding hazard ratio for overall survival was 0.56 (95% confidence

Patients with advanced-stage MAC had a worse prognosis than women with nonmucinous EOC [73]. The authors matched 27 patients with MAC to 54 patients with nonmucinous ovarian

serous tumors but in only 16% of mucinous tumors.

of ovarian cancer.

than those with SAC (Fig 4).

interval, 0.48-0.64).

#### **7. Clinicopathological features**

Seidman, et al. [60] carefully reviewed the pathology of 220 consecutive cases of epithelial ovarian cancer. After excluding carcinosarcomas and primary peritoneal cancers, they found the incidence of primary MAC to be 3.4%. Other authors reviewed 1400 cases of EOC from 14 centers in Japan [58]. In this large group, 16% patients had an initial diagnosis of invasive primary mucinous ovarian cancer. However, after a careful pathologic review, only 4.9% had invasive primary ovarian cancer, with the remainder reclassified as either mucinous intraepi‐ thelial carcinoma, mucinous borderline tumors, or metastases from another site. Seidman and colleagues argue that these lower estimates are likely a more accurate reflection of the incidence of mucinous ovarian cancer because the following problems were likely in the literature: (1) misclassification of a gastrointestinal primary tumor as an ovarian primary tumor (80% of mucinous epithelial tumors found in the ovary are extraovarian in origin); (2) misclassification of a mucinous borderline tumor as an invasive cancer; and (3) classification of pseudomyxoma peritoneii as being of ovarian origin when it is now standard to consider all such cases as intestinal in origin.

Mutations in KRAS, BRCA, and p53 are the most frequently studied single gene alterations in ovarian cancer pathogenesis. Some investigators have gone beyond analysis of single gene mutations and have used gene expression analysis to evaluate differences between serous and mucinous ovarian carcinomas. Marchini, et al.[61] carried out genomic analyses using a microarray chip with 16,000 genes and found that serous and mucinous tumors were easily distinguished on the basis of expression profiles. Using a probe set of 59,000 genes, Heinzel‐ mann-Schwarz, et al. [62] likewise found clear separation in expression profiles between serous and mucinous tumors of the ovary.

The role of the KRAS oncogene has been explored extensively in EOC. The RAS family of G proteins is part of the pathway that signals cell division. Mutations in the RAS genes have been found to stimulate cell growth [63]. In the literature, 50% of MAC had KRAS mutations, compared to only 5% of SAC, 10% of endometrioid ovarian caricinomas, and no CCC [64]. Interestingly, the same KRAS mutations found in invasive mucinous tumors also are found in adjacent borderline and benign mucinous lesions in the same specimens [65].

Mutations of BRCA1 and BRCA2 are thought to play a significant role in developing SAC but not MAC. BRCA1 and BRCA2 are tumor suppressor genes that help to repair damaged DNA and are commonly mutated not only in inherited SAC but also in many cases of sporadic SAC. Tonin, et al. [66] reviewed the histopathologic subtypes of ovarian carcinomas in 58 families with hereditary breast and ovarian carcinomas. In those patients with known BRCA mutations, 64% had SAC, and only 2% had MAC. In contrast, among women with ovarian cancer negative for BRCA mutation, 29% had MAC, and this proportion was significantly higher than among women with BRCA mutation. Similarly, in a review of the literature that included 636 BRCA mutation-positive women with ovarian cancer, only 2% were found to have mucinous subtypes. p53 also seems to play a prominent role in carcinogenesis of serous ovarian tumors but not mucinous ovarian tumors [70]. Mutations in p53 have been found in almost 60% of serous tumors but in only 16% of mucinous tumors.

(p<0.001) [69]. The relative risk of death from MAC for compared to SAC was 4.14 (p<0.001). Shimada, et al. [58] compared 24 women with primary MAC to 189 women with SAC and found response rates to platinum-based regimens of 12.5 % and 37.7% respectively. Pectasides, et al. [59] compared 47 women with advanced stage primary MAC to 94 with advanced-stage SAC, all of whom had received a platinum-based regimen in 1 of 9 Hellenic Cooperative Oncology Group studies. The authors found a better response rate in women with SAC (70% for serous vs 38.5 % for mucinous), although this did not translate into survival differences

Seidman, et al. [60] carefully reviewed the pathology of 220 consecutive cases of epithelial ovarian cancer. After excluding carcinosarcomas and primary peritoneal cancers, they found the incidence of primary MAC to be 3.4%. Other authors reviewed 1400 cases of EOC from 14 centers in Japan [58]. In this large group, 16% patients had an initial diagnosis of invasive primary mucinous ovarian cancer. However, after a careful pathologic review, only 4.9% had invasive primary ovarian cancer, with the remainder reclassified as either mucinous intraepi‐ thelial carcinoma, mucinous borderline tumors, or metastases from another site. Seidman and colleagues argue that these lower estimates are likely a more accurate reflection of the incidence of mucinous ovarian cancer because the following problems were likely in the literature: (1) misclassification of a gastrointestinal primary tumor as an ovarian primary tumor (80% of mucinous epithelial tumors found in the ovary are extraovarian in origin); (2) misclassification of a mucinous borderline tumor as an invasive cancer; and (3) classification of pseudomyxoma peritoneii as being of ovarian origin when it is now standard to consider all such cases as

Mutations in KRAS, BRCA, and p53 are the most frequently studied single gene alterations in ovarian cancer pathogenesis. Some investigators have gone beyond analysis of single gene mutations and have used gene expression analysis to evaluate differences between serous and mucinous ovarian carcinomas. Marchini, et al.[61] carried out genomic analyses using a microarray chip with 16,000 genes and found that serous and mucinous tumors were easily distinguished on the basis of expression profiles. Using a probe set of 59,000 genes, Heinzel‐ mann-Schwarz, et al. [62] likewise found clear separation in expression profiles between serous

The role of the KRAS oncogene has been explored extensively in EOC. The RAS family of G proteins is part of the pathway that signals cell division. Mutations in the RAS genes have been found to stimulate cell growth [63]. In the literature, 50% of MAC had KRAS mutations, compared to only 5% of SAC, 10% of endometrioid ovarian caricinomas, and no CCC [64]. Interestingly, the same KRAS mutations found in invasive mucinous tumors also are found in

Mutations of BRCA1 and BRCA2 are thought to play a significant role in developing SAC but not MAC. BRCA1 and BRCA2 are tumor suppressor genes that help to repair damaged DNA

adjacent borderline and benign mucinous lesions in the same specimens [65].

between the 2 groups.

intestinal in origin.

and mucinous tumors of the ovary.

**7. Clinicopathological features**

104 Ovarian Cancer - A Clinical and Translational Update

The expression of multiple individual proteins has been examined in serous and mucinous tumor specimens using immunohistochemical stains. Compared to serous tumors, mucinous tumors are more likely to express E-cadherin (62% vs. 4%, p<0.001) and less likely to stain positive for N-cadherin (8% vs. 68%, p<0.001). The cadherin family of glycoproteins helps cells establish contact with other cells and stabilize tissue architecture. The matrix metalloprotei‐ nases, which also play a role on cell migration and adhesion, have also been found to be expressed differently between serous and mucinous tumors. Kobel, et al. evaluated 21 proteins with immunohistochemistry in 500 ovarian cancer specimens. They found different expression between serous and mucinous subtypes in 20 of the 21 biomarkers they examined, including p53, cadherin, metalloproteinase, CA125, and WT-1. Collectively, these and other molecular studies point toward a distinct pathogenesis of MAC compared to other histological subtypes of ovarian cancer.

The majority of MAC are either well or moderately differentiated and this contributes to the low risk of relapse for FIGO stage I tumors. It also is known that patients at an early stage show good outcomes. On the other hand, patients with advanced mucinous adenocarcinoma are recognized to have poorer outcomes. There was no significant difference in survival between mucinous invasive adenocarcinoma and SAC in patients with optimal surgical management [58]. In contrast, patients with suboptimal therapy showed a significantly worse prognosis than those with SAC (Fig 4).

Whenamucinous tumorisgrosslylimitedtotheovary,thereis littlechanceofoccultlymphnode metastasis. Cho, et al. [71] reviewed 26 cases of MAC noted to be grossly stage I intraoperative‐ ly. All of these patients underwent lymphadenectomy as part of their staging procedures, and none were found to have lymph node disease. In contrast, 10% of patients with apparent stage I SAC of the ovary have been reported to have occult nodal metastasis at the time of diagnosis. Using the Swedish Family Canter Database of over 6,000 women with a diagnosis of ovarian cancer showed that the average overall survival was 34 months in women with serous sub‐ types, compared to 70 months for women with mucinous subtypes [72]. In addition, the hazard ratio for cause-specific survival from MAC compared to SAC was 0.49 (95% confidence inter‐ val, 0.41-0.57); the corresponding hazard ratio for overall survival was 0.56 (95% confidence interval, 0.48-0.64).

Patients with advanced-stage MAC had a worse prognosis than women with nonmucinous EOC [73]. The authors matched 27 patients with MAC to 54 patients with nonmucinous ovarian

presented above and the status of the ovarian surface should be used clinically with caution: in one study, up to 24 % of the cases of colonic adenocarcinoma metastatic to the ovary showed unilateral ovarian involvement with tumor measurements of at least 10 cm [77]. In the same study, 46% of the cases with available information on gross intraoperative appearance had a

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Although gross examination of the adnexae can often predict the site of origin, both ovarian and extraovarian sources of primary disease should be explored. Intraoperatively, the surgeon should perform a careful exploration of potential gatrointestinal sources, including palpating the pancreas and running the entire small and large bowel. Postoperatively, the surgeon should consider a colonoscopy and mammogram if these screening tests have not been

Although the presence of certain histological features can favor a diagnosis of primary MAC over metastasis, there are cases where a definitive diagnosis cannot be provided due to the presence of discordant or overlapping features. Microscopic features that favor the diagnosis of primary ovarian MAC include a coexisting borderline and benign mucinous components, an expansile pattern of invasion, and a coexisting ovarian teratoma, Brenner tumor, or mural nodule. In contrast, the following microscopic features favor the diagnosis of metastatic adenocarcinoma to the ovary: (1) prominent desmoplastic response, (2) nodular pattern of invasion (i.e., tumor nodules among structures indigenous to the ovarian parenchyma), (3) small clusters of tumor cell within the corpora lutea or albicantia, (4) numerous pools of mucin dissecting the ovarian stroma (i.e., pseudomyxoma ovarii) in the absence of a coexistent ovarian teratoma, (5) an extensive signet ring cell pattern, (6) ovarian surface involvement, (7) vascular invasion, (8) hilar involvement, and (9) an extensive infiltrative pattern of invasion. Immunohistochemistry may help determine the primary site of a mucinous carcinoma. Primary ovarian mucinous carcinomas tend to be positive for CK7 and CK20 with a predom‐ inance of CK7 expression, while colorectal primaries tend to express CK20 only. In addition, colorectal cancers usually express racemase and beta-catenin while primary mucinous ovarian cancers do not. In regard to other gynecological primaries metastatic to the ovary, it is worth mentioning that Human Papilloma Virus (HPV) in situ hybridization can confirm an endo‐ cervical origin because most of the endocervical adenocarcinoma are related to HPV. p16 immunostagining is useful only in well differentiated adenocarcinoma cases where a diffuse

Attention must be paid to the fact that high-grade ovarian mucinous or endometrioid adeno‐ carcinomas can be positive for p16. Estrogen and progesterone receptors usually are expressed in endometrioid carcinomas, metastasizing from the endometrium or primary to the ovary. Metastatic endocervical adenocarcinomas in ovaries cannot be distinguished from a primary mucinous carcinoma of the ovary because both tumors are progesterone receptor negative and usually negative for estrogen receptors, although they can have variable expression for the latter (weak/diffuse or strong/focal staining) [78]. The presence of mesothelin, fascin, and prostate stem cell antigen (PSCA) favor a pancreatic primary, while the presence of expressed Dpc4 favors an ovarian primary for differentiating primary ovarian tumors from metastasis from the pancreas [79]. Most breast cancers are CK7-positive/CK20-negative, unlike ovarian

smooth capsule.

performed within the year prior to diagnosis.

staining will be in keeping with an endocercvical origin.

**Figure 4.** Overall survival and residual tumor in mucinous adenocarcinoma (MAC). There was no significant difference in survival between mucinous invasive adenocarcinoma and serous adenocarcinoma (SAC) in patients with optimal surgical management. In contrast, patients with suboptimal surgery showed a significantly worse prognosis than those with serous adenocarcinoma.

cancer (2:1), all of whom had stage III or IV disease and had undergone primary therapy. There was no difference between patients with the mucinous tumors and nonmucinous with regard to histological grade, stage, optimal or suboptimal debulking, chemotherapy regimen, or length of follow-up. Patients with advanced MAC had a progression-free survival of 5.7 months, compared to 14.1 for patients with nonmucinous ovarian cancer, and an overall survival of 12.0 compared to 36.7 months.

#### **8. Pathological diagnosis for MAC**

Most MAC involving the ovary prove to be metastases, as opposed to ovarian primary carcinomas. Therefore, surgeons and pathologists must have a high suspicion toward meta‐ static disease when considering the origin of MAC found in the ovary. Seidman, et al.[74] found that only 23% of invasive mucinous carcinomas of the ovary were primary ovarian cancer. Most clinicians assume that metastases to the ovary have a gastrointestinal origin. However, although gastrointestinal tract tumors are the most common source of ovarian metastases, accounting for 45% of such tumors, they also see ovarian metastases from primary tumors of the pancreas (accounting for 20% of ovarian metastases), cervix (13%), breast (8%), and uterus (5%). The remaining 10% of ovarian metastases are from unknown primary tumors [68].

At surgical exploration a working differential diagnosis can be developed on the basis of tumor size and laterality. Among unilateral tumors, more than 80% of those larger than 10 cm are ovarian primary tumors, while 88% of those smaller than 10 cm are metastases. This division has been retrospectively validated by other investigators, who showed it to be correct 84% of the time in differentiating primary from metastatic mucinous carcinomas of the ovary [74]. Other authors also have found this algorithm is useful in predicting the site of origin for ovarian carcinomas [75, 76]. In addition, primary ovarian carcinomas tend to have a smooth capsule, while ovarian metastases often involve the gross ovarian surface. However, the algorithm presented above and the status of the ovarian surface should be used clinically with caution: in one study, up to 24 % of the cases of colonic adenocarcinoma metastatic to the ovary showed unilateral ovarian involvement with tumor measurements of at least 10 cm [77]. In the same study, 46% of the cases with available information on gross intraoperative appearance had a smooth capsule.

Although gross examination of the adnexae can often predict the site of origin, both ovarian and extraovarian sources of primary disease should be explored. Intraoperatively, the surgeon should perform a careful exploration of potential gatrointestinal sources, including palpating the pancreas and running the entire small and large bowel. Postoperatively, the surgeon should consider a colonoscopy and mammogram if these screening tests have not been performed within the year prior to diagnosis.

Although the presence of certain histological features can favor a diagnosis of primary MAC over metastasis, there are cases where a definitive diagnosis cannot be provided due to the presence of discordant or overlapping features. Microscopic features that favor the diagnosis of primary ovarian MAC include a coexisting borderline and benign mucinous components, an expansile pattern of invasion, and a coexisting ovarian teratoma, Brenner tumor, or mural nodule. In contrast, the following microscopic features favor the diagnosis of metastatic adenocarcinoma to the ovary: (1) prominent desmoplastic response, (2) nodular pattern of invasion (i.e., tumor nodules among structures indigenous to the ovarian parenchyma), (3) small clusters of tumor cell within the corpora lutea or albicantia, (4) numerous pools of mucin dissecting the ovarian stroma (i.e., pseudomyxoma ovarii) in the absence of a coexistent ovarian teratoma, (5) an extensive signet ring cell pattern, (6) ovarian surface involvement, (7) vascular invasion, (8) hilar involvement, and (9) an extensive infiltrative pattern of invasion.

cancer (2:1), all of whom had stage III or IV disease and had undergone primary therapy. There was no difference between patients with the mucinous tumors and nonmucinous with regard to histological grade, stage, optimal or suboptimal debulking, chemotherapy regimen, or length of follow-up. Patients with advanced MAC had a progression-free survival of 5.7 months, compared to 14.1 for patients with nonmucinous ovarian cancer, and an overall

**1 2 3 4 5 (year)**

**Figure 4.** Overall survival and residual tumor in mucinous adenocarcinoma (MAC). There was no significant difference in survival between mucinous invasive adenocarcinoma and serous adenocarcinoma (SAC) in patients with optimal surgical management. In contrast, patients with suboptimal surgery showed a significantly worse prognosis than

**p 0.013**

**Optimal operation (residual tumor 1cm)**

**Suboptimal operation**

**SAC**

**MAC**

Most MAC involving the ovary prove to be metastases, as opposed to ovarian primary carcinomas. Therefore, surgeons and pathologists must have a high suspicion toward meta‐ static disease when considering the origin of MAC found in the ovary. Seidman, et al.[74] found that only 23% of invasive mucinous carcinomas of the ovary were primary ovarian cancer. Most clinicians assume that metastases to the ovary have a gastrointestinal origin. However, although gastrointestinal tract tumors are the most common source of ovarian metastases, accounting for 45% of such tumors, they also see ovarian metastases from primary tumors of the pancreas (accounting for 20% of ovarian metastases), cervix (13%), breast (8%), and uterus (5%). The remaining 10% of ovarian metastases are from unknown primary tumors [68].

At surgical exploration a working differential diagnosis can be developed on the basis of tumor size and laterality. Among unilateral tumors, more than 80% of those larger than 10 cm are ovarian primary tumors, while 88% of those smaller than 10 cm are metastases. This division has been retrospectively validated by other investigators, who showed it to be correct 84% of the time in differentiating primary from metastatic mucinous carcinomas of the ovary [74]. Other authors also have found this algorithm is useful in predicting the site of origin for ovarian carcinomas [75, 76]. In addition, primary ovarian carcinomas tend to have a smooth capsule, while ovarian metastases often involve the gross ovarian surface. However, the algorithm

survival of 12.0 compared to 36.7 months.

106 Ovarian Cancer - A Clinical and Translational Update

those with serous adenocarcinoma.

**8. Pathological diagnosis for MAC**

Immunohistochemistry may help determine the primary site of a mucinous carcinoma. Primary ovarian mucinous carcinomas tend to be positive for CK7 and CK20 with a predom‐ inance of CK7 expression, while colorectal primaries tend to express CK20 only. In addition, colorectal cancers usually express racemase and beta-catenin while primary mucinous ovarian cancers do not. In regard to other gynecological primaries metastatic to the ovary, it is worth mentioning that Human Papilloma Virus (HPV) in situ hybridization can confirm an endo‐ cervical origin because most of the endocervical adenocarcinoma are related to HPV. p16 immunostagining is useful only in well differentiated adenocarcinoma cases where a diffuse staining will be in keeping with an endocercvical origin.

Attention must be paid to the fact that high-grade ovarian mucinous or endometrioid adeno‐ carcinomas can be positive for p16. Estrogen and progesterone receptors usually are expressed in endometrioid carcinomas, metastasizing from the endometrium or primary to the ovary. Metastatic endocervical adenocarcinomas in ovaries cannot be distinguished from a primary mucinous carcinoma of the ovary because both tumors are progesterone receptor negative and usually negative for estrogen receptors, although they can have variable expression for the latter (weak/diffuse or strong/focal staining) [78]. The presence of mesothelin, fascin, and prostate stem cell antigen (PSCA) favor a pancreatic primary, while the presence of expressed Dpc4 favors an ovarian primary for differentiating primary ovarian tumors from metastasis from the pancreas [79]. Most breast cancers are CK7-positive/CK20-negative, unlike ovarian primaries which typically express both. In addition, breast cancers almost always express estrogen receptors as well as gross cystic disease fluid protein (GCDFP)- 15 [80]. Mucinous ovarian carcinomas are unlikely to express these markers.

Carcinoembryonic antigen (CEA) is a well known serum tumor marker for gastrointestinal carcinomas. CEA has been noted to be elevated in almost one third of all ovarian carcinomas. However, CEA is much more likely to be elevated in mucinous ovarian carcinomas than in nonmucinous ovarian carcinomas (88% vs 19%) [81, 82]. Nolen, et al. [83] compared the levels of 58 serum biomarkers in serous ovarian carcinomas to mucinous, clear cell, and endoterioid ovarian carcinomas. Using immunoassays, they found significant differences between the 2 groups for 10 of the biomarkers examined. SAC had significantly higher levels of CA125, follicle-stimulating hormone, luteinizing hormone, and SMRP. Mucinous tumors had higher levels of CA72-4, matrixmetalloproteinase-9, CD40L, insulin-like growth factor-binding protein-1, myeloperoxidase, and tissue plasminogen activator-1.

#### **9. Future directions**

Realizing that MAC is a disease distinct from SAC, several collaborative groups have proposed innovative prospective chemotherapy protocols for patients with advanced or recurrent MAC. Sato, et al. [84] evaluated 6 different cytotoxic agents in 5 different primary mucinous ovarian cancer cell lines. All 5 cell lines resisted platinum agents and taxanes given as single agents. However, 2 of the 5 cell lines were sensitive to oxaliplatin, VP-16, and 5-fluorouracil (5-FU) as single agents. The investigators then treated the cell lines with oxaliplatin plus VP-16 and oxaliplatin plus 5-FU. They found that oxaliplatin with 5-FU had was significant inhibition in 4 of the 5 cell lines, whereas the combination of oxaliplatin plus VP-16 was active in only 1 of the 5 cell lines. Moreover, the combination of oxaliplatin plus 5-FU appeared to be synergistic by providing significantly more inhibition than either drug alone. The authors then applied the cell line results to a mouse model of mucinous ovarian cancer xenograft and found that mice treated with oxaliplatin plus 5-FU survived significantly longer than mice treated with either agent alone or control mice treated with placebo (Fig 5).

primary endpoint will be overall survival; secondary endpoints will be progression-free survival, response rate, toxicity, and quality of life. The study also assesses the translational endpoint, KRAS mutations and expression of vascular endothelial growth factor and epider‐

**Figure 5.** Survival in mice with mucinous adenocarcinoma cells (TU-OM-1 cell). Mice treated with oxaliplatin and 5-FU

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Based on the similarity of biological characteristics, the standard chemotherapy regimen for colorectal cancer has been given in patients with MAC. Therefore, the first phase-II chemo‐ therapy study of oral S-1, a 5FU derivative, combined with oxaliplatin (SOX) for advanced or recurrent patients with MAC was conducted in the Japan ovarian mucinous adenocarcinoma

We hope that future research in this field will enable to develop an effective strategy for

mal growth factor. The targeted accrual for the study is 322 patients.

survived significantly longer than mice treated with either agent alone or control mice.

**Clear cell Serous**

III 10 (9.9%) 145 (61.7%) *P<0.0001*

study group.

FIGO stage

**Table 1.** Patients Characteristics

conquest of chemoresistance in EOC.

No 101 235 Median age 51 (31-72) 54 (23-82)

Ia 49 (48.5%) 39 (16.6%) Ib 11 (10.9%) 15 ( 6.5%) Ic 0 2 ( 0.9%) II 38 (37.6%) 22 (9.4%)

IV 31 (30.7%) 38 (16.2%)

These basic studies led to a single-arm phase II trial of S-1 and oxaliplatin that currently is enrolling women with advanced or recurrent mucinous ovarian cancer in. S-1 is an orally active drug made by Taiho Pharmaceuticals that combines 3 separate molecules. The first is tegafur, a prodrug that is converted to fluorouracil in cells. Next is gimeracil, an inhibitor of dihydro‐ purimidine dehydrogenase, an enzyme that degrades fluorouracil. The third component is oteracil, a molecule that inhibits the phosphorylation of fluorouracil in the gastrointestinal tract, reducing gastrointestinal toxicities. The primary endpoint of the study is response rate; secondary endpoints are toxicity, progression-free survival, and overall survival.

The GOG and the Gynecologic Cancer Intergroup (GCIG) are about to begin accrual to a 4 arm, phase III, randomized study comparing carboplatin and PTX with and without bevaci‐ zumab to oxaliplatin and capecitabine with and without bevacizumab in women with stage II-IV or recurrent, untreated, stage I, primary, mucinous ovarian or fallopian tube cancer. The

**Figure 5.** Survival in mice with mucinous adenocarcinoma cells (TU-OM-1 cell). Mice treated with oxaliplatin and 5-FU survived significantly longer than mice treated with either agent alone or control mice.

primary endpoint will be overall survival; secondary endpoints will be progression-free survival, response rate, toxicity, and quality of life. The study also assesses the translational endpoint, KRAS mutations and expression of vascular endothelial growth factor and epider‐ mal growth factor. The targeted accrual for the study is 322 patients.

Based on the similarity of biological characteristics, the standard chemotherapy regimen for colorectal cancer has been given in patients with MAC. Therefore, the first phase-II chemo‐ therapy study of oral S-1, a 5FU derivative, combined with oxaliplatin (SOX) for advanced or recurrent patients with MAC was conducted in the Japan ovarian mucinous adenocarcinoma study group.


We hope that future research in this field will enable to develop an effective strategy for conquest of chemoresistance in EOC.

**Table 1.** Patients Characteristics

primaries which typically express both. In addition, breast cancers almost always express estrogen receptors as well as gross cystic disease fluid protein (GCDFP)- 15 [80]. Mucinous

Carcinoembryonic antigen (CEA) is a well known serum tumor marker for gastrointestinal carcinomas. CEA has been noted to be elevated in almost one third of all ovarian carcinomas. However, CEA is much more likely to be elevated in mucinous ovarian carcinomas than in nonmucinous ovarian carcinomas (88% vs 19%) [81, 82]. Nolen, et al. [83] compared the levels of 58 serum biomarkers in serous ovarian carcinomas to mucinous, clear cell, and endoterioid ovarian carcinomas. Using immunoassays, they found significant differences between the 2 groups for 10 of the biomarkers examined. SAC had significantly higher levels of CA125, follicle-stimulating hormone, luteinizing hormone, and SMRP. Mucinous tumors had higher levels of CA72-4, matrixmetalloproteinase-9, CD40L, insulin-like growth factor-binding

Realizing that MAC is a disease distinct from SAC, several collaborative groups have proposed innovative prospective chemotherapy protocols for patients with advanced or recurrent MAC. Sato, et al. [84] evaluated 6 different cytotoxic agents in 5 different primary mucinous ovarian cancer cell lines. All 5 cell lines resisted platinum agents and taxanes given as single agents. However, 2 of the 5 cell lines were sensitive to oxaliplatin, VP-16, and 5-fluorouracil (5-FU) as single agents. The investigators then treated the cell lines with oxaliplatin plus VP-16 and oxaliplatin plus 5-FU. They found that oxaliplatin with 5-FU had was significant inhibition in 4 of the 5 cell lines, whereas the combination of oxaliplatin plus VP-16 was active in only 1 of the 5 cell lines. Moreover, the combination of oxaliplatin plus 5-FU appeared to be synergistic by providing significantly more inhibition than either drug alone. The authors then applied the cell line results to a mouse model of mucinous ovarian cancer xenograft and found that mice treated with oxaliplatin plus 5-FU survived significantly longer than mice treated with

These basic studies led to a single-arm phase II trial of S-1 and oxaliplatin that currently is enrolling women with advanced or recurrent mucinous ovarian cancer in. S-1 is an orally active drug made by Taiho Pharmaceuticals that combines 3 separate molecules. The first is tegafur, a prodrug that is converted to fluorouracil in cells. Next is gimeracil, an inhibitor of dihydro‐ purimidine dehydrogenase, an enzyme that degrades fluorouracil. The third component is oteracil, a molecule that inhibits the phosphorylation of fluorouracil in the gastrointestinal tract, reducing gastrointestinal toxicities. The primary endpoint of the study is response rate;

The GOG and the Gynecologic Cancer Intergroup (GCIG) are about to begin accrual to a 4 arm, phase III, randomized study comparing carboplatin and PTX with and without bevaci‐ zumab to oxaliplatin and capecitabine with and without bevacizumab in women with stage II-IV or recurrent, untreated, stage I, primary, mucinous ovarian or fallopian tube cancer. The

secondary endpoints are toxicity, progression-free survival, and overall survival.

ovarian carcinomas are unlikely to express these markers.

108 Ovarian Cancer - A Clinical and Translational Update

protein-1, myeloperoxidase, and tissue plasminogen activator-1.

either agent alone or control mice treated with placebo (Fig 5).

**9. Future directions**

#### **Author details**

Jun Naniwa, Hiroaki Itamochi and Junzo Kigawa

Tottori University Hospital Cancer Center, Japan

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**Chapter 6**

**Demographic and Clinical Characteristics of Mucinous**

Ovarian cancer is the fifth leading cause of cancer death in the United States, and contrib‐ utes significantly to the worldwide cancer burden [1]. The lack of gynecologic-specific symptoms and effective early detection methods for ovarian cancer leads to a preponder‐ ance of late-stage diagnoses. Ovarian cancer is a surgically-staged and treated disease, and the application of appropriate, guidelines-based treatment is currently the only option to re‐

The National Comprehensive Cancer Network (NCCN) [3] and the National Institutes of Health (NIH) [4] publish widely used treatment guidelines for ovarian cancer cases in the United States. Both NCCN and NIH's Physician Data Query (PDQ) incorporate tumor his‐ tology into treatment guidelines. While the NCCN publishes guidelines for the three main types of ovarian cancer, epithelial, sex cord-stromal, and germ cell tumors [5]; the PDQ of‐ fers guidelines only for the most common epithelial tumors. While epithelial tumors account for about 90% of all ovarian neoplasms, they are not a homogenous group [5]. The four main epithelial subtypes (serous, mucinous, clear cell, and endometrioid) can have very different

Among epithelial ovarian cancer subtypes is mucinous epithelial ovarian cancer (mEOC), a relatively rare subtype accounting for approximately 14% of invasive ovarian

and reproduction in any medium, provided the original work is properly cited.

© 2013 Stewart et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Epithelial Ovarian Cancer, and Survival Following a**

**Mucinous Epithelial Ovarian Cancer Diagnosis**

Sherri L. Stewart, Jennifer M. Wike,

Maria J. Schymura

**1. Introduction**

http://dx.doi.org/10.5772/53740

duce ovarian cancer mortality [2].

clinical and pathologic patterns.

Trevor D. Thompson, Rosemary D. Cress, Amy R. Kahn, Cynthia D. O'Malley and

Additional information is available at the end of the chapter


## **Demographic and Clinical Characteristics of Mucinous Epithelial Ovarian Cancer, and Survival Following a Mucinous Epithelial Ovarian Cancer Diagnosis**

Sherri L. Stewart, Jennifer M. Wike, Trevor D. Thompson, Rosemary D. Cress, Amy R. Kahn, Cynthia D. O'Malley and Maria J. Schymura

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53740

**1. Introduction**

[78] Ronnett, B. M, Yemelyanova, A. V, Vang, R, et al. Endocervical adenocarcinomas with ovarian metastases: analysis of 29 cases with emphasis on minimally invasive cervical tumors and the ability of the metastases to simulate primary ovarian neo‐

[79] Cao, D, Ji, H, & Ronnett, B. M. Expression of mesothelin, fascin, and prostate stem cell antigen in primary ovarian mucinous tumors and their utility in differentiating primary ovarian mucinous tumors from metastatic pancreatic mucinous carcinomas

[80] Oien, K. A. Pathologic evaluation of unknown primary cancer. Semin Oncol (2009). ,

[81] Tholander, B, Taube, A, Lindgren, A, et al. Pretreatment serum levels of CA-125, car‐ cinoembryonic antigen, tissue polypeptide antigen, and placental alkaline phospha‐ tase, in patients with ovarian carcinoma, borderline tumors, or benign adnexal masses: relevance for differential diagnosis. Gynecol Oncol (1990). , 39(1), 16-25. [82] Tuxen, M. K, Soletormos, G, & Dombernowsky, P. Tumor markers in the manage‐ ment of patients with ovarian cancer. Cancer Treat Rev (1995). , 21(3), 215-245. [83] Nolen, B, Marrangoni, A, Velikokhatnaya, L, et al. A serum based analysis of ovarian

[84] Sato, S, Itamochi, H, Kigawa, J, et al. Combination chemotherapy of oxaliplatin and 5-fluorouracil may be an effective regimen for mucinous adenocarcinoma of the ova‐

plasms. Am J Surg Pathol (2008). , 32(12), 1835-1853.

in the ovary. Int J Gynecol Pathol (2005). , 24(1), 67-72.

epithelial tumorigenesis. Gynecol Oncol (2009). , 112(1), 47-54.

ry: a potential treatment strategy. Cancer Sci (2009). , 100(3), 546-551.

36(1), 8-37.

116 Ovarian Cancer - A Clinical and Translational Update

Ovarian cancer is the fifth leading cause of cancer death in the United States, and contrib‐ utes significantly to the worldwide cancer burden [1]. The lack of gynecologic-specific symptoms and effective early detection methods for ovarian cancer leads to a preponder‐ ance of late-stage diagnoses. Ovarian cancer is a surgically-staged and treated disease, and the application of appropriate, guidelines-based treatment is currently the only option to re‐ duce ovarian cancer mortality [2].

The National Comprehensive Cancer Network (NCCN) [3] and the National Institutes of Health (NIH) [4] publish widely used treatment guidelines for ovarian cancer cases in the United States. Both NCCN and NIH's Physician Data Query (PDQ) incorporate tumor his‐ tology into treatment guidelines. While the NCCN publishes guidelines for the three main types of ovarian cancer, epithelial, sex cord-stromal, and germ cell tumors [5]; the PDQ of‐ fers guidelines only for the most common epithelial tumors. While epithelial tumors account for about 90% of all ovarian neoplasms, they are not a homogenous group [5]. The four main epithelial subtypes (serous, mucinous, clear cell, and endometrioid) can have very different clinical and pathologic patterns.

Among epithelial ovarian cancer subtypes is mucinous epithelial ovarian cancer (mEOC), a relatively rare subtype accounting for approximately 14% of invasive ovarian

© 2013 Stewart et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

cancer cases [6]. mEOC has a distinct natural history compared to other epithelial sub‐ types, especially the most common serous subtype. mEOCs are more often diagnosed in younger women [6] than other epithelial tumors, and epidemiologic studies have shown a lack of protective effect from parity and oral contraceptive use [7-11]. Pathologic stud‐ ies have determined that mutations in the K-ras oncogene are more common in mEOC compared to other subtypes [12], while mutations in the BRCA1 tumor suppressor gene are less common [13]. Despite their distinctive nature, mEOCs are included in overall epithelial ovarian cancer treatment guidelines, as standard care for all epithelial sub‐ types is defined in the same manner [3,4].

characteristics and treatment data as part of state-mandated cancer surveillance. For this ret‐ rospective study, additional detailed patient, tumor, and treatment data were collected by these registries from multiple sources including hospital, outpatient facility and physician records. Vital status was determined by linkage with the National Death Index http:// www.cdc.gov/nchs/ndi.htm. The study population included patients with invasive epithe‐ lial ovarian cancer diagnosed between 1998 and 2000. Only invasive cases of epithelial ovari‐ an cancer were included; benign and low malignant potential tumors were excluded. Primary peritoneal cancers and fallopian tube cancers were also excluded. Subjects diag‐ nosed at autopsy or by death certificate were ineligible. All cases included were histological‐

Demographic and Clinical Characteristics of Mucinous Epithelial Ovarian Cancer, and…

http://dx.doi.org/10.5772/53740

119

Histology was collected according to World Health Organization International Classification of Diseases for Oncology, third edition (ICD-O-3) morphology codes [21]. All epithelial his‐

Race and ethnicity was categorized as white non-Hispanic, black non-Hispanic, Asian non-Hispanic, and Hispanic. A total of 34 cases were excluded from the analysis on the basis of race or ethnicity data. Three of the 34 cases were classified as American Indian/Alaska Na‐ tive race; these were excluded because of the inability to draw any conclusions from this race because of the very small number. The remaining 31 cases were excluded because race or ethnicity information was unspecified or missing. Stage was defined using the Interna‐ tional Federation of Gynecology and Obstetrics (FIGO) system, with categories I, II, III IV, or unknown. Grade was collapsed into four categories defined as Grade I (well differentiated tumors), Grade II (moderately differentiated tumors), Grade III/IV (poorly differentiated and undifferentiated tumors) and unknown grade. Laterality was collapsed into unilateral (single ovary involved at diagnosis: right, left, or unspecified), or bilateral (both ovaries in‐ volved at diagnosis) categories. Comorbidity was defined using the Deyo-Charlson Comor‐ bidity Index [22, 23], a commonly used measure of disease burden. Comorbidity information was collected via linkage with state hospital discharge data. Any comorbidity present in the 12 months prior to or 4 months following an ovarian cancer diagnosis was included. Type of treatment was defined to distinguish patients who received various com‐

8380,8940,8950,8951,8310,9000,8323,8020,8050,8052,

8330,8340,8440,8450,8490,8560,8570,8980,8981

8070,8120,8130,8140,8260,

ly confirmed. Cases were followed up for six years for vital status information.

tologies collected were collapsed into categories for analysis according to Table 1.

**Epithelial Ovarian Cancer (EOC) Subtype ICD-O-3 Codes**

(includes endometrioid, clear cell, Brenner, mixed,

undifferentiated, and unspecified or other epithelial tumors)

**Table 1.** Histologic definitions by epithelial ovarian cancer subtype

Mucinous (mEOC) 8470, 8471, 8480, 8481 Serous 8441,8442,8460,8461,8462

**2.2. Data classification**

Other

Because of the differences in risk factors and presentation, a few studies have examined dif‐ ferences in outcomes of mEOC compared to other epithelial subtypes. Many have found lower response rates to chemotherapy and inferior outcomes compared to other subtypes [14-16]. Based on these results, it has been suggested that mEOC be treated as a different en‐ tity and not grouped along with epithelial tumors in standard treatment and also in clinical trials for epithelial ovarian cancer [17]; however, these suggestions have yet to be widely adopted or implemented. While the existing evidence seems consistent, studies producing this evidence have contained small numbers and generally represent the experience of indi‐ vidual institutions.

#### **1.1. Objectives**

The objective of this chapter is to fully characterize mEOC using a population-based ap‐ proach. We add to the paucity of existing literature on mEOC with an analysis that utilizes ovarian cancer medical record data from two large populations in the United States, New York and Northern California. We comprehensively examine demographics, pathologic characteristics, and the outcomes of treatment for mEOC. We compare these characteristics to other epithelial subtypes in order to determine whether clinical presentation or outcomes differ among epithelial subtypes. Finally, we discuss the results of this research in the con‐ text of published studies on mEOC.

#### **2. Study design**

#### **2.1. Setting and population**

The data presented and analyzed here are from the Ovarian Cancer Treatment Patterns and Outcomes (OCTPO) study, funded by the Centers for Disease Control and Prevention (CDC), and conducted by the New York State and the California Cancer Registries [18-20]. The New York State Cancer Registry (NYSCR) conducts surveillance on all 19 million New York state residents, and the two components of the California Cancer Registry (CCR) that were funded for this study serve the contiguous geographical area of Greater San Francisco-San Jose and Sacramento regions, providing surveillance for a population of 9 million resi‐ dents in California. Both the NYSCR and the CCR conduct high quality, population-based cancer surveillance, and routinely review medical records to abstract demographics, tumor characteristics and treatment data as part of state-mandated cancer surveillance. For this ret‐ rospective study, additional detailed patient, tumor, and treatment data were collected by these registries from multiple sources including hospital, outpatient facility and physician records. Vital status was determined by linkage with the National Death Index http:// www.cdc.gov/nchs/ndi.htm. The study population included patients with invasive epithe‐ lial ovarian cancer diagnosed between 1998 and 2000. Only invasive cases of epithelial ovari‐ an cancer were included; benign and low malignant potential tumors were excluded. Primary peritoneal cancers and fallopian tube cancers were also excluded. Subjects diag‐ nosed at autopsy or by death certificate were ineligible. All cases included were histological‐ ly confirmed. Cases were followed up for six years for vital status information.

#### **2.2. Data classification**

cancer cases [6]. mEOC has a distinct natural history compared to other epithelial sub‐ types, especially the most common serous subtype. mEOCs are more often diagnosed in younger women [6] than other epithelial tumors, and epidemiologic studies have shown a lack of protective effect from parity and oral contraceptive use [7-11]. Pathologic stud‐ ies have determined that mutations in the K-ras oncogene are more common in mEOC compared to other subtypes [12], while mutations in the BRCA1 tumor suppressor gene are less common [13]. Despite their distinctive nature, mEOCs are included in overall epithelial ovarian cancer treatment guidelines, as standard care for all epithelial sub‐

Because of the differences in risk factors and presentation, a few studies have examined dif‐ ferences in outcomes of mEOC compared to other epithelial subtypes. Many have found lower response rates to chemotherapy and inferior outcomes compared to other subtypes [14-16]. Based on these results, it has been suggested that mEOC be treated as a different en‐ tity and not grouped along with epithelial tumors in standard treatment and also in clinical trials for epithelial ovarian cancer [17]; however, these suggestions have yet to be widely adopted or implemented. While the existing evidence seems consistent, studies producing this evidence have contained small numbers and generally represent the experience of indi‐

The objective of this chapter is to fully characterize mEOC using a population-based ap‐ proach. We add to the paucity of existing literature on mEOC with an analysis that utilizes ovarian cancer medical record data from two large populations in the United States, New York and Northern California. We comprehensively examine demographics, pathologic characteristics, and the outcomes of treatment for mEOC. We compare these characteristics to other epithelial subtypes in order to determine whether clinical presentation or outcomes differ among epithelial subtypes. Finally, we discuss the results of this research in the con‐

The data presented and analyzed here are from the Ovarian Cancer Treatment Patterns and Outcomes (OCTPO) study, funded by the Centers for Disease Control and Prevention (CDC), and conducted by the New York State and the California Cancer Registries [18-20]. The New York State Cancer Registry (NYSCR) conducts surveillance on all 19 million New York state residents, and the two components of the California Cancer Registry (CCR) that were funded for this study serve the contiguous geographical area of Greater San Francisco-San Jose and Sacramento regions, providing surveillance for a population of 9 million resi‐ dents in California. Both the NYSCR and the CCR conduct high quality, population-based cancer surveillance, and routinely review medical records to abstract demographics, tumor

types is defined in the same manner [3,4].

118 Ovarian Cancer - A Clinical and Translational Update

vidual institutions.

**2. Study design**

**2.1. Setting and population**

text of published studies on mEOC.

**1.1. Objectives**

Histology was collected according to World Health Organization International Classification of Diseases for Oncology, third edition (ICD-O-3) morphology codes [21]. All epithelial his‐ tologies collected were collapsed into categories for analysis according to Table 1.


**Table 1.** Histologic definitions by epithelial ovarian cancer subtype

Race and ethnicity was categorized as white non-Hispanic, black non-Hispanic, Asian non-Hispanic, and Hispanic. A total of 34 cases were excluded from the analysis on the basis of race or ethnicity data. Three of the 34 cases were classified as American Indian/Alaska Na‐ tive race; these were excluded because of the inability to draw any conclusions from this race because of the very small number. The remaining 31 cases were excluded because race or ethnicity information was unspecified or missing. Stage was defined using the Interna‐ tional Federation of Gynecology and Obstetrics (FIGO) system, with categories I, II, III IV, or unknown. Grade was collapsed into four categories defined as Grade I (well differentiated tumors), Grade II (moderately differentiated tumors), Grade III/IV (poorly differentiated and undifferentiated tumors) and unknown grade. Laterality was collapsed into unilateral (single ovary involved at diagnosis: right, left, or unspecified), or bilateral (both ovaries in‐ volved at diagnosis) categories. Comorbidity was defined using the Deyo-Charlson Comor‐ bidity Index [22, 23], a commonly used measure of disease burden. Comorbidity information was collected via linkage with state hospital discharge data. Any comorbidity present in the 12 months prior to or 4 months following an ovarian cancer diagnosis was included. Type of treatment was defined to distinguish patients who received various com‐ binations of surgery and chemotherapy. In descriptive analyses, chemotherapy was further categorized by receipt of specific agents. These categories consisted of surgery and platinum agent (cisplatin or carboplatin) receipt, surgery and platinum agent and paclitaxel receipt (standard treatment for EOC) [24], and surgery and any chemotherapy agent or combination of agents other than cisplatin, carboplatin, or paclitaxel.

(8.0% vs. 5.9 and 6.7%) and Asian non-Hispanic (14.2% vs. 5.4 and 9.8%) populations com‐ pared to serous and other EOCs. Lower percentages of mEOCs (5.8%) were found among Hispanics compared to serous and mEOCs (7.5 and 7.7%). mEOCS were more likely to be diagnosed at FIGO stage I (45.2%) compared to serous (10.0%) and other mEOCs (24.9%). Higher percentages of low grade tumors and unilateral ovarian involvement at diagnosis were also present with mEOCs compared to other EOC types. A little under half of mEOC patients (46.3%) were treated with surgery only and 39.0% were treated with surgery plus a

> Serous (n=1195)

Demographic and Clinical Characteristics of Mucinous Epithelial Ovarian Cancer, and…

Other Epithelial (n=1211)

http://dx.doi.org/10.5772/53740

121

**P-value**

Mucinous (n=230)

White Non-Hispanic 162 (72.0%) 956 (81.3%) 910 (75.8%) Black Non-Hispanic 18 (8.0%) 69 (5.9%) 80 (6.7%) Asian Non-Hispanic 32 (14.2%) 63 (5.4%) 118 (9.8%) Hispanic 13 (5.8%) 88 (7.5%) 93 (7.7%)

I 104 (45.2%) 119 (10.0%) 302 (24.9%) II 26 (11.3%) 66 (5.5%) 125 (10.3%) III 62 (27.0%) 747 (62.5%) 380 (31.4%) IV 19 (8.3%) 153 (12.8%) 225 (18.6%) Unknown 19 (8.3%) 110 (9.2%) 179 (14.8%)

I 73 (31.7%) 80 (6.7%) 92 (7.6%) II 69 (30.0%) 223 (18.7%) 193 (15.9%) III/IV 30 (13.0%) 730 (61.1%) 493 (40.7%) Unknown 58 (25.2%) 162 (13.6%) 433 (35.8%)

Unilateral 167 (77.7%) 410 (36.7%) 634 (67.7%) Bilateral 48 (22.3%) 707 (63.3%) 302 (32.3%)

None 161 (74.5%) 847 (77.6%) 814 (74.9%) 1 38 (17.6%) 181 (16.6%) 170 (15.6%) 2 or more 17 (7.9%) 63 (5.8%) 103 (9.5%)

**Age at diagnosis\*** 57 (45, 72) 62 (52, 72) 63 (51, 75) <0.001 Race/Ethnicity <0.001

**FIGO Stage** <0.001

**Grade** <0.001

**Laterality** <0.001

**Comorbidity** 0.0257

platinum agent and paclitaxel.

**Characteristic**

#### **2.3. Analyses**

Statistical testing was performed using the likelihood ratio chi-square test for discrete varia‐ bles. The Kruskal-Wallis test was used to test for differences among continuous variables. A generalized logits model was fit to determine the characteristics associated with epithelial subtype. Variables included in the model were age, race/ethnicity, stage, grade, and laterali‐ ty. Age was transformed in all models using restricted cubic spline functions to allow for nonlinearity [25]. Due to the lack of availability of grade and stage information for some cas‐ es (31% for grade; 15% for stage), missing indicator variables were included for each varia‐ ble in all models. Because of potential issues with using missing indicator variables, separate models that imputed missing data were fit (data not shown) [26,27]. These models yielded consistent results with the un-imputed models. Six-year survival curves are presented as Kaplan-Meier estimates. Statistical testing for differences in unadjusted survival rates across epithelial subtypes was performed using the log-rank test. For adjusted survival, a time-de‐ pendent Cox model was used to determine the predictors of six-year survival. Age, race/ ethnicity, stage, grade, epithelial subtype, comorbidity, laterality, surgery, and chemothera‐ py were included as covariates in the survival model. Time-dependent covariates for sur‐ gery and chemotherapy were used to prevent an artificial inflation of the association between treatment and survival. Cases were considered as not receiving treatment until the date of the procedure; they were considered as having received treatment after the date of the procedure. Interactions between epithelial subtype and treatment were included to de‐ termine if the effects of surgery and chemotherapy varied across subtypes. The proportional hazards assumption was assessed using time-dependent covariates and the Schoenfeld re‐ sidual correlation test. Laterality was found to violate the proportional hazards (PH) as‐ sumption. Stratified log[-log S(t)] plots were used to help determine time intervals within which the PH assumption held. An interaction between laterality and time was included in the final model to satisfy the PH assumption.

#### **3. Results**

#### **3.1. Demographic and clinical characteristics of mucinous epithelial ovarian cancer**

The characteristics of ovarian cancer cases in New York and Northern California are pre‐ sented by epithelial subtype in Table 2. Overall, 230 (8.7%) tumors were mEOC, 1195 (45.3%) tumors were serous EOC, and 1211 (45.9%) were other EOC. mEOCs were diagnosed at younger ages (57 years) compared to other subtypes (62 years for serous, 63 years for other EOCs). Relatively higher percentages of mEOCs were found among black non-Hispanic (8.0% vs. 5.9 and 6.7%) and Asian non-Hispanic (14.2% vs. 5.4 and 9.8%) populations com‐ pared to serous and other EOCs. Lower percentages of mEOCs (5.8%) were found among Hispanics compared to serous and mEOCs (7.5 and 7.7%). mEOCS were more likely to be diagnosed at FIGO stage I (45.2%) compared to serous (10.0%) and other mEOCs (24.9%). Higher percentages of low grade tumors and unilateral ovarian involvement at diagnosis were also present with mEOCs compared to other EOC types. A little under half of mEOC patients (46.3%) were treated with surgery only and 39.0% were treated with surgery plus a platinum agent and paclitaxel.

binations of surgery and chemotherapy. In descriptive analyses, chemotherapy was further categorized by receipt of specific agents. These categories consisted of surgery and platinum agent (cisplatin or carboplatin) receipt, surgery and platinum agent and paclitaxel receipt (standard treatment for EOC) [24], and surgery and any chemotherapy agent or combination

Statistical testing was performed using the likelihood ratio chi-square test for discrete varia‐ bles. The Kruskal-Wallis test was used to test for differences among continuous variables. A generalized logits model was fit to determine the characteristics associated with epithelial subtype. Variables included in the model were age, race/ethnicity, stage, grade, and laterali‐ ty. Age was transformed in all models using restricted cubic spline functions to allow for nonlinearity [25]. Due to the lack of availability of grade and stage information for some cas‐ es (31% for grade; 15% for stage), missing indicator variables were included for each varia‐ ble in all models. Because of potential issues with using missing indicator variables, separate models that imputed missing data were fit (data not shown) [26,27]. These models yielded consistent results with the un-imputed models. Six-year survival curves are presented as Kaplan-Meier estimates. Statistical testing for differences in unadjusted survival rates across epithelial subtypes was performed using the log-rank test. For adjusted survival, a time-de‐ pendent Cox model was used to determine the predictors of six-year survival. Age, race/ ethnicity, stage, grade, epithelial subtype, comorbidity, laterality, surgery, and chemothera‐ py were included as covariates in the survival model. Time-dependent covariates for sur‐ gery and chemotherapy were used to prevent an artificial inflation of the association between treatment and survival. Cases were considered as not receiving treatment until the date of the procedure; they were considered as having received treatment after the date of the procedure. Interactions between epithelial subtype and treatment were included to de‐ termine if the effects of surgery and chemotherapy varied across subtypes. The proportional hazards assumption was assessed using time-dependent covariates and the Schoenfeld re‐ sidual correlation test. Laterality was found to violate the proportional hazards (PH) as‐ sumption. Stratified log[-log S(t)] plots were used to help determine time intervals within which the PH assumption held. An interaction between laterality and time was included in

**3.1. Demographic and clinical characteristics of mucinous epithelial ovarian cancer**

The characteristics of ovarian cancer cases in New York and Northern California are pre‐ sented by epithelial subtype in Table 2. Overall, 230 (8.7%) tumors were mEOC, 1195 (45.3%) tumors were serous EOC, and 1211 (45.9%) were other EOC. mEOCs were diagnosed at younger ages (57 years) compared to other subtypes (62 years for serous, 63 years for other EOCs). Relatively higher percentages of mEOCs were found among black non-Hispanic

of agents other than cisplatin, carboplatin, or paclitaxel.

120 Ovarian Cancer - A Clinical and Translational Update

the final model to satisfy the PH assumption.

**2.3. Analyses**

**3. Results**



compared to serous tumors (odds ratio [OR] 1.94, 95% confidence interval [CI] 1.13-3.35). The relationship between age and epithelial subtype was nonlinear; ages 55 years and younger were more often associated with mEOC compared to both serous and other EOCs (Figures 1 and 2). Less advanced stage was associated with mEOCs compared to serous EOC (OR 0.29, 95% CI 0.18-0.47 for stage III and 0.39, 0.19-0.78 for stage IV). mEOCs were less likely to be grade III/IV compared to serous (OR 0.11, 95% CI 0.07-0.20) and other EOC (OR 0.10, 95% CI 0.06-0.16). Bilateral ovarian cancer at diagnosis was less often associated

> Mucinous vs. Serous Odds Ratio 95% CI

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with mEOCs compared to serous EOC (OR = 0.32, 95% CI 0.22-0.49).

**Age at diagnosis\*** 0.0008 Nonlinear Nonlinear

White non-Hispanic 1.00 1.00 Black non-Hispanic 1.36 (0.73-2.54) 1.52 (0.82-2.80) Asian non-Hispanic 1.94 (1.13-3.35) 1.17 (0.72-1.90) Hispanic 0.77 (0.40-1.50) 0.76 (0.40-1.44)

I 1.00 1.00 II 0.89 (0.50-1.60) 0.96 (0.57-1.63) III 0.29 (0.18-0.47) 1.10 (0.70-1.72) IV 0.39 (0.19-0.78) 0.68 (0.35-1.32) Unknown 0.41 (0.21-0.83) 0.80 (0.41-1.57)

I 1.00 1.00 II 0.62 (0.38-1.00) 0.49 (0.31-0.77) III/IV 0.11 (0.07-0.20) 0.10 (0.06-0.16) Unknown 0.92 (0.55-1.55) 0.31 (0.19-0.49)

Unilateral 1.00 1.00 Bilateral 0.32 (0.22-0.49) 0.80 (0.53-1.21)

**Table 3.** Adjusted odds ratios and 95% confidence intervals for demographic and clinical characteristics of invasive epithelial ovarian cases by subtype, New York and Northern California. \*The relationship between age and histologic

**Characteristic P-value**

Nonlinear 0.0002 **Race/Ethnicity** 0.0723

**FIGO Stage** <.0001

**Grade** <.0001

**Laterality** <.0001

subtype is shown in Figures 1 and 2.CI=confidence interval

**Table 2.** Demographic and clinical characteristics of invasive epithelial ovarian cancer cases by subtype, New York and Northern California. \* Continuous variable presented as median (25th percentile, 75th percentile).

**Figure 1.** Adjusted relationship between age and risk of mucinous epithelial compared to serous epithelial ovarian cancer. Solid line indicates log-odds ratio, dotted lines indicated confidence intervals.

Table 3, Figure 1 and Figure 2 show the demographic and clinical characteristics significant‐ ly associated with mEOCs compared to other epithelial ovarian cancers, after adjusting for other factors. mEOCs were more often associated with Asian non-Hispanic race/ethnicity compared to serous tumors (odds ratio [OR] 1.94, 95% confidence interval [CI] 1.13-3.35). The relationship between age and epithelial subtype was nonlinear; ages 55 years and younger were more often associated with mEOC compared to both serous and other EOCs (Figures 1 and 2). Less advanced stage was associated with mEOCs compared to serous EOC (OR 0.29, 95% CI 0.18-0.47 for stage III and 0.39, 0.19-0.78 for stage IV). mEOCs were less likely to be grade III/IV compared to serous (OR 0.11, 95% CI 0.07-0.20) and other EOC (OR 0.10, 95% CI 0.06-0.16). Bilateral ovarian cancer at diagnosis was less often associated with mEOCs compared to serous EOC (OR = 0.32, 95% CI 0.22-0.49).

**Characteristic**

122 Ovarian Cancer - A Clinical and Translational Update

Mucinous (n=230)

Surgery only 95 (46.3%) 132 (12.2%) 196 (17.9%) Surgery+Platinum 7 (3.4%) 46 (4.3%) 28 (2.6%) Surgery+Platinum+ Paclitaxel 80 (39.0%) 826 (76.5%) 565 (51.6%) Surgery+other chemotherapy 2 (1.0%) 21 (1.9%) 12 (1.1%) Chemotherapy only 12 (5.9%) 32 (3.0%) 169 (15.4%) No surgery/no chemotherapy 9 (4.4%) 23 (2.1%) 124 (11.3%)

Northern California. \* Continuous variable presented as median (25th percentile, 75th percentile).

**Treatment** <0.001

**Table 2.** Demographic and clinical characteristics of invasive epithelial ovarian cancer cases by subtype, New York and

**Figure 1.** Adjusted relationship between age and risk of mucinous epithelial compared to serous epithelial ovarian

Table 3, Figure 1 and Figure 2 show the demographic and clinical characteristics significant‐ ly associated with mEOCs compared to other epithelial ovarian cancers, after adjusting for other factors. mEOCs were more often associated with Asian non-Hispanic race/ethnicity

cancer. Solid line indicates log-odds ratio, dotted lines indicated confidence intervals.

Serous (n=1195) Other Epithelial (n=1211)

**P-value**


**Table 3.** Adjusted odds ratios and 95% confidence intervals for demographic and clinical characteristics of invasive epithelial ovarian cases by subtype, New York and Northern California. \*The relationship between age and histologic subtype is shown in Figures 1 and 2.CI=confidence interval

associations occurred among those who received both surgery and chemotherapy; women with serous EOC and other EOC had better survival than those with mEOC in this group (serous HR 0.45, 05% CI 0.33-0.62; other EOC HR 0.44, 95% CI 0.32-0.61). This was also the case for women who were treated with chemotherapy alone (serous EOC HR 0.16, 95% CI 0.07-0.38; other EOC HR 0.40, 95% CI 0.20-0.81). In women who received only surgery or did not receive treatment, those with serous EOC had better survival than those with mEOC

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**Figure 3.** Six-year survival following an invasive epithelial ovarian cancer diagnosis by subtype, New York and North‐

Hazard Ratio (95% CI)

**Characteristic Wald χ<sup>2</sup> DF\* P-value**

**Age at diagnosis\*** 99.68 2 <0.0001 Nonlinear 10.75 1 0.0010 **Race/Ethnicity** 26.63 3 <0.0001

White non-Hispanic 1.00

Black non-Hispanic 1.47 (1.17-1.86)

ern California

(HR 0.65, 95% CI 0.42-1.00, HR 0.23, 95% CI 0.10-0.53, respectively).

**Figure 2.** Adjusted relationship between age and risk of and mucinous epithelial ovarian cancer compared to other epithelial ovarian cancer. Solid line indicates log-odds ratio, dotted lines indicated confidence intervals.

#### **3.2. Survival following a mucinous ovarian cancer diagnosis**

Unadjusted Kaplan-Meier estimates showed that survival following an epithelial ovarian cancer diagnosis was initially worse for mEOC and other EOC compared to serous EOC (Figure 3). At approximately 38-40 months post-diagnosis, mEOC and other EOC tumor sur‐ vival rates stabilized, whereas survival from serous EOC continually decreased. At the end of the 6 year follow-up period, survival was significantly different among the epithelial sub‐ types (log rank p<0.001). Unadjusted survival was 49.8% among women with mEOC, 39.0% among women with other EOC, and 30.8% among women serous EOC.

The results of the multivariable Cox model predicting 6-year survival are shown in Table 4 and Figure 4. After adjustment, black race, advanced stage, higher grade, and the presence of comorbidities were all associated with increased mortality from EOC (Table 4), as was in‐ creasing age (especially age > 60, Figure 4). By epithelial subtype, mEOC conferred a worse prognosis and was associated with increased mortality compared to both serous EOC (Haz‐ ard ratio [HR] 0.51, 95% CI 0.40-0.65), and other EOC (HR 0.56, 95% CI 0.44-0.72). Significant interactions were found between epithelial subtype and both surgery (p=0.0064) and chemo‐ therapy (p=0.0340). In all cases, mEOC was associated with increased mortality. Significant associations occurred among those who received both surgery and chemotherapy; women with serous EOC and other EOC had better survival than those with mEOC in this group (serous HR 0.45, 05% CI 0.33-0.62; other EOC HR 0.44, 95% CI 0.32-0.61). This was also the case for women who were treated with chemotherapy alone (serous EOC HR 0.16, 95% CI 0.07-0.38; other EOC HR 0.40, 95% CI 0.20-0.81). In women who received only surgery or did not receive treatment, those with serous EOC had better survival than those with mEOC (HR 0.65, 95% CI 0.42-1.00, HR 0.23, 95% CI 0.10-0.53, respectively).

**Figure 2.** Adjusted relationship between age and risk of and mucinous epithelial ovarian cancer compared to other

Unadjusted Kaplan-Meier estimates showed that survival following an epithelial ovarian cancer diagnosis was initially worse for mEOC and other EOC compared to serous EOC (Figure 3). At approximately 38-40 months post-diagnosis, mEOC and other EOC tumor sur‐ vival rates stabilized, whereas survival from serous EOC continually decreased. At the end of the 6 year follow-up period, survival was significantly different among the epithelial sub‐ types (log rank p<0.001). Unadjusted survival was 49.8% among women with mEOC, 39.0%

The results of the multivariable Cox model predicting 6-year survival are shown in Table 4 and Figure 4. After adjustment, black race, advanced stage, higher grade, and the presence of comorbidities were all associated with increased mortality from EOC (Table 4), as was in‐ creasing age (especially age > 60, Figure 4). By epithelial subtype, mEOC conferred a worse prognosis and was associated with increased mortality compared to both serous EOC (Haz‐ ard ratio [HR] 0.51, 95% CI 0.40-0.65), and other EOC (HR 0.56, 95% CI 0.44-0.72). Significant interactions were found between epithelial subtype and both surgery (p=0.0064) and chemo‐ therapy (p=0.0340). In all cases, mEOC was associated with increased mortality. Significant

epithelial ovarian cancer. Solid line indicates log-odds ratio, dotted lines indicated confidence intervals.

**3.2. Survival following a mucinous ovarian cancer diagnosis**

124 Ovarian Cancer - A Clinical and Translational Update

among women with other EOC, and 30.8% among women serous EOC.

**Figure 3.** Six-year survival following an invasive epithelial ovarian cancer diagnosis by subtype, New York and North‐ ern California



**Characteristic Wald χ<sup>2</sup> DF\* P-value**

**Surgery/No chemotherapy**

**Chemotherapy/No Surgery**

**No Surgery/ No Chemotherapy**

**Chemotherapy (Yes vs. No) 11.05 3 0.0115**

interactions.

Mucinous 1.0

Mucinous 1.0

Mucinous 1.0

Mucinous 1.0

**Surgery (Yes vs. No)** 34.93 3 <0.0001

Serous 0.23 (0.10-0.53) Other Epithelial 0.75 (0.38-1.49)

Mucinous 0.38 (0.20-0.75) Serous 1.06 (0.63-1.79) Other Epithelial 0.42 (0.31-0.58)

Mucinous 1.25 (0.79-1.97) Serous 0.88 (0.68-1.14) Other Epithelial 0.67 (0.51-0.87)

**Table 4.** Multivariate proportional hazards results of invasive epithelial ovarian cancer cases, New York and Northern California. \*The relationship between age and risk of death is shown in Figure 4. \*\*The overall epithelial subtype comparisons are from a model excluding the subtype and treatment interactions. These are presented to show the "average" effect across treatments. All other hazard ratios in the model are calculated from the model including the

Serous 0.16 (0.07-0.38) Other Epithelial 0.40 (0.20-0.81)

Serous 0.65 (0.42-1.00) Other Epithelial 0.83 (0.55-1.26)

Serous 0.45 (0.33-0.62) Other Epithelial 0.44 (0.32-0.61)

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**Characteristic Wald χ<sup>2</sup> DF\* P-value**

126 Ovarian Cancer - A Clinical and Translational Update

**FIGO Stage** 239.24 4 <0.0001

**Grade** 12.83 3 0.0050

**Laterality** 38.41 2 <0.0001

**Comorbidity** 20.04 2 <0.0001

**Epithelial subtype\*\*** 48.68 6 <0.0001

0 – 2 years

"/>2 years – 6 years

**Surgery+Chemotherapy**

Asian non-Hispanic 0.62 (0.45-0.84) Hispanic 0.76 (0.59-0.98)

I 1.00

I 1.00

II 1.36 (1.01-1.84) III/IV 1.50 (1.13-2.00) Unknown 1.69 (1.25-2.29)

Bilateral vs. Unilateral 1.04 (0.87-1.23)

Bilateral vs. Unilateral 1.86 (1.53-2.27)

1 1.26 (1.08-1.48) 2 or more 1.59 (1.26-2.01)

0 1.00

Mucinous 1.0

Serous 0.51 (0.40-0.65) Other Epithelial 0.56 (0.44-0.72)

II 1.40 (0.96- 2.04) III 4.96 (3.77- 6.51) IV 8.02 (5.93-10.84) Unknown 5.36 (3.93- 7.31)

Hazard Ratio (95% CI)

> **Table 4.** Multivariate proportional hazards results of invasive epithelial ovarian cancer cases, New York and Northern California. \*The relationship between age and risk of death is shown in Figure 4. \*\*The overall epithelial subtype comparisons are from a model excluding the subtype and treatment interactions. These are presented to show the "average" effect across treatments. All other hazard ratios in the model are calculated from the model including the interactions.

#### **4. Discussion**

This large, population-based study adds further, definitive evidence for demographic and clinical characteristics previously associated with mEOC: Asian race, early stage, low grade, and unilateral ovarian involvement at diagnosis [9,16,28]. Regardless of the large proportion of stage I diagnoses (about 45%), mEOC appears to be a particularly deadly subtype of ovar‐ ian cancer. These patterns seen in mEOC are consistent with clear cell EOC, which also tends to be diagnosed at early stages [29], and has poor overall survival compared to other EOCs [29,30]. Patterns of other epithelial subtypes vary: endometrioid EOC is often diag‐ nosed at early stages, but generally has better overall survival compared to other EOCs [31]; serous EOC is most often diagnosed at late stages (stage III and IV), and survival from these tumors appears to be significantly associated with grade [32]. These divergent patterns sug‐ gest that EOC is an extremely heterogeneous group, and histologic subtype should be con‐ sidered in addition to stage before and during treatment.

tent with these findings, in that chemotherapy did not appear to have a beneficial effect in women with mEOC. Evidence suggests that mEOC response rates to platinum-based chemotherapy are low overall (13-26%) [14,34]. This decreased response could be related to a lack of sensitivity of mEOCs to standard platinum-containing chemotherapy regi‐ mens [17]. It is well-established that platinum sensitivity varies by pathologic and clini‐ cal characteristics including tumor type [34,35]. Relatively recently, some groups in the United States have suggested that different treatment strategies should be considered for mEOC, and that future clinical trials should be redesigned to 1) exclude women with mEOC and other rare EOC types [36], and 2) assist with the development of novel agents more targeted to mEOC that can be used in the front-line and recurrent settings [17, 37]. Several barriers exist to such clinical trials, including decreased availability of funding [38], as well as potential lack of enrollment and participation due to the rarity and deadly nature of mEOC. Despite these limitations however, a phase III clinical trial comparing standard carboplatin and paclitaxel regimen (with and without bevacizumab) to oxalitlatin and capecitabine (with and without bevacizumab) in women with stages II-

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IV or recurrent untreated stage I primary mEOC was recently announced [39].

to substantiate the usefulness of this approach.

Some groups have suggested additional chemotherapeutic agents that may be more effec‐ tive in the treatment of mEOC. Based on studies with mEOC cell lines, combination chemo‐ therapy consisting of oxaliplatin and 5-fluorouracil may be beneficial for mEOCs [40]. The suggested use of fluorouracil, a chemotherapy agent used in the treatment of colon cancer [41], has gained additional support because of the similarities between mEOC and mucinous tumors of the colon [16,42]. A recent review comparing characteristics of these two tumor types concluded that there are multiple similarities with respect to mutational patterns, clin‐ ical presentation, therapy response, and outcomes [43]. The review further proposes that the search for new and more effective chemotherapeutic agents for mucinous tumors might be more successful if comparisons are made across organs [43]. However, there are clear differ‐ ences in the cellular localization of mucin in these two types, and further research is needed

Regardless of the availability of and evidence for alternative treatment regimens, the results presented here underscore the need for precise pathologic assessment of all EOCs with re‐ spect to site of tumor origin, histologic type and subtype, behavior and grade. The vast ma‐ jority of histologic-specific analyses using medical record data (including this one) are limited by the fact that there is no central pathology review of included cases. Because of this, a few studies have retrospectively reviewed stored specimens and medical records to examine concordance of pathologic characteristics of ovarian cancer. In a population-based study using Surveillance Epidemiology and End Results (SEER) data, there was 98% con‐ cordance on site of origin, and 97% concordance on overall epithelial histologic type [44]. Concordance varied by histologic subtype; it was 100% for clear cell EOC, 87% for mEOC, 80% for serous EOC, and 73% for endometrioid EOC. For tumor behavior, there was 85% concordance for invasive ovarian tumors. In most cases (90%), tumors originally diagnosed as invasive were thought to be low malignant potential upon review. Another study exam‐ ining pathology in the Gilda Radner Familial Ovarian Cancer Tumor Registry reported

**Figure 4.** Adjusted relationship between age and risk of death within six years following an epithelial ovarian cancer diagnosis. Solid line indicates log-hazard ratio, dotted lines indicate 95% confidence intervals.

The poor survival from mEOC shown here is consistent with other studies [14,16,33] and may be due to a decreased response to chemotherapy. Our study results are consis‐ tent with these findings, in that chemotherapy did not appear to have a beneficial effect in women with mEOC. Evidence suggests that mEOC response rates to platinum-based chemotherapy are low overall (13-26%) [14,34]. This decreased response could be related to a lack of sensitivity of mEOCs to standard platinum-containing chemotherapy regi‐ mens [17]. It is well-established that platinum sensitivity varies by pathologic and clini‐ cal characteristics including tumor type [34,35]. Relatively recently, some groups in the United States have suggested that different treatment strategies should be considered for mEOC, and that future clinical trials should be redesigned to 1) exclude women with mEOC and other rare EOC types [36], and 2) assist with the development of novel agents more targeted to mEOC that can be used in the front-line and recurrent settings [17, 37]. Several barriers exist to such clinical trials, including decreased availability of funding [38], as well as potential lack of enrollment and participation due to the rarity and deadly nature of mEOC. Despite these limitations however, a phase III clinical trial comparing standard carboplatin and paclitaxel regimen (with and without bevacizumab) to oxalitlatin and capecitabine (with and without bevacizumab) in women with stages II-IV or recurrent untreated stage I primary mEOC was recently announced [39].

**4. Discussion**

128 Ovarian Cancer - A Clinical and Translational Update

This large, population-based study adds further, definitive evidence for demographic and clinical characteristics previously associated with mEOC: Asian race, early stage, low grade, and unilateral ovarian involvement at diagnosis [9,16,28]. Regardless of the large proportion of stage I diagnoses (about 45%), mEOC appears to be a particularly deadly subtype of ovar‐ ian cancer. These patterns seen in mEOC are consistent with clear cell EOC, which also tends to be diagnosed at early stages [29], and has poor overall survival compared to other EOCs [29,30]. Patterns of other epithelial subtypes vary: endometrioid EOC is often diag‐ nosed at early stages, but generally has better overall survival compared to other EOCs [31]; serous EOC is most often diagnosed at late stages (stage III and IV), and survival from these tumors appears to be significantly associated with grade [32]. These divergent patterns sug‐ gest that EOC is an extremely heterogeneous group, and histologic subtype should be con‐

**Figure 4.** Adjusted relationship between age and risk of death within six years following an epithelial ovarian cancer

The poor survival from mEOC shown here is consistent with other studies [14,16,33] and may be due to a decreased response to chemotherapy. Our study results are consis‐

diagnosis. Solid line indicates log-hazard ratio, dotted lines indicate 95% confidence intervals.

sidered in addition to stage before and during treatment.

Some groups have suggested additional chemotherapeutic agents that may be more effec‐ tive in the treatment of mEOC. Based on studies with mEOC cell lines, combination chemo‐ therapy consisting of oxaliplatin and 5-fluorouracil may be beneficial for mEOCs [40]. The suggested use of fluorouracil, a chemotherapy agent used in the treatment of colon cancer [41], has gained additional support because of the similarities between mEOC and mucinous tumors of the colon [16,42]. A recent review comparing characteristics of these two tumor types concluded that there are multiple similarities with respect to mutational patterns, clin‐ ical presentation, therapy response, and outcomes [43]. The review further proposes that the search for new and more effective chemotherapeutic agents for mucinous tumors might be more successful if comparisons are made across organs [43]. However, there are clear differ‐ ences in the cellular localization of mucin in these two types, and further research is needed to substantiate the usefulness of this approach.

Regardless of the availability of and evidence for alternative treatment regimens, the results presented here underscore the need for precise pathologic assessment of all EOCs with re‐ spect to site of tumor origin, histologic type and subtype, behavior and grade. The vast ma‐ jority of histologic-specific analyses using medical record data (including this one) are limited by the fact that there is no central pathology review of included cases. Because of this, a few studies have retrospectively reviewed stored specimens and medical records to examine concordance of pathologic characteristics of ovarian cancer. In a population-based study using Surveillance Epidemiology and End Results (SEER) data, there was 98% con‐ cordance on site of origin, and 97% concordance on overall epithelial histologic type [44]. Concordance varied by histologic subtype; it was 100% for clear cell EOC, 87% for mEOC, 80% for serous EOC, and 73% for endometrioid EOC. For tumor behavior, there was 85% concordance for invasive ovarian tumors. In most cases (90%), tumors originally diagnosed as invasive were thought to be low malignant potential upon review. Another study exam‐ ining pathology in the Gilda Radner Familial Ovarian Cancer Tumor Registry reported 95.3% concordance on primary site [45]. The agreement by histologic subtype was lower, with disagreement on 38.3% of cases. The vast majority of differences were related to differ‐ ences in classification of serous EOC, either by the initial or reviewing pathologist. Concord‐ ance by grade was slightly better than that by histologic subtype, with disagreement on 31.2% of cases. The majority of differences centered on the differential assignment of grade II versus grade III. Few cases (a total of 7.6%) were upgraded or downgraded in a way that would have potential implications for treatment. While these pathologic review findings are encouraging overall and provide support for analyses of mEOC such as this one, they may not be exact enough to support the prescription of alternative treatment regimens based solely on histologic subtype.

those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention, the State of California Department of Public Health, and

Demographic and Clinical Characteristics of Mucinous Epithelial Ovarian Cancer, and…

, Trevor D. Thompson1

1 Division of Cancer Prevention and Control, Centers for Disease Control and Prevention,

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[2] Stewart SL, Rim SH, Richards TB. Gynecologic oncologists and ovarian cancer treat‐ ment: avenues for improved survival. J Womens Health (Larchmt) 2011; 20(9):

[3] National Comprehensive Cancer Network Guidelines. Epithelial Ovarian Cancer (in‐ cluding Fallopian Tube Cancer and Primary Peritoneal Cancer). www.nccn.com/

[4] National Cancer Institute. Ovarian Epithelial Cancer Treatment (PDQ®). http:// www.cancer.gov/cancertopics/pdq/treatment/ovarianepithelial/HealthProfessional/

files/cancer-guidelines/ovarian/index.html (accessed Sept 5, 2012).

[5] Scully RE. Ovarian tumors. Am. J of Pathol. 1977; 87(3): 686-720.

2 Centers for Disease Control and Prevention, NPCR-Contractor, Atlanta, GA, USA

3 Public Health Institute, Sacramento, CA and University of California, Davis, USA

and Maria J. Schymura5

, Rosemary D. Cress3

,

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the New York State Department of Health.

, Jennifer M. Wike2

, Cynthia D. O'Malley4

\*Address all correspondence to: Sstewart2@cdc.gov

4 Amgen, Inc, South San Francisco, CA, USA

5 New York State Registry, Albany, NY, USA

rent-and-comprehensive-statistics-

page1 (accessed Sept 5, 2012)

**Author details**

Sherri L. Stewart1

Atlanta, GA, USA

**References**

1257-60.

Amy R. Kahn5

#### **5. Conclusions**

The results presented here provide definitive evidence that mEOC is associated with differ‐ ent demographic and clinical characteristics than other EOC subtypes, and women diag‐ nosed with mEOC have worse adjusted survival compared to those with other EOC subtypes. A particular strength of this study is the population-based approach, which re‐ flects the experience of two U.S. populations of women with ovarian cancer, as opposed to that of a single institution or those participating in clinical research. This study yields sever‐ al implications for future research. First and foremost, the continued characterization of the heterogeneity of ovarian cancer through basic, clinical, and population research is necessary. Second, the need for precision in pathologic assessments is paramount, and pathologists, on‐ cologists and scientists all have a role in assisting with this through research and education. Finally, assessment of provider knowledge and awareness regarding treatment recommen‐ dations, and proposed or enacted changes to these recommendations, would be beneficial for ensuring appropriate use of evidence-based practices in the treatment of ovarian cancer.

#### **Acknowledgments**

This work was supported by cooperative agreements (U58/CCU920352 and U58/ CCU220322) with the Centers of Disease Control and Prevention. The collection of cancer in‐ cidence data used in this study was supported by the California Department of Public Health as part of the statewide cancer reporting program mandated by California Health and Safety Code Section 103885; the National Cancer Institute's Surveillance, Epidemiology and End Results Program under contract N01-PC-35136 awarded to the Northern California Cancer Center and contract N01-PC-45025 awarded to the Public Health Institute; the Cen‐ ters for Disease Control and Prevention's National Program of Cancer Registries, under agreement #U58/DP00080 awarded to the Public Health Institute, and agreement #U58/ DP000783 awarded to the New York State Department of Health; and the New York State Department of Health as part of the statewide cancer reporting mandate specified in New York State Public Health Law Section 2401. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention, the State of California Department of Public Health, and the New York State Department of Health.

#### **Author details**

95.3% concordance on primary site [45]. The agreement by histologic subtype was lower, with disagreement on 38.3% of cases. The vast majority of differences were related to differ‐ ences in classification of serous EOC, either by the initial or reviewing pathologist. Concord‐ ance by grade was slightly better than that by histologic subtype, with disagreement on 31.2% of cases. The majority of differences centered on the differential assignment of grade II versus grade III. Few cases (a total of 7.6%) were upgraded or downgraded in a way that would have potential implications for treatment. While these pathologic review findings are encouraging overall and provide support for analyses of mEOC such as this one, they may not be exact enough to support the prescription of alternative treatment regimens based

The results presented here provide definitive evidence that mEOC is associated with differ‐ ent demographic and clinical characteristics than other EOC subtypes, and women diag‐ nosed with mEOC have worse adjusted survival compared to those with other EOC subtypes. A particular strength of this study is the population-based approach, which re‐ flects the experience of two U.S. populations of women with ovarian cancer, as opposed to that of a single institution or those participating in clinical research. This study yields sever‐ al implications for future research. First and foremost, the continued characterization of the heterogeneity of ovarian cancer through basic, clinical, and population research is necessary. Second, the need for precision in pathologic assessments is paramount, and pathologists, on‐ cologists and scientists all have a role in assisting with this through research and education. Finally, assessment of provider knowledge and awareness regarding treatment recommen‐ dations, and proposed or enacted changes to these recommendations, would be beneficial for ensuring appropriate use of evidence-based practices in the treatment of ovarian cancer.

This work was supported by cooperative agreements (U58/CCU920352 and U58/ CCU220322) with the Centers of Disease Control and Prevention. The collection of cancer in‐ cidence data used in this study was supported by the California Department of Public Health as part of the statewide cancer reporting program mandated by California Health and Safety Code Section 103885; the National Cancer Institute's Surveillance, Epidemiology and End Results Program under contract N01-PC-35136 awarded to the Northern California Cancer Center and contract N01-PC-45025 awarded to the Public Health Institute; the Cen‐ ters for Disease Control and Prevention's National Program of Cancer Registries, under agreement #U58/DP00080 awarded to the Public Health Institute, and agreement #U58/ DP000783 awarded to the New York State Department of Health; and the New York State Department of Health as part of the statewide cancer reporting mandate specified in New York State Public Health Law Section 2401. The findings and conclusions in this report are

solely on histologic subtype.

130 Ovarian Cancer - A Clinical and Translational Update

**5. Conclusions**

**Acknowledgments**

Sherri L. Stewart1 , Jennifer M. Wike2 , Trevor D. Thompson1 , Rosemary D. Cress3 , Amy R. Kahn5 , Cynthia D. O'Malley4 and Maria J. Schymura5

\*Address all correspondence to: Sstewart2@cdc.gov

1 Division of Cancer Prevention and Control, Centers for Disease Control and Prevention, Atlanta, GA, USA

2 Centers for Disease Control and Prevention, NPCR-Contractor, Atlanta, GA, USA

3 Public Health Institute, Sacramento, CA and University of California, Davis, USA

4 Amgen, Inc, South San Francisco, CA, USA

5 New York State Registry, Albany, NY, USA

#### **References**


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[30] Pectasides D, Pectasides E, Psyrri A, et al. Treatment issues in clear cell carcinoma of

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[32] Diaz-Padilla I, Malpica AL, Minig L, et al. Ovarian low-grade serous carcinoma: a

[33] Bamias A, Psaltopoulou T, Sotiropoulou M, et al. Mucinous but not clear cell histolo‐ gy is associated with inferior survival in patients with advanced stage ovarian carci‐ noma treated with platinum-paclitaxel chemotherapy. Cancer. 2010 15;116(6):1462-8.

[34] Shimada M, Kigawa J, Ohishi Y, et al. Clinicopathological characteristics of mucinous

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[7] Soegaard M, Jensen A, Høgdall E, et al. Different risk factor profiles for mucinous and nonmucinous ovarian cancer: results from the Danish MALOVA study. Cancer

[8] Kurian AW, Balise RR, McGuire V, et al. Histologic types of epithelial ovarian cancer:

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[11] Risch HA, Marrett LD, Jain M, et al. Differences in risk factors for epithelial ovarian cancer by histologic type. Results of a case-control study. Am J Epidemiol.

[12] Gemignani ML, Schlaerth AC, Bogomolniy F, et al. Role of KRAS and BRAF gene mutations in mucinous ovarian carcinoma. Gynecol Oncol. 2003;90(2):378-81.

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[14] Pectasides D, Fountzilas G, Aravantinos G, et al. Advanced stage mucinous epithelial ovarian cancer: the Hellenic Cooperative Oncology Group experience. Gynecol On‐

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[37] Harrison ML, Jameson C, Gore ME. Mucinous ovarian cancer. Int J Gynecol Cancer 2008;18(2):209-14.

**Chapter 7**

**The Genetics of Ovarian Cancer**

Additional information is available at the end of the chapter

same time to provide the best possible advice to their relatives.

Health Care providers managing patients with Epithelial Ovarian Cancer (EOC) must be fa‐ miliar not only with the diagnosis, staging, treatment and follow-up of this disease, but also with the current knowledge on carcinogenesis, genetics and prevention/early diagnosis. This knowledge is needed in order to provide the best possible care to the patients and at the

For the general population of women, the lifetime risk of developing ovarian cancer is 1.4%, which means that a woman's average risk of developing ovarian cancer during her lifetime is about one in 70. The lifetime risk of dying from ovarian cancer is 1.04%. Ovarian Cancer can be called a rare disease but at the same time it is the ninth most common cancer in the USA, with an estimated 22,280 new cases in 2012, and the fifth most deadly, with an estimat‐ ed 15,500 deaths in 2012.The median age at diagnosis is 63 years. The poor ratio of survival to incidence in EOC results from the high percentage of cases diagnosed at an advanced stage. It is hard to find ovarian cancer early, as it may not cause any symptoms. When symptoms do appear, disease is often advanced and it is well known that the prognosis largely depends on its extent at diagnosis. Less than one-fourth of women present with lo‐ calized disease. Despite advances in surgery and chemotherapy, survival of patients with EOC stands at about 31-45% at 5 years. Despite the efficacy of platinum-based chemothera‐ py, over 75% of women with stage III/IV EOC ultimately relapse and die from their disease. Median survival for women whose disease does not respond or in whom duration of re‐ sponse is short is less than 12 months. Although new drugs hold the potential of improved responses in advanced and recurrent EOC, a greater impact could be made by recognition of high-risk patients and by offering the proper advice and risk-reducing surgery when indi‐ cated. It is important that health care professionals can recognize women with possible he‐ reditary Ovarian Cancer and have the basic knowledge to inform them of their management

> © 2013 Gennatas; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Constantine Gennatas

http://dx.doi.org/10.5772/54541

**1. Introduction**


#### **Chapter 7**

### **The Genetics of Ovarian Cancer**

#### Constantine Gennatas

[37] Harrison ML, Jameson C, Gore ME. Mucinous ovarian cancer. Int J Gynecol Cancer

[38] Kramer JM, Schulman KA. Transforming the Economics of Clinical Trials. A tale of two citations. Institute of Medicine; 2012. http://www.iom.edu/~/media/Files/ Perspectives-Files/2012/Discussion-Papers/HSP-Drugs-Transforming-the-Economics.

[39] Frumovitz M, Schmeler KM, Malpica A, et al. Unmasking the complexities of muci‐

[40] Sato S, Itamochi H, Kigawa J, et al. Combination chemotherapy of oxaliplatin and 5 fluorouracil may be an effective regimen for mucinous adenocarcinoma of the ovary:

[41] Andre T, de Gramont A, et al. An overview of adjuvant systemic chemotherapy for

[42] Zaino RJ, Brady MF, Lele SM, et al. Advanced stage mucinous adenocarcinoma of the ovary is both rare and highly lethal: a Gynecologic Oncology Group study. Cancer

[43] Kelemen LE, Köbel M. Mucinous carcinomas of the ovary and colorectum: different

[44] Tyler CW Jr, Lee NC, Robboy SJ, et al. The diagnosis of ovarian cancer by patholo‐ gists: how often do diagnoses by contributing pathologists agree with a panel of gy‐

[45] Piver MS, Tsukada Y, Werness BA, et al. Comparative study of ovarian cancer histo‐ pathology by registry pathologists and referral pathologists: a study by the Gilda

Radner Familial Ovarian Cancer Registry. Gynecol Oncol. 2000;78(2):166-70.

necologic pathologists? Am J Obstet Gynecol. 1991;164(1 Pt 1):65-70.

nous ovarian carcinoma. Gynecol Oncol. 2010;117(3):491-6.

a potential treatment strategy. Cancer Sci. 2009;100(3):546-51.

colon cancer. Clin Colorectal Cancer 2004; 4(Suppl 1):S22-8.

organ, same dilemma. Lancet Oncol. 2011;12(11):1071-80.

2008;18(2):209-14.

134 Ovarian Cancer - A Clinical and Translational Update

(accessed Sept 5, 2012)

2011;117(3):554-62

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54541

#### **1. Introduction**

Health Care providers managing patients with Epithelial Ovarian Cancer (EOC) must be fa‐ miliar not only with the diagnosis, staging, treatment and follow-up of this disease, but also with the current knowledge on carcinogenesis, genetics and prevention/early diagnosis. This knowledge is needed in order to provide the best possible care to the patients and at the same time to provide the best possible advice to their relatives.

For the general population of women, the lifetime risk of developing ovarian cancer is 1.4%, which means that a woman's average risk of developing ovarian cancer during her lifetime is about one in 70. The lifetime risk of dying from ovarian cancer is 1.04%. Ovarian Cancer can be called a rare disease but at the same time it is the ninth most common cancer in the USA, with an estimated 22,280 new cases in 2012, and the fifth most deadly, with an estimat‐ ed 15,500 deaths in 2012.The median age at diagnosis is 63 years. The poor ratio of survival to incidence in EOC results from the high percentage of cases diagnosed at an advanced stage. It is hard to find ovarian cancer early, as it may not cause any symptoms. When symptoms do appear, disease is often advanced and it is well known that the prognosis largely depends on its extent at diagnosis. Less than one-fourth of women present with lo‐ calized disease. Despite advances in surgery and chemotherapy, survival of patients with EOC stands at about 31-45% at 5 years. Despite the efficacy of platinum-based chemothera‐ py, over 75% of women with stage III/IV EOC ultimately relapse and die from their disease. Median survival for women whose disease does not respond or in whom duration of re‐ sponse is short is less than 12 months. Although new drugs hold the potential of improved responses in advanced and recurrent EOC, a greater impact could be made by recognition of high-risk patients and by offering the proper advice and risk-reducing surgery when indi‐ cated. It is important that health care professionals can recognize women with possible he‐ reditary Ovarian Cancer and have the basic knowledge to inform them of their management

© 2013 Gennatas; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

options. Details about the Genetics of Breast and Ovarian Cancer are available at the Nation‐ al Cancer Institute site and other relevant sites and publications [1-6].

how a cell functions, including how quickly it grows, how often it divides, and how long it lives. To control these functions, genes produce proteins that perform specific tasks and act as messengers for the cell. Therefore, it is essential that each gene have the correct instruc‐ tions or "code" for making its protein so that the protein can perform the proper function for

The Genetics of Ovarian Cancer http://dx.doi.org/10.5772/54541 137

Many cancers begin when one or more genes in a cell are mutated creating an abnormal pro‐ tein or no protein at all. The information provided by an abnormal protein is different from that of a normal protein, which can cause cells to multiply uncontrollably and become can‐ cerous. A person may either be born with the genetic mutation in all of their cells (germline mutation) or acquire a genetic mutation in a single cell during his or her lifetime. An ac‐ quired mutation is passed on to all cells that develop from that single cell (somatic muta‐ tion). A germline mutation or a hereditary mutation, according to the NCI definition, is a gene change in a body's reproductive cell that becomes incorporated into the DNA of every cell in the body of the offspring. Germline mutations are passed on from parents to off‐ spring. Somatic mutations, according to the NCI, are alterations in DNA that occur after conception. Somatic mutations can occur in any of the cells of the body except the germ cells and therefore are not passed on to children. These alterations can (but do not always) cause cancer or other diseases. If the mutant cell continues to divide, the individual will come to contain a patch of tissue of genotype different from the cells of the rest of the body. So this is a change in the genetic structure that is neither inherited nor passed to offspring. These changes can be caused by environmental factors such as ultraviolet radiation from the sun and cigarette smoke or can occur if a mistake is made as DNA copies itself during cell divi‐ sion. Mutations may also occur in a single cell within an early embryo. As all the cells divide during growth and development, the individual will have some cells with the mutation and some cells without the genetic change. This situation is called mosaicism. Some genetic changes are very rare; others are common in the population. Genetic changes that occur in more than 1 percent of the population are called polymorphisms. They are common enough to be considered a normal variation in the DNA. Polymorphisms are responsible for many of the normal differences between people such as eye color, hair color, and blood type. Al‐ though many polymorphisms have no negative effects on a person's health, some of these

variations may influence the risk of developing certain disorders [1-5, 9-17].

Most ovarian cancers (about 85% to 90%) are considered sporadic, meaning that the damage to the genes occurs by chance after a person is born and there is no risk of passing on the gene to a person's children. Inherited ovarian cancers are less common (about 10% to 15%) and occur when gene mutations are passed within a family, from one generation to the next. Every cell usually has two copies of each gene: one inherited from a person's mother and one inherited from a person's father. Most types of hereditary ovarian cancer follow an au‐ tosomal dominant inheritance pattern, in which a mutation needs to happen in only one copy of the gene for the person to have an increased risk of getting the disease. This means that a parent with a gene mutation may pass on a copy of the normal gene or a copy of the gene with a mutation. Therefore, a child who has a parent with a mutation has a 50% chance of inheriting that mutation. A brother, sister, or parent of a person who has a gene mutation

the cell [1-5].

#### **2. The pathogenesis of ovarian cancer – The role of genes**

The pathogenesis of ovarian carcinoma remains unclear and represents a fascinating re‐ search area. It is possible that several pathways lead to ovarian cancer. Certain theories have been proposed to explain its epidemiology including the theory of incessant ovulation, go‐ nadotropin stimulation, excess androgenic stimulation, and inflammation. Associated risk factors for ovarian cancer support some or all of these hypotheses. Multiparity, oral contra‐ ceptive use, and breastfeeding are associated with a decreased risk of ovarian cancer. Oophorectomy reduces but does not completely eliminate the risk of ovarian cancer. A his‐ tory of tubal ligation or hysterectomy with ovarian conservation is also associated with a de‐ creased risk of ovarian cancer. Risk is increased in women with a family history of ovarian cancer, with the postmenopausal use of hormone therapy, and among women who have used fertility drugs. Obesity, tall height, and high body mass index have also been associat‐ ed with increased risk of ovarian cancer. Perineal exposure to talcum powder has been in‐ vestigated as possible risk factor for ovarian cancer. It is very important to note that some women are at an increased risk due to an inherited susceptibility to ovarian cancer with the magnitude of that risk depending on the affected gene and specific mutation [1-5,7,8].

There is significant heterogeneity within the EOC group. Histologically defined subtypes such as serous, endometrioid, mucinous, and low- and high-grade malignancies all have variable clinical manifestations and underlying molecular signatures. Substantial advan‐ ces have been made in understanding the genetic alterations and biologic processes in ovarian cancer; however, the etiology remains poorly understood. According to a recent publication by S Vaughan et al the term ovarian cancer is misleading. Ovarian Cancer is not a single disease, and a considerable proportion of tumors do not arise from ovarian tissue. "The unifying clinical feature of all ovarian cancers is frequent loco-regional dis‐ semination to the ovary and related pelvic organs. We considered whether the term ovarian cancer should be replaced with the terms pelvic or peritoneal cancer but we rec‐ ognized the confusion that might ensue for patients and physicians, as well as in the sci‐ entific literature, especially during a transition period. Before the term ovarian cancer is abandoned, the disparate origins of this disease need to be more widely understood by patients, physicians and scientists."[1-5, 7, 8]

While approximately 90% of ovarian cancers occur sporadically, 10% of women with ovari‐ an cancer have inherited genetic changes that predisposed them to ovarian cancer. It is very important to identify these persons and properly manage them. The following information is very useful for the candidates of genetic testing: Genes carry information in the form of DNA within each cell of the human body. There are 30,000 different genes in each cell's chromosomes and there are 23 pairs of chromosomes in each cell. One chromosome of each pair is inherited from the person's father and one from the person's mother. Genes control how a cell functions, including how quickly it grows, how often it divides, and how long it lives. To control these functions, genes produce proteins that perform specific tasks and act as messengers for the cell. Therefore, it is essential that each gene have the correct instruc‐ tions or "code" for making its protein so that the protein can perform the proper function for the cell [1-5].

options. Details about the Genetics of Breast and Ovarian Cancer are available at the Nation‐

The pathogenesis of ovarian carcinoma remains unclear and represents a fascinating re‐ search area. It is possible that several pathways lead to ovarian cancer. Certain theories have been proposed to explain its epidemiology including the theory of incessant ovulation, go‐ nadotropin stimulation, excess androgenic stimulation, and inflammation. Associated risk factors for ovarian cancer support some or all of these hypotheses. Multiparity, oral contra‐ ceptive use, and breastfeeding are associated with a decreased risk of ovarian cancer. Oophorectomy reduces but does not completely eliminate the risk of ovarian cancer. A his‐ tory of tubal ligation or hysterectomy with ovarian conservation is also associated with a de‐ creased risk of ovarian cancer. Risk is increased in women with a family history of ovarian cancer, with the postmenopausal use of hormone therapy, and among women who have used fertility drugs. Obesity, tall height, and high body mass index have also been associat‐ ed with increased risk of ovarian cancer. Perineal exposure to talcum powder has been in‐ vestigated as possible risk factor for ovarian cancer. It is very important to note that some women are at an increased risk due to an inherited susceptibility to ovarian cancer with the magnitude of that risk depending on the affected gene and specific mutation [1-5,7,8].

There is significant heterogeneity within the EOC group. Histologically defined subtypes such as serous, endometrioid, mucinous, and low- and high-grade malignancies all have variable clinical manifestations and underlying molecular signatures. Substantial advan‐ ces have been made in understanding the genetic alterations and biologic processes in ovarian cancer; however, the etiology remains poorly understood. According to a recent publication by S Vaughan et al the term ovarian cancer is misleading. Ovarian Cancer is not a single disease, and a considerable proportion of tumors do not arise from ovarian tissue. "The unifying clinical feature of all ovarian cancers is frequent loco-regional dis‐ semination to the ovary and related pelvic organs. We considered whether the term ovarian cancer should be replaced with the terms pelvic or peritoneal cancer but we rec‐ ognized the confusion that might ensue for patients and physicians, as well as in the sci‐ entific literature, especially during a transition period. Before the term ovarian cancer is abandoned, the disparate origins of this disease need to be more widely understood by

While approximately 90% of ovarian cancers occur sporadically, 10% of women with ovari‐ an cancer have inherited genetic changes that predisposed them to ovarian cancer. It is very important to identify these persons and properly manage them. The following information is very useful for the candidates of genetic testing: Genes carry information in the form of DNA within each cell of the human body. There are 30,000 different genes in each cell's chromosomes and there are 23 pairs of chromosomes in each cell. One chromosome of each pair is inherited from the person's father and one from the person's mother. Genes control

patients, physicians and scientists."[1-5, 7, 8]

al Cancer Institute site and other relevant sites and publications [1-6].

136 Ovarian Cancer - A Clinical and Translational Update

**2. The pathogenesis of ovarian cancer – The role of genes**

Many cancers begin when one or more genes in a cell are mutated creating an abnormal pro‐ tein or no protein at all. The information provided by an abnormal protein is different from that of a normal protein, which can cause cells to multiply uncontrollably and become can‐ cerous. A person may either be born with the genetic mutation in all of their cells (germline mutation) or acquire a genetic mutation in a single cell during his or her lifetime. An ac‐ quired mutation is passed on to all cells that develop from that single cell (somatic muta‐ tion). A germline mutation or a hereditary mutation, according to the NCI definition, is a gene change in a body's reproductive cell that becomes incorporated into the DNA of every cell in the body of the offspring. Germline mutations are passed on from parents to off‐ spring. Somatic mutations, according to the NCI, are alterations in DNA that occur after conception. Somatic mutations can occur in any of the cells of the body except the germ cells and therefore are not passed on to children. These alterations can (but do not always) cause cancer or other diseases. If the mutant cell continues to divide, the individual will come to contain a patch of tissue of genotype different from the cells of the rest of the body. So this is a change in the genetic structure that is neither inherited nor passed to offspring. These changes can be caused by environmental factors such as ultraviolet radiation from the sun and cigarette smoke or can occur if a mistake is made as DNA copies itself during cell divi‐ sion. Mutations may also occur in a single cell within an early embryo. As all the cells divide during growth and development, the individual will have some cells with the mutation and some cells without the genetic change. This situation is called mosaicism. Some genetic changes are very rare; others are common in the population. Genetic changes that occur in more than 1 percent of the population are called polymorphisms. They are common enough to be considered a normal variation in the DNA. Polymorphisms are responsible for many of the normal differences between people such as eye color, hair color, and blood type. Al‐ though many polymorphisms have no negative effects on a person's health, some of these variations may influence the risk of developing certain disorders [1-5, 9-17].

Most ovarian cancers (about 85% to 90%) are considered sporadic, meaning that the damage to the genes occurs by chance after a person is born and there is no risk of passing on the gene to a person's children. Inherited ovarian cancers are less common (about 10% to 15%) and occur when gene mutations are passed within a family, from one generation to the next. Every cell usually has two copies of each gene: one inherited from a person's mother and one inherited from a person's father. Most types of hereditary ovarian cancer follow an au‐ tosomal dominant inheritance pattern, in which a mutation needs to happen in only one copy of the gene for the person to have an increased risk of getting the disease. This means that a parent with a gene mutation may pass on a copy of the normal gene or a copy of the gene with a mutation. Therefore, a child who has a parent with a mutation has a 50% chance of inheriting that mutation. A brother, sister, or parent of a person who has a gene mutation also has a 50% chance of having the same mutation. Autosomal dominant inheritance of breast/ovarian cancer is characterized by transmission of cancer predisposition from genera‐ tion to generation, through either the mother's or the father's side of the family, with an in‐ heritance risk of 50%. Although the risk of inheriting the predisposition is 50%, not everyone with the predisposition will develop cancer because of incomplete penetrance and/or gen‐ der-restricted or gender-related expression. Both males and females can inherit and transmit an autosomal dominant cancer predisposition. A male who inherits a cancer predisposition can still pass the altered gene on to his sons and daughters [1-5, 9-17].

for by inherited mutations in BRCA1 and BRCA2. Overall, it has been estimated that inherited BRCA1 and BRCA2 mutations account for 5 to 10 percent of breast cancers and 10 to 15 percent of ovarian cancers among white women in the United States. When examining consecutive series of patients with ovarian cancer who have been unselected for family history, approximately 10% to 15% of patients have a deleterious mutation in either of these genes. When studying patients with ovarian cancer who have a family history of ovarian cancer or early onset breast cancer, the likelihood of finding a BRCA1 or BRCA2 mutation rises considerably. In fact, it is generally stated that the majority of hereditary ovarian cancer is explained by BRCA1 or BRCA2 abnormalities. The Gyneco‐ logic Oncology Group conducted a prospective study of women with ovarian cancer and a positive family history. Specifically, they enrolled patients with ovarian cancer who had any of the following features: a first degree relative with ovarian cancer, a sec‐ ond-degree relative with ovarian cancer plus a first-degree relative with early-onset breast cancer (defined as younger than 50 years), or a first- and second-degree relative with early onset breast cancer. Of 26 eligible patients screened for mutations, 12 had deleterious alterations, eight in BRCA1 and four in BRCA2 [1-5, 7, 8]. Although repro‐ ductive, demographic, and lifestyle factors affect risk of ovarian cancer, the single most important ovarian cancer risk factor is a family history of the disease. A large metaanalysis of 15 published studies estimated an odds ratio of 3.1 for the risk of ovarian cancer associated with at least one first degree relative with ovarian cancer. The family characteristics that suggest hereditary breast and ovarian cancer predisposition include the following: 1) Multiple cancers within a family. 2) Cancers that are usually diagnosed at an earlier age than in sporadic. 3) History of two or more primary cancers in a single particular individual. The Claus and the Gail models are widely used in research stud‐ ies and clinical counseling. Both have limitations, and the risk estimates derived from the two models may differ for an individual patient. Several other models, which in‐ clude more detailed family history information, are also in use. The use of these models requires specific knowledge and expertise. 4) Cases of male breast cancer are definitely

The Genetics of Ovarian Cancer http://dx.doi.org/10.5772/54541 139

**2. Lynch syndrome or hereditary nonpolyposis colon cancer (HNPCC).** Lynch syndrome increases a woman's risk of ovarian cancer. It is caused by mutations in several differ‐ ent genes and it also increases the risk of colorectal cancer, as well as cancers of the stomach, small intestine, liver, bile duct, urinary tract, endometrium, the brain and cen‐ tral nervous system, and possibly breast cancer. Defects in mismatch repair in patients with Lynch syndrome account for approximately 10% of hereditary ovarian cancers and for 1% to 2% of overall cases. Patients with this syndrome, however, individually carry an approximately 12% risk of developing ovarian cancer. The mechanism of increased risk is through defects in the mismatch-repair machinery and its resulting genetic insta‐ bility that places cells at risk of multiple mutations; however, carcinogenesis in ovarian cancer has not been well studied beyond a description of mismatch repair defects.

Genetic conditions that are also associated with a small increased risk of ovarian cancer are

indications for genetic testing [1-5, 9-17].

the following:

#### **3. The BRCA1 and BRCA2 genes**

Breast and ovarian cancer are components of several autosomal syndromes but most strong‐ ly associated with both cancers are the BRCA1 or BRCA2 mutation syndromes, which ac‐ count for about 90% of hereditary cases. The BRCA1 gene is located on chromosome 17q21, while BRCA2 is located on chromosome 13q12. BRCA1 and BRCA2 play major roles in the repair of DNA doublestrand breaks by homologous recombination. Homologus recombina‐ tion repairs doublestrand breaks that occur in late S and G2 phase of the cell cycle and also has a key role in repairing doublestrand breaks that result from unrepaired single-strand break. BRCA1 signals the presence of doublestrand breaks, while BRCA2 is directly in‐ volved in the mechanism of homologous recombination. So the BRCA1 and BRCA2 proteins are considered caretakers of the genome, and play key roles in the signaling of DNA dam‐ age, the activation of DNA repair, the induction of apoptosis, and the monitoring of cell cy‐ cle checkpoints. Cells that lack functional BRCA have increased aneuploidy, centrosome amplification, and chromosomal aberrations, which make them susceptible to further muta‐ tions. BRCA appears to function as a cofactor for a variety of transcription factors, and the associated ovarian cancers are more likely to be high grade and of serous histopathology. In the absence of BRCA1 or BRCA2, alternative DNA repair pathways are used, which result in chromosomal instability and cell death. Normal cell of carriers are usually heterozygote with loss of the second allele occurring during tumorigenesis in the tumor cells of these women. [1-5, 7,8].

There are several genetic conditions linked to an increased risk of ovarian cancer involving mutations in several other genes, including TP53, PTEN, STK11/LKB1, CDH1, CHEK2, ATM, MLH1, and MSH2.Some of the most common hereditary cancer syndromes associated with ovarian cancer risk are the following:

**1. Hereditary breast and ovarian cancer syndrome.** This syndrome is associated with mu‐ tations in the BRCA1 or BRCA2 genes (BRCA stands for BReast CAncer) and it is relat‐ ed with an increased risk of breast cancer and ovarian cancer. There have also been reports of a small number of families with an excess of ovarian cancer, but no breast cancer, called site-specific ovarian cancer families. These families have been linked to mutations in BRCA1 and are thought to represent a unique phenotype of the hereditary breast-ovarian syndrome. The majority of hereditary breast cancers can be accounted for by inherited mutations in BRCA1 and BRCA2. Overall, it has been estimated that inherited BRCA1 and BRCA2 mutations account for 5 to 10 percent of breast cancers and 10 to 15 percent of ovarian cancers among white women in the United States. When examining consecutive series of patients with ovarian cancer who have been unselected for family history, approximately 10% to 15% of patients have a deleterious mutation in either of these genes. When studying patients with ovarian cancer who have a family history of ovarian cancer or early onset breast cancer, the likelihood of finding a BRCA1 or BRCA2 mutation rises considerably. In fact, it is generally stated that the majority of hereditary ovarian cancer is explained by BRCA1 or BRCA2 abnormalities. The Gyneco‐ logic Oncology Group conducted a prospective study of women with ovarian cancer and a positive family history. Specifically, they enrolled patients with ovarian cancer who had any of the following features: a first degree relative with ovarian cancer, a sec‐ ond-degree relative with ovarian cancer plus a first-degree relative with early-onset breast cancer (defined as younger than 50 years), or a first- and second-degree relative with early onset breast cancer. Of 26 eligible patients screened for mutations, 12 had deleterious alterations, eight in BRCA1 and four in BRCA2 [1-5, 7, 8]. Although repro‐ ductive, demographic, and lifestyle factors affect risk of ovarian cancer, the single most important ovarian cancer risk factor is a family history of the disease. A large metaanalysis of 15 published studies estimated an odds ratio of 3.1 for the risk of ovarian cancer associated with at least one first degree relative with ovarian cancer. The family characteristics that suggest hereditary breast and ovarian cancer predisposition include the following: 1) Multiple cancers within a family. 2) Cancers that are usually diagnosed at an earlier age than in sporadic. 3) History of two or more primary cancers in a single particular individual. The Claus and the Gail models are widely used in research stud‐ ies and clinical counseling. Both have limitations, and the risk estimates derived from the two models may differ for an individual patient. Several other models, which in‐ clude more detailed family history information, are also in use. The use of these models requires specific knowledge and expertise. 4) Cases of male breast cancer are definitely indications for genetic testing [1-5, 9-17].

also has a 50% chance of having the same mutation. Autosomal dominant inheritance of breast/ovarian cancer is characterized by transmission of cancer predisposition from genera‐ tion to generation, through either the mother's or the father's side of the family, with an in‐ heritance risk of 50%. Although the risk of inheriting the predisposition is 50%, not everyone with the predisposition will develop cancer because of incomplete penetrance and/or gen‐ der-restricted or gender-related expression. Both males and females can inherit and transmit an autosomal dominant cancer predisposition. A male who inherits a cancer predisposition

Breast and ovarian cancer are components of several autosomal syndromes but most strong‐ ly associated with both cancers are the BRCA1 or BRCA2 mutation syndromes, which ac‐ count for about 90% of hereditary cases. The BRCA1 gene is located on chromosome 17q21, while BRCA2 is located on chromosome 13q12. BRCA1 and BRCA2 play major roles in the repair of DNA doublestrand breaks by homologous recombination. Homologus recombina‐ tion repairs doublestrand breaks that occur in late S and G2 phase of the cell cycle and also has a key role in repairing doublestrand breaks that result from unrepaired single-strand break. BRCA1 signals the presence of doublestrand breaks, while BRCA2 is directly in‐ volved in the mechanism of homologous recombination. So the BRCA1 and BRCA2 proteins are considered caretakers of the genome, and play key roles in the signaling of DNA dam‐ age, the activation of DNA repair, the induction of apoptosis, and the monitoring of cell cy‐ cle checkpoints. Cells that lack functional BRCA have increased aneuploidy, centrosome amplification, and chromosomal aberrations, which make them susceptible to further muta‐ tions. BRCA appears to function as a cofactor for a variety of transcription factors, and the associated ovarian cancers are more likely to be high grade and of serous histopathology. In the absence of BRCA1 or BRCA2, alternative DNA repair pathways are used, which result in chromosomal instability and cell death. Normal cell of carriers are usually heterozygote with loss of the second allele occurring during tumorigenesis in the tumor cells of these

There are several genetic conditions linked to an increased risk of ovarian cancer involving mutations in several other genes, including TP53, PTEN, STK11/LKB1, CDH1, CHEK2, ATM, MLH1, and MSH2.Some of the most common hereditary cancer syndromes associated

**1. Hereditary breast and ovarian cancer syndrome.** This syndrome is associated with mu‐ tations in the BRCA1 or BRCA2 genes (BRCA stands for BReast CAncer) and it is relat‐ ed with an increased risk of breast cancer and ovarian cancer. There have also been reports of a small number of families with an excess of ovarian cancer, but no breast cancer, called site-specific ovarian cancer families. These families have been linked to mutations in BRCA1 and are thought to represent a unique phenotype of the hereditary breast-ovarian syndrome. The majority of hereditary breast cancers can be accounted

can still pass the altered gene on to his sons and daughters [1-5, 9-17].

**3. The BRCA1 and BRCA2 genes**

138 Ovarian Cancer - A Clinical and Translational Update

women. [1-5, 7,8].

with ovarian cancer risk are the following:

**2. Lynch syndrome or hereditary nonpolyposis colon cancer (HNPCC).** Lynch syndrome increases a woman's risk of ovarian cancer. It is caused by mutations in several differ‐ ent genes and it also increases the risk of colorectal cancer, as well as cancers of the stomach, small intestine, liver, bile duct, urinary tract, endometrium, the brain and cen‐ tral nervous system, and possibly breast cancer. Defects in mismatch repair in patients with Lynch syndrome account for approximately 10% of hereditary ovarian cancers and for 1% to 2% of overall cases. Patients with this syndrome, however, individually carry an approximately 12% risk of developing ovarian cancer. The mechanism of increased risk is through defects in the mismatch-repair machinery and its resulting genetic insta‐ bility that places cells at risk of multiple mutations; however, carcinogenesis in ovarian cancer has not been well studied beyond a description of mismatch repair defects.

Genetic conditions that are also associated with a small increased risk of ovarian cancer are the following:

**1. Peutz - Jeghers syndrome.** This syndrome is caused by a specific genetic mutation in the STK11 gene and is associated with multiple polyps in the digestive tract that be‐ come noncancerous tumors, increased pigmentation on the face and hands and with an increased risk of ovarian, breast, uterine, and lung cancers.

genetic changes alternate splicing, and other genetic factors may affect BRCA function in as

The Genetics of Ovarian Cancer http://dx.doi.org/10.5772/54541 141

An analysis of genomic changes in ovarian cancer has provided the most comprehen‐ sive and integrated view of cancer genes for any cancer type to date. Ovarian serous ad‐ enocarcinoma tumors from 500 patients were examined by The Cancer Genome Atlas (TCGA) Research Network and analyses were reported in 2011.These findings confirm that mutations in a single gene, TP53, are present in more than 96 percent of all such cancers. TP53 encodes a tumor suppressor protein that normally prevents cancer forma‐ tion. Mutations in the gene disrupt this protein's function, which contributes to uncon‐ trolled growth of ovarian cells. Several less-frequent mutations in other genes have also been identified and was also established how sets of genes are expressed in a fashion that can predict patient survival, identifying patterns for 108 genes associated with poor survival and 85 genes associated with better survival. Patients whose tumors had a gene-expression signature associated with poor survival lived for a period that was 23 percent shorter than patients whose tumors did not have such a signature. To identify opportunities for targeted treatment, the investigators searched for existing drugs that might inhibit amplified or over-expressed genes that were suggested to play a role in ovarian cancer. Sixty-eight genes have been identified that could be targeted by existing or experimental therapeutic compounds. One type of drug, a PARP (Poly ADP ribose polymerase) inhibitor, might be able to counteract the DNA repair gene observed in half of the ovarian tumors studied. These drugs could be effective against the disease, this study revealed that 50 percent of tumors might be responsive to drugs that exploit the genetic instability of the tumors and induce the cancer cells to die. The results of this study support the existence of four distinct subtypes of the disease, based on the pat‐ terns seen in the transcription of RNA from DNA. They also support the existence of four related subtypes based on the patterns of DNA methylation—a chemical reaction in which a small molecule called a methyl group is added to DNA, changing the activity of individual genes. These patterns likely reflect the functional changes associated with ovarian serous adenocarcinoma, but are not strongly associated with survival duration. In this study, approximately 21 percent of the tumors showed mutations in BRCA1 and BRCA2 genes. Analysis of these tumors confirmed observations that patients with mutat‐ ed BRCA1 and BRCA2 genes have better survival odds than patients without mutations in these genes. If either of the BRCA1 and BRCA2 genes is mutated, there is improved survival duration. However, if BRCA1 activity is instead reduced by methylation, there

Only genetic testing can determine whether a person has a genetic mutation. Most experts strongly recommend that people considering genetic testing first consult a genetic counselor if possible. Genetic counselors are trained to explain the risks and benefits of genetic testing. If a Genetic counselor is not available the clinician treating a patient with Ovarian Cancer

many as 82% of sporadic occurrences.

is no improved survival duration [1-5, 9-17].

**4. Genetic testing**


A study of genetic disorders can provide great insight into the etiology and early events in carcinogenesis. Evaluation of BRCA1 and BRCA2 mutant and sporadic tumors with gene expression profiling has demonstrated that the greatest contrast in expression patterns was between that of BRCA1 and BRCA2 mutant tumors and that sporadic tumors shared charac‐ teristics of both. This intriguing finding suggests that BRCA1 and BRCA2 tumors may have variable pathways in carcinogenesis and that even sporadic tumors may develop as a result of alterations in either pathway. Ovarian carcinogenesis, as in most cancers, involves multi‐ ple genetic alterations. A great deal has been learned about proteins and pathways impor‐ tant in the early stages of malignant transformation and metastasis, as derived from studies of individual tumors, microarray data, animal models, and inherited disorders that confer susceptibility. However, a full understanding of the earliest recognizable events in epithelial ovarian carcinogenesis is limited by the lack of a well-defined premalignant state common to all ovarian subtypes and by the paucity of data from early-stage cancers. Evidence sug‐ gests that ovarian cancers can progress both through a stepwise mutation process (lowgrade pathway) and through greater genetic instability that leads to rapid metastasis without an identifiable precursor lesion (high-grade pathway). In an interesting review, CN Landen et al. discuss many of the genetic and molecular disorders in each key process that is altered in cancer cells, and present a model of ovarian pathogenesis that incorporates the role of tumor cell mutations and factors in the host microenvironment important to tumor initiation and progression [1-5, 9-17]. Borderline tumors have a much less frequent incidence of BRCA mutations, which also suggests a different molecular origin. Other than in heredi‐ tary syndromes, BRCA genes are rarely mutated in sporadic ovarian cancers, although epi‐ genetic changes alternate splicing, and other genetic factors may affect BRCA function in as many as 82% of sporadic occurrences.

An analysis of genomic changes in ovarian cancer has provided the most comprehen‐ sive and integrated view of cancer genes for any cancer type to date. Ovarian serous ad‐ enocarcinoma tumors from 500 patients were examined by The Cancer Genome Atlas (TCGA) Research Network and analyses were reported in 2011.These findings confirm that mutations in a single gene, TP53, are present in more than 96 percent of all such cancers. TP53 encodes a tumor suppressor protein that normally prevents cancer forma‐ tion. Mutations in the gene disrupt this protein's function, which contributes to uncon‐ trolled growth of ovarian cells. Several less-frequent mutations in other genes have also been identified and was also established how sets of genes are expressed in a fashion that can predict patient survival, identifying patterns for 108 genes associated with poor survival and 85 genes associated with better survival. Patients whose tumors had a gene-expression signature associated with poor survival lived for a period that was 23 percent shorter than patients whose tumors did not have such a signature. To identify opportunities for targeted treatment, the investigators searched for existing drugs that might inhibit amplified or over-expressed genes that were suggested to play a role in ovarian cancer. Sixty-eight genes have been identified that could be targeted by existing or experimental therapeutic compounds. One type of drug, a PARP (Poly ADP ribose polymerase) inhibitor, might be able to counteract the DNA repair gene observed in half of the ovarian tumors studied. These drugs could be effective against the disease, this study revealed that 50 percent of tumors might be responsive to drugs that exploit the genetic instability of the tumors and induce the cancer cells to die. The results of this study support the existence of four distinct subtypes of the disease, based on the pat‐ terns seen in the transcription of RNA from DNA. They also support the existence of four related subtypes based on the patterns of DNA methylation—a chemical reaction in which a small molecule called a methyl group is added to DNA, changing the activity of individual genes. These patterns likely reflect the functional changes associated with ovarian serous adenocarcinoma, but are not strongly associated with survival duration. In this study, approximately 21 percent of the tumors showed mutations in BRCA1 and BRCA2 genes. Analysis of these tumors confirmed observations that patients with mutat‐ ed BRCA1 and BRCA2 genes have better survival odds than patients without mutations in these genes. If either of the BRCA1 and BRCA2 genes is mutated, there is improved survival duration. However, if BRCA1 activity is instead reduced by methylation, there is no improved survival duration [1-5, 9-17].

#### **4. Genetic testing**

**1. Peutz - Jeghers syndrome.** This syndrome is caused by a specific genetic mutation in the STK11 gene and is associated with multiple polyps in the digestive tract that be‐ come noncancerous tumors, increased pigmentation on the face and hands and with an

**2. Nevoid basal cell carcinoma syndrome or Gorlin syndrome** is associated with a muta‐ tion in PTCH and a 20% life time risk of developing stromal tumors and fibromas of the ovaries. There is a small risk that these fibromas could develop into fibrosarcoma. Peo‐ ple with Gorlin syndrome often have multiple basal cell carcinomas and jaw cysts and

**3. Li-Fraumeni syndrome.** The Li-Fraumeni syndrome is a rare condition associated with a specific genetic mutation. People with Li-Fraumeni syndrome have a higher risk of developing osteosarcoma, soft tissue sarcoma, leukemia, breast cancer, brain cancer,

**4. Ataxia telangiectasia.** Ataxia telangiectasia is a rare disorder associated with a specific genetic mutation. It causes progressive neurological problems and an increased risk of leukemia, lymphoma, and possibly sarcoma, breast, ovarian and stomach cancer. Germ‐ line mutations in the genes responsible for those syndromes produce different clinical phenotypes of characteristic malignancies and, in some instances, associated nonmalig‐

A study of genetic disorders can provide great insight into the etiology and early events in carcinogenesis. Evaluation of BRCA1 and BRCA2 mutant and sporadic tumors with gene expression profiling has demonstrated that the greatest contrast in expression patterns was between that of BRCA1 and BRCA2 mutant tumors and that sporadic tumors shared charac‐ teristics of both. This intriguing finding suggests that BRCA1 and BRCA2 tumors may have variable pathways in carcinogenesis and that even sporadic tumors may develop as a result of alterations in either pathway. Ovarian carcinogenesis, as in most cancers, involves multi‐ ple genetic alterations. A great deal has been learned about proteins and pathways impor‐ tant in the early stages of malignant transformation and metastasis, as derived from studies of individual tumors, microarray data, animal models, and inherited disorders that confer susceptibility. However, a full understanding of the earliest recognizable events in epithelial ovarian carcinogenesis is limited by the lack of a well-defined premalignant state common to all ovarian subtypes and by the paucity of data from early-stage cancers. Evidence sug‐ gests that ovarian cancers can progress both through a stepwise mutation process (lowgrade pathway) and through greater genetic instability that leads to rapid metastasis without an identifiable precursor lesion (high-grade pathway). In an interesting review, CN Landen et al. discuss many of the genetic and molecular disorders in each key process that is altered in cancer cells, and present a model of ovarian pathogenesis that incorporates the role of tumor cell mutations and factors in the host microenvironment important to tumor initiation and progression [1-5, 9-17]. Borderline tumors have a much less frequent incidence of BRCA mutations, which also suggests a different molecular origin. Other than in heredi‐ tary syndromes, BRCA genes are rarely mutated in sporadic ovarian cancers, although epi‐

increased risk of ovarian, breast, uterine, and lung cancers.

may develop medulloblastoma in childhood.

and adrenal cortical tumors.

140 Ovarian Cancer - A Clinical and Translational Update

nant abnormalities.

Only genetic testing can determine whether a person has a genetic mutation. Most experts strongly recommend that people considering genetic testing first consult a genetic counselor if possible. Genetic counselors are trained to explain the risks and benefits of genetic testing. If a Genetic counselor is not available the clinician treating a patient with Ovarian Cancer has the duty to take its role. He/she must consider if each patient with ovarian cancer is a candidate for genetic testing. Hereditary cancer syndromes have a major ethical, legal and psychological impact on the individual as well as family members and the caring physician. As a result, a careful counseling before, during and after the testing is necessary. There are many issues that one has to know before proceeding with the genetic testing.

carriers is about 40-50% and for BRCA2 carriers about 10-20%. The following information must be provided to genetic testing candidates according to the NCI. Women must know that in addition to family history, other environmental and lifestyle factors may increase their risk of ovarian cancer. Discussing their family history and personal risk factors with a doctor helps them to better understand their risk. People with a higher than average risk may benefit from genetic counseling, and the implementation of early detection and preven‐

The Genetics of Ovarian Cancer http://dx.doi.org/10.5772/54541 143

There can be benefits to genetic testing, whether a person receives a positive or a nega‐ tive result. The potential benefits of a negative result include a sense of relief and the possibility that special preventive checkups, tests, or surgeries may not be needed. A positive test result can bring relief from uncertainty and allow people to make informed decisions about their future, including taking steps to reduce their cancer risk. In addi‐ tion, many people who have a positive test result may be able to participate in medical research that could, in the long run, help reduce deaths from breast cancer. The direct medical risks, or harms, of genetic testing are very small, but test results may have an effect on a person's emotions, social relationships, finances, and medical choices. People who receive a positive test result may feel anxious, depressed, or angry. They may choose to undergo preventive measures, such as prophylactic surgery, that have serious long-term implications and whose effectiveness is uncertain. People who receive a nega‐ tive test result may experience "survivor guilt," caused by the knowledge that they like‐ ly do not have an increased risk of developing a disease that affects one or more loved ones. Because genetic testing can reveal information about more than one family mem‐ ber, the emotions caused by test results can create tension within families. Test results can also affect personal choices, such as marriage and childbearing. Issues surrounding the privacy and confidentiality of genetic test results are additional potential risks.

Ovarian cancer may run in the family if first-degree relatives (mother, sisters, daughters) or many other family members (grandmothers, aunts, nieces, granddaughters) have had ovari‐ an cancer. If a woman's first-degree relatives developed ovarian cancer, her risk of ovarian cancer is about three times higher than the average woman's risk of ovarian cancer. The risk increases if other close relatives have had ovarian cancer. When using family history to as‐ sess risk, the accuracy and completeness of family history data must be taken into account. A reported family history may be erroneous, or a person may be unaware of relatives affect‐ ed with cancer. In addition, small family sizes, premature deaths, immigration and poor medical records may limit the information obtained from a family history. Breast or ovarian cancer on the paternal side of the family usually involves more distant relatives than on the maternal side and thus may be more difficult to obtain. When comparing self-reported in‐ formation with independently verified cases, the sensitivity of a history of breast cancer is

relatively high, at 83% to 97%, but lower for ovarian cancer, at 60%. [1-5, 10,11]

However, **a positive test** result provides information only about a person's risk of develop‐ ing cancer. It cannot tell whether an individual will actually develop cancer or when. It must be stressed that not all women who inherit a harmful BRCA1 or BRCA2 mutation will devel‐ op breast or ovarian cancer. A positive genetic test result may have important health and

tion strategies.

**Criteria for recommending genetic testing:** Currently, there are no standard criteria for rec‐ ommending or referring someone for BRCA1 orBRCA2 mutation testing American Society of Clinical Oncology (ASCO) has published some guidelines for Genetic Testing of cancer patients and their families. ASCO also encourages Oncologists to assume the responsibility of genetics counseling with patients and their families. ASCO General recommendation as to indications for genetic testing in generally are the following: 1) When a person has a strong family history of cancer or very early age of onset of disease. 2) Test can be adequate‐ ly interpreted. 3) Result will influence medical management of the patient/family member. In a family with a history of breast and/or ovarian cancer, it may be most informative to first test a family member who has breast or ovarian cancer. If that person is found to have a harmful BRCA1 or BRCA2 mutation, then other family members can be tested to see if they also have the mutation. Women who have a relative with a harmful BRCA1 or BRCA2 mu‐ tation and women who appear to be at increased risk of breast and/or ovarian cancer be‐ cause of their family history should consider genetic counseling to learn more about their potential risks and about BRCA1 and BRCA2 genetic tests.

ASCO Recommendation as to indications for genetic testing for Breast and Ovarian cancers are the following: 1) Family with more than two breast cancer cases and one or more cases of ovarian cancer diagnosed at any age. 2) Family with more than three breast cancer cases diagnosed before age 50. 3) Sister pairs with two of the following cancers diagnosed before age 50: two breast cancers; two ovarian cancers; or a breast and ovarian cancer.4) Relatives of individuals with breast cancer diagnosed before the age of 30.Despite the above recom‐ mendations, there are individuals who do not fit any of the above categories and yet like to be tested. Such individuals need to be counseled to determine the appropriateness of genetic testing [1-5, 9-17].

**Genetic counseling:** Genetic counseling is generally recommended before and after a genet‐ ic test. This counseling should be performed by a health care professional experienced in cancer genetics. Genetic counseling usually involves a risk assessment based on the individ‐ ual's personal and family medical history and discussions about the appropriateness of ge‐ netic testing, the specific test(s) that might be used and the technical accuracy of the test(s), the medical implications of a positive or a negative test result, the possibility that a test re‐ sult might not be informative (an ambiguous result), the psychological risks and benefits of genetic test results, and the risk of passing a mutation to children. In case genetic testing turns positive health care professional must explain to her that a positive test result indi‐ cates that a person has inherited a known harmful mutation in BRCA1 or BRCA2 and, there‐ fore, has an increased risk of developing cancer. Women considering genetic testing must know in advance certain facts about the risk to develop Ovarian Cancer if the tests are posi‐ tive as well as the available prevention options. The lifetime risk for women who are BRCA1 carriers is about 40-50% and for BRCA2 carriers about 10-20%. The following information must be provided to genetic testing candidates according to the NCI. Women must know that in addition to family history, other environmental and lifestyle factors may increase their risk of ovarian cancer. Discussing their family history and personal risk factors with a doctor helps them to better understand their risk. People with a higher than average risk may benefit from genetic counseling, and the implementation of early detection and preven‐ tion strategies.

has the duty to take its role. He/she must consider if each patient with ovarian cancer is a candidate for genetic testing. Hereditary cancer syndromes have a major ethical, legal and psychological impact on the individual as well as family members and the caring physician. As a result, a careful counseling before, during and after the testing is necessary. There are

**Criteria for recommending genetic testing:** Currently, there are no standard criteria for rec‐ ommending or referring someone for BRCA1 orBRCA2 mutation testing American Society of Clinical Oncology (ASCO) has published some guidelines for Genetic Testing of cancer patients and their families. ASCO also encourages Oncologists to assume the responsibility of genetics counseling with patients and their families. ASCO General recommendation as to indications for genetic testing in generally are the following: 1) When a person has a strong family history of cancer or very early age of onset of disease. 2) Test can be adequate‐ ly interpreted. 3) Result will influence medical management of the patient/family member. In a family with a history of breast and/or ovarian cancer, it may be most informative to first test a family member who has breast or ovarian cancer. If that person is found to have a harmful BRCA1 or BRCA2 mutation, then other family members can be tested to see if they also have the mutation. Women who have a relative with a harmful BRCA1 or BRCA2 mu‐ tation and women who appear to be at increased risk of breast and/or ovarian cancer be‐ cause of their family history should consider genetic counseling to learn more about their

ASCO Recommendation as to indications for genetic testing for Breast and Ovarian cancers are the following: 1) Family with more than two breast cancer cases and one or more cases of ovarian cancer diagnosed at any age. 2) Family with more than three breast cancer cases diagnosed before age 50. 3) Sister pairs with two of the following cancers diagnosed before age 50: two breast cancers; two ovarian cancers; or a breast and ovarian cancer.4) Relatives of individuals with breast cancer diagnosed before the age of 30.Despite the above recom‐ mendations, there are individuals who do not fit any of the above categories and yet like to be tested. Such individuals need to be counseled to determine the appropriateness of genetic

**Genetic counseling:** Genetic counseling is generally recommended before and after a genet‐ ic test. This counseling should be performed by a health care professional experienced in cancer genetics. Genetic counseling usually involves a risk assessment based on the individ‐ ual's personal and family medical history and discussions about the appropriateness of ge‐ netic testing, the specific test(s) that might be used and the technical accuracy of the test(s), the medical implications of a positive or a negative test result, the possibility that a test re‐ sult might not be informative (an ambiguous result), the psychological risks and benefits of genetic test results, and the risk of passing a mutation to children. In case genetic testing turns positive health care professional must explain to her that a positive test result indi‐ cates that a person has inherited a known harmful mutation in BRCA1 or BRCA2 and, there‐ fore, has an increased risk of developing cancer. Women considering genetic testing must know in advance certain facts about the risk to develop Ovarian Cancer if the tests are posi‐ tive as well as the available prevention options. The lifetime risk for women who are BRCA1

many issues that one has to know before proceeding with the genetic testing.

142 Ovarian Cancer - A Clinical and Translational Update

potential risks and about BRCA1 and BRCA2 genetic tests.

testing [1-5, 9-17].

There can be benefits to genetic testing, whether a person receives a positive or a nega‐ tive result. The potential benefits of a negative result include a sense of relief and the possibility that special preventive checkups, tests, or surgeries may not be needed. A positive test result can bring relief from uncertainty and allow people to make informed decisions about their future, including taking steps to reduce their cancer risk. In addi‐ tion, many people who have a positive test result may be able to participate in medical research that could, in the long run, help reduce deaths from breast cancer. The direct medical risks, or harms, of genetic testing are very small, but test results may have an effect on a person's emotions, social relationships, finances, and medical choices. People who receive a positive test result may feel anxious, depressed, or angry. They may choose to undergo preventive measures, such as prophylactic surgery, that have serious long-term implications and whose effectiveness is uncertain. People who receive a nega‐ tive test result may experience "survivor guilt," caused by the knowledge that they like‐ ly do not have an increased risk of developing a disease that affects one or more loved ones. Because genetic testing can reveal information about more than one family mem‐ ber, the emotions caused by test results can create tension within families. Test results can also affect personal choices, such as marriage and childbearing. Issues surrounding the privacy and confidentiality of genetic test results are additional potential risks.

Ovarian cancer may run in the family if first-degree relatives (mother, sisters, daughters) or many other family members (grandmothers, aunts, nieces, granddaughters) have had ovari‐ an cancer. If a woman's first-degree relatives developed ovarian cancer, her risk of ovarian cancer is about three times higher than the average woman's risk of ovarian cancer. The risk increases if other close relatives have had ovarian cancer. When using family history to as‐ sess risk, the accuracy and completeness of family history data must be taken into account. A reported family history may be erroneous, or a person may be unaware of relatives affect‐ ed with cancer. In addition, small family sizes, premature deaths, immigration and poor medical records may limit the information obtained from a family history. Breast or ovarian cancer on the paternal side of the family usually involves more distant relatives than on the maternal side and thus may be more difficult to obtain. When comparing self-reported in‐ formation with independently verified cases, the sensitivity of a history of breast cancer is relatively high, at 83% to 97%, but lower for ovarian cancer, at 60%. [1-5, 10,11]

However, **a positive test** result provides information only about a person's risk of develop‐ ing cancer. It cannot tell whether an individual will actually develop cancer or when. It must be stressed that not all women who inherit a harmful BRCA1 or BRCA2 mutation will devel‐ op breast or ovarian cancer. A positive genetic test result may have important health and social implications for family members, including future generations. Unlike most other medical tests, genetic tests can reveal information not only about the person being tested but also about that person's relatives. Both men and women who inherit harmful BRCA1 or BRCA2 mutations, whether they develop cancer themselves or not, may pass the mutations on to their sons and daughters. However, not all children of people who have a harmful mu‐ tation will inherit the mutation. How a **negative test** result will be interpreted depends on whether or not someone in the tested person's family is known to carry a harmful BRCA1 or BRCA2 mutation. If someone in the family has a known mutation, testing other family mem‐ bers for the same mutation can provide information about their cancer risk. If a person tests negative for a known mutation in his or her family, it is unlikely that they have an inherited susceptibility to cancer associated with BRCA1 orBRCA2. Such a test result is called **a "true negative."** On the other hand having a true negative test result does not mean that a person will not develop cancer; it means that the person's risk of cancer is probably the same as that of people in the general population. In cases in which a family has a history of breast and/or ovarian cancer and no known mutation in BRCA1 or BRCA2 has been previously identified, a negative test result is not informative. It is not possible to tell whether an individual has a harmful BRCA1 or BRCA2 mutation that was not detected by testing and this is called a **"false negative test"** or whether the result is a true negative. In addition, it is possible for people to have a mutation in a gene other than BRCA1 or BRCA2 that increases their cancer risk but is not detectable by the test(s) used. If genetic testing shows a change in BRCA1 or BRCA2 that has not been previously associated with cancer in other people, the person's test result may be interpreted as **"ambiguous"** and the result is considered as uncertain. It is es‐ timated that 10 percent of women who underwent BRCA1 and BRCA2 mutation testing had this type of ambiguous result. Because everyone has genetic differences that are not associat‐ ed with an increased risk of disease, it is sometimes not known whether a specific DNA change affects a person's risk of developing cancer. As more research is conducted and more people are tested for BRCA1 or BRCA2 changes, we expect to learn more about these changes and cancer risk [1-5, 9-17].

primary prevention. Not all methods are appropriate for all patients, and potential ad‐ verse effects, complications, cost, and efficacy of these interventions must be considered and reviewed with patients before implementation. It must be stressed that having a particular genetic mutation linked to ovarian cancer cannot predict that a person will

The Genetics of Ovarian Cancer http://dx.doi.org/10.5772/54541 145

**Cancer prevention** is action taken to lower the chance of getting cancer. By preventing can‐ cer, the number of new cases of cancer in a group or population is lowered. Hopefully, this will lower the number of deaths caused by cancer. To prevent new cancers from starting, we must consider risk and protective factors. Anything that increases one person's chance of developing cancer is called a cancer risk factor; anything that decreases the chance of devel‐ oping cancer is called a cancer protective factor. Some risk factors for cancer can be avoided, but many cannot. For example, both smoking and inheriting certain genes are risk factors for some types of cancer, but only smoking can be avoided. Regular exercise and a healthy diet may be protective factors for some types of cancer. Avoiding risk factors and increasing protective factors may lower the risk but it does not mean that cancer will be avoided. Dif‐ ferent ways to prevent cancer are being studied, including: Changing lifestyle or eating hab‐ its, avoiding things known to cause cancer or taking medicines to treat a precancerous condition or to keep cancer from starting[1-5,18-29]. According to the NCI's PDQ cancer in‐ formation about **Ovarian cancer prevention** the following risk factors may **increase** the risk of ovarian cancer: Family history of ovarian cancer, inherited risk, hormone replacement therapy, fertility drugs, talc and obesity. Factors associated with a **decreased** risk of ovarian cancer include: (a) using oral contraceptives, (b) having and breastfeeding children, (c) hav‐ ing a bilateral tubal ligation or hysterectomy, and (d) having a prophylactic oophorectomy. Multiple studies have consistently demonstrated a decrease in ovarian cancer risk in women who take oral contraceptives. The protective association increases with the duration of oral contraceptive use and persists up to 25 years after discontinuing oral contraceptives. A re‐ view of the literature demonstrated a 10% to 12% decrease in risk associated with use for 1 year and an approximate 50% decrease after 5 years of use. This reduced risk was present among both nulliparous and parous women. A protective association between oral contra‐ ceptives and risk of ovarian cancer has been observed in most studies among women who carry a mutation in BRCA1 and BRCA2 genes but a population-based study did not observe an association between oral contraceptives and ovarian cancer, while parity was protective. There may be a slight increase in a woman's risk of breast cancer during the time she is tak‐ ing oral contraceptives. This risk decreases over time. Pregnancy and breastfeeding are linked to a decreased risk of ovarian cancer. Ovulation stops or occurs less often in women who are pregnant or breastfeeding and women who ovulate less often have a decreased risk of ovarian cancer. Factors that increase risk for ovarian cancer include increasing age and nulliparity, while those that decrease risk include surgical history and use of Oral contracep‐ tives. Relatively few studies have addressed the effect of these risk factors in women who are genetically susceptible to ovarian cancer. Ovarian cancer incidence rises in a linear fash‐ ion from age 30 years to age 50 years and continues to increase, though at a slower rate, thereafter. Before age 30 years, the risk of developing epithelial ovarian cancer is remote, even in hereditary cancer families. Nulliparity is consistently associated with an increased

develop cancer. [1-5, 18-29].

**Genetic tests:** Several methods are available to test for BRCA1 and BRCA2 mutations. Most of these methods look for changes in BRCA1 and BRCA2 DNA. At least one method looks for changes in the proteins produced by these genes. Frequently, a combination of methods is used. A blood sample is needed for these tests. The blood is drawn in a laboratory, doc‐ tor's office, hospital, or clinic and then sent to a laboratory that specializes in the tests. It usu‐ ally takes several weeks or longer to get the test results. Genetic tests are expensive and this represents a major problem in every day practice.

#### **5. Management of women with mutated genes**

The options available today for women who have tested positive can be divided into secondary and primary prevention. Methods of secondary prevention, such as surveil‐ lance, attempt to diagnose cancers at an early stage, while primary prevention prevents cancer development. Chemoprevention and prophylactic oophorectomy are examples of primary prevention. Not all methods are appropriate for all patients, and potential ad‐ verse effects, complications, cost, and efficacy of these interventions must be considered and reviewed with patients before implementation. It must be stressed that having a particular genetic mutation linked to ovarian cancer cannot predict that a person will develop cancer. [1-5, 18-29].

social implications for family members, including future generations. Unlike most other medical tests, genetic tests can reveal information not only about the person being tested but also about that person's relatives. Both men and women who inherit harmful BRCA1 or BRCA2 mutations, whether they develop cancer themselves or not, may pass the mutations on to their sons and daughters. However, not all children of people who have a harmful mu‐ tation will inherit the mutation. How a **negative test** result will be interpreted depends on whether or not someone in the tested person's family is known to carry a harmful BRCA1 or BRCA2 mutation. If someone in the family has a known mutation, testing other family mem‐ bers for the same mutation can provide information about their cancer risk. If a person tests negative for a known mutation in his or her family, it is unlikely that they have an inherited susceptibility to cancer associated with BRCA1 orBRCA2. Such a test result is called **a "true negative."** On the other hand having a true negative test result does not mean that a person will not develop cancer; it means that the person's risk of cancer is probably the same as that of people in the general population. In cases in which a family has a history of breast and/or ovarian cancer and no known mutation in BRCA1 or BRCA2 has been previously identified, a negative test result is not informative. It is not possible to tell whether an individual has a harmful BRCA1 or BRCA2 mutation that was not detected by testing and this is called a **"false negative test"** or whether the result is a true negative. In addition, it is possible for people to have a mutation in a gene other than BRCA1 or BRCA2 that increases their cancer risk but is not detectable by the test(s) used. If genetic testing shows a change in BRCA1 or BRCA2 that has not been previously associated with cancer in other people, the person's test result may be interpreted as **"ambiguous"** and the result is considered as uncertain. It is es‐ timated that 10 percent of women who underwent BRCA1 and BRCA2 mutation testing had this type of ambiguous result. Because everyone has genetic differences that are not associat‐ ed with an increased risk of disease, it is sometimes not known whether a specific DNA change affects a person's risk of developing cancer. As more research is conducted and more people are tested for BRCA1 or BRCA2 changes, we expect to learn more about these

**Genetic tests:** Several methods are available to test for BRCA1 and BRCA2 mutations. Most of these methods look for changes in BRCA1 and BRCA2 DNA. At least one method looks for changes in the proteins produced by these genes. Frequently, a combination of methods is used. A blood sample is needed for these tests. The blood is drawn in a laboratory, doc‐ tor's office, hospital, or clinic and then sent to a laboratory that specializes in the tests. It usu‐ ally takes several weeks or longer to get the test results. Genetic tests are expensive and this

The options available today for women who have tested positive can be divided into secondary and primary prevention. Methods of secondary prevention, such as surveil‐ lance, attempt to diagnose cancers at an early stage, while primary prevention prevents cancer development. Chemoprevention and prophylactic oophorectomy are examples of

changes and cancer risk [1-5, 9-17].

144 Ovarian Cancer - A Clinical and Translational Update

represents a major problem in every day practice.

**5. Management of women with mutated genes**

**Cancer prevention** is action taken to lower the chance of getting cancer. By preventing can‐ cer, the number of new cases of cancer in a group or population is lowered. Hopefully, this will lower the number of deaths caused by cancer. To prevent new cancers from starting, we must consider risk and protective factors. Anything that increases one person's chance of developing cancer is called a cancer risk factor; anything that decreases the chance of devel‐ oping cancer is called a cancer protective factor. Some risk factors for cancer can be avoided, but many cannot. For example, both smoking and inheriting certain genes are risk factors for some types of cancer, but only smoking can be avoided. Regular exercise and a healthy diet may be protective factors for some types of cancer. Avoiding risk factors and increasing protective factors may lower the risk but it does not mean that cancer will be avoided. Dif‐ ferent ways to prevent cancer are being studied, including: Changing lifestyle or eating hab‐ its, avoiding things known to cause cancer or taking medicines to treat a precancerous condition or to keep cancer from starting[1-5,18-29]. According to the NCI's PDQ cancer in‐ formation about **Ovarian cancer prevention** the following risk factors may **increase** the risk of ovarian cancer: Family history of ovarian cancer, inherited risk, hormone replacement therapy, fertility drugs, talc and obesity. Factors associated with a **decreased** risk of ovarian cancer include: (a) using oral contraceptives, (b) having and breastfeeding children, (c) hav‐ ing a bilateral tubal ligation or hysterectomy, and (d) having a prophylactic oophorectomy. Multiple studies have consistently demonstrated a decrease in ovarian cancer risk in women who take oral contraceptives. The protective association increases with the duration of oral contraceptive use and persists up to 25 years after discontinuing oral contraceptives. A re‐ view of the literature demonstrated a 10% to 12% decrease in risk associated with use for 1 year and an approximate 50% decrease after 5 years of use. This reduced risk was present among both nulliparous and parous women. A protective association between oral contra‐ ceptives and risk of ovarian cancer has been observed in most studies among women who carry a mutation in BRCA1 and BRCA2 genes but a population-based study did not observe an association between oral contraceptives and ovarian cancer, while parity was protective. There may be a slight increase in a woman's risk of breast cancer during the time she is tak‐ ing oral contraceptives. This risk decreases over time. Pregnancy and breastfeeding are linked to a decreased risk of ovarian cancer. Ovulation stops or occurs less often in women who are pregnant or breastfeeding and women who ovulate less often have a decreased risk of ovarian cancer. Factors that increase risk for ovarian cancer include increasing age and nulliparity, while those that decrease risk include surgical history and use of Oral contracep‐ tives. Relatively few studies have addressed the effect of these risk factors in women who are genetically susceptible to ovarian cancer. Ovarian cancer incidence rises in a linear fash‐ ion from age 30 years to age 50 years and continues to increase, though at a slower rate, thereafter. Before age 30 years, the risk of developing epithelial ovarian cancer is remote, even in hereditary cancer families. Nulliparity is consistently associated with an increased risk of ovarian cancer, including among BRCA1/BRCA2mutation carriers. Risk may also be increased among women who have used fertility drugs, especially those who remain nulli‐ gravid. Evidence is growing that the use of menopausal HRT is associated with an increased risk of ovarian cancer, particularly in long-time users and users of sequential estrogen-pro‐ gesterone schedules [1-5].

mended as appropriate interventions for women at high risk of ovarian cancer in a National Institutes of Health consensus conference although they did concede that there was no evi‐ dence of efficacy. Indeed, multiple investigations have been performed that cast doubt on the efficacy of these interventions. For example, a recent study prospectively screened 1,110 women with increased risk of ovarian cancer with pelvic ultrasound and CA-125 measure‐ ments. About half of patients were at moderate risk of developing ovarian cancer, with a 4% to 10% lifetime risk and half were at high risk with more than 10% lifetime risk. Invasive ovarian cancer was diagnosed in 12 patients. Two patients had stage I disease, one had stage II, four had stage III, and one had stage IV. These screening techniques missed an additional two patients with stage III disease and one patient with stage IV ovarian cancer. Based on abnormal ultrasound findings, 29 additional women underwent surgery for what turned out to be benign processes. The positive predictive value was 17%, and the sensitivity was less than 50%. These screening techniques are especially problematic for premenopausal women (the cohort with BRCA mutations is of highest interest) in which the false-positive rate was 79%. The conclusion is that the use of pelvic ultrasound and CA-125 does not meet World Health Organization screening standards for women with an increased risk for ovari‐ an cancer. The advantages of surveillance include avoidance of premature menopause and the fact that there is no intervention for those without disease. It allows management with other techniques, which may be available in the future. However, surveillance does not pre‐ vent disease, and an objective assessment of the data on screening for ovarian cancer does not support the use of these modalities, even in patients at elevated risk. For women who have not finished childbearing or are deferring prophylactic oophorectomy for other rea‐ sons, current practice guidelines from the National Comprehensive Cancer Network recom‐ mends concurrent transvaginal ultrasound and CA-125 every 6 months starting at age 35 or 5 to 10 years earlier than the earliest ovarian cancer diagnosis in the family (and preferably days 1 to 10 of cycle for premenopausal women). If initiated, it is important for these women to understand the shortcomings of surveillance. They should be aware of the high likelihood of an abnormal scan in ovulating women, and also understand that a normal scan does not

The Genetics of Ovarian Cancer http://dx.doi.org/10.5772/54541 147

guarantee absence of disease, even in the advanced stages [1-5, 18-29].

**Chemoprevention** involves the use of natural or synthetic substances to reduce the risk of developing cancer or to reduce the chance that cancer will come back. It has been postulated that incessant ovulation may be one mechanism by which ovarian cancer develops. Consis‐ tent with this theory is the observation that parity is associated with a reduction in risk. **The use of oral contraceptives** has also been shown to reduce ovarian cancer risk by as much as 50% in the general population. However, there have been relatively few investigations studying the effect of oral contraceptive use on ovarian cancer risk in women with BRCA mutations. Unfortunately, the available data are conflicting. In one retrospective investiga‐ tion of 451 patients with BRCA mutations, women who used oral contraceptives for 6 or more years had an odds ratio of ovarian cancer of 0.62 (95% confidence interval [CI], 0.35– 1.09). Although not a statistically significant reduction in risk, this study suggests that oral contraceptives may be an effective form of chemoprevention in carriers. In contrast, Modan et al performed a case-control study of 1,591 Jewish women, 257 of whom underwent genet‐ ic testing and were found to have a BRCA mutation. They did not find clear evidence of a

**Surveillance** means cancer **screening**, or a way of detecting the disease early. Screening does not, however, change the risk of developing cancer. The goal is to find cancer early, when it may be most treatable. **Screening,** looking for cancer before a person has any symp‐ toms, can help find cancer at an early stage and increase the chances for cure or prolong sur‐ vival. By the time symptoms appear, the disease may have begun to spread and treatment results are usually disappointing. Before recommending screening it is important to esti‐ mate women who have increased risk to develop ovarian cancer in order to suggest the proper screening tests, when to start screening and how often to repeat it. If screening tests are abnormal then the physician has to proceed to diagnostic tests. There are unfortunately no satisfactory standard screening tests for ovarian cancer. Family members of ovarian can‐ cer patients must be informed that tests that may detect ovarian cancer are the following: Pelvic examination, transvaginal ultrasound and CA-125 assay. Although screening for ovarian cancer has not been proven to decrease the death rate from the disease, this ap‐ proach is the only available screening today for the possible early diagnosis for Ovarian Cancer and this is what we must follow. Several biomarkers with potential application to ovarian cancer screening are under development but have not yet been validated or evaluat‐ ed for efficacy in early detection and mortality reduction. The Pap test, which is considered by many women as the "screening for Gynecological Cancer", may occasionally detect ma‐ lignant ovarian cells, but it is not sensitive, the reported sensitivity is about 10%–30%, and has not been evaluated for the early detection of ovarian cancer. Other methods of detection, including cytologic examination of peritoneal lavage obtained by culdocentesis and proteo‐ mics used to identify patterns or specific serum markers that may be used in place of, or in conjunction with, CA 125 measurements remain under study. Given the low incidence of ovarian cancer in the general population, the use of these modalities has not been adopted for screening purposes in the general population. To be cost effective and avoid unnecessary surgical interventions, the use of transvaginal ultrasound and CA-125 would need to be nearly 100% specific and sensitive. Premenopausal women in particular have a high inci‐ dence of benign ovarian cysts. Although CA-125 can be a reliable marker for recurrence in women with a previous diagnosis of ovarian cancer, only 50% of early-stage ovarian cancers are associated with an abnormal CA-125. It must be noted that CA-125 can also show spuri‐ ous elevations in association with any process, which irritates the peritoneal or pleural cavi‐ ty, such as endometriosis, pneumonia, pulmonary embolism, or even normal menses. Prospective screening trials, using ultrasound and CA-125, in women in the general popula‐ tion have resulted in approximately 30 surgeries for every cancer diagnosed and have failed to detect disease at an early stage. Given the higher prevalence of ovarian cancer in patients with BRCA mutations, there has been speculation that pelvic ultrasound and CA-125 may be useful screening strategies for these patients. In fact, annual or semiannual screening with pelvic examination, transvaginal ultrasonography, and serum CA-125 was recom‐ mended as appropriate interventions for women at high risk of ovarian cancer in a National Institutes of Health consensus conference although they did concede that there was no evi‐ dence of efficacy. Indeed, multiple investigations have been performed that cast doubt on the efficacy of these interventions. For example, a recent study prospectively screened 1,110 women with increased risk of ovarian cancer with pelvic ultrasound and CA-125 measure‐ ments. About half of patients were at moderate risk of developing ovarian cancer, with a 4% to 10% lifetime risk and half were at high risk with more than 10% lifetime risk. Invasive ovarian cancer was diagnosed in 12 patients. Two patients had stage I disease, one had stage II, four had stage III, and one had stage IV. These screening techniques missed an additional two patients with stage III disease and one patient with stage IV ovarian cancer. Based on abnormal ultrasound findings, 29 additional women underwent surgery for what turned out to be benign processes. The positive predictive value was 17%, and the sensitivity was less than 50%. These screening techniques are especially problematic for premenopausal women (the cohort with BRCA mutations is of highest interest) in which the false-positive rate was 79%. The conclusion is that the use of pelvic ultrasound and CA-125 does not meet World Health Organization screening standards for women with an increased risk for ovari‐ an cancer. The advantages of surveillance include avoidance of premature menopause and the fact that there is no intervention for those without disease. It allows management with other techniques, which may be available in the future. However, surveillance does not pre‐ vent disease, and an objective assessment of the data on screening for ovarian cancer does not support the use of these modalities, even in patients at elevated risk. For women who have not finished childbearing or are deferring prophylactic oophorectomy for other rea‐ sons, current practice guidelines from the National Comprehensive Cancer Network recom‐ mends concurrent transvaginal ultrasound and CA-125 every 6 months starting at age 35 or 5 to 10 years earlier than the earliest ovarian cancer diagnosis in the family (and preferably days 1 to 10 of cycle for premenopausal women). If initiated, it is important for these women to understand the shortcomings of surveillance. They should be aware of the high likelihood of an abnormal scan in ovulating women, and also understand that a normal scan does not guarantee absence of disease, even in the advanced stages [1-5, 18-29].

risk of ovarian cancer, including among BRCA1/BRCA2mutation carriers. Risk may also be increased among women who have used fertility drugs, especially those who remain nulli‐ gravid. Evidence is growing that the use of menopausal HRT is associated with an increased risk of ovarian cancer, particularly in long-time users and users of sequential estrogen-pro‐

**Surveillance** means cancer **screening**, or a way of detecting the disease early. Screening does not, however, change the risk of developing cancer. The goal is to find cancer early, when it may be most treatable. **Screening,** looking for cancer before a person has any symp‐ toms, can help find cancer at an early stage and increase the chances for cure or prolong sur‐ vival. By the time symptoms appear, the disease may have begun to spread and treatment results are usually disappointing. Before recommending screening it is important to esti‐ mate women who have increased risk to develop ovarian cancer in order to suggest the proper screening tests, when to start screening and how often to repeat it. If screening tests are abnormal then the physician has to proceed to diagnostic tests. There are unfortunately no satisfactory standard screening tests for ovarian cancer. Family members of ovarian can‐ cer patients must be informed that tests that may detect ovarian cancer are the following: Pelvic examination, transvaginal ultrasound and CA-125 assay. Although screening for ovarian cancer has not been proven to decrease the death rate from the disease, this ap‐ proach is the only available screening today for the possible early diagnosis for Ovarian Cancer and this is what we must follow. Several biomarkers with potential application to ovarian cancer screening are under development but have not yet been validated or evaluat‐ ed for efficacy in early detection and mortality reduction. The Pap test, which is considered by many women as the "screening for Gynecological Cancer", may occasionally detect ma‐ lignant ovarian cells, but it is not sensitive, the reported sensitivity is about 10%–30%, and has not been evaluated for the early detection of ovarian cancer. Other methods of detection, including cytologic examination of peritoneal lavage obtained by culdocentesis and proteo‐ mics used to identify patterns or specific serum markers that may be used in place of, or in conjunction with, CA 125 measurements remain under study. Given the low incidence of ovarian cancer in the general population, the use of these modalities has not been adopted for screening purposes in the general population. To be cost effective and avoid unnecessary surgical interventions, the use of transvaginal ultrasound and CA-125 would need to be nearly 100% specific and sensitive. Premenopausal women in particular have a high inci‐ dence of benign ovarian cysts. Although CA-125 can be a reliable marker for recurrence in women with a previous diagnosis of ovarian cancer, only 50% of early-stage ovarian cancers are associated with an abnormal CA-125. It must be noted that CA-125 can also show spuri‐ ous elevations in association with any process, which irritates the peritoneal or pleural cavi‐ ty, such as endometriosis, pneumonia, pulmonary embolism, or even normal menses. Prospective screening trials, using ultrasound and CA-125, in women in the general popula‐ tion have resulted in approximately 30 surgeries for every cancer diagnosed and have failed to detect disease at an early stage. Given the higher prevalence of ovarian cancer in patients with BRCA mutations, there has been speculation that pelvic ultrasound and CA-125 may be useful screening strategies for these patients. In fact, annual or semiannual screening with pelvic examination, transvaginal ultrasonography, and serum CA-125 was recom‐

gesterone schedules [1-5].

146 Ovarian Cancer - A Clinical and Translational Update

**Chemoprevention** involves the use of natural or synthetic substances to reduce the risk of developing cancer or to reduce the chance that cancer will come back. It has been postulated that incessant ovulation may be one mechanism by which ovarian cancer develops. Consis‐ tent with this theory is the observation that parity is associated with a reduction in risk. **The use of oral contraceptives** has also been shown to reduce ovarian cancer risk by as much as 50% in the general population. However, there have been relatively few investigations studying the effect of oral contraceptive use on ovarian cancer risk in women with BRCA mutations. Unfortunately, the available data are conflicting. In one retrospective investiga‐ tion of 451 patients with BRCA mutations, women who used oral contraceptives for 6 or more years had an odds ratio of ovarian cancer of 0.62 (95% confidence interval [CI], 0.35– 1.09). Although not a statistically significant reduction in risk, this study suggests that oral contraceptives may be an effective form of chemoprevention in carriers. In contrast, Modan et al performed a case-control study of 1,591 Jewish women, 257 of whom underwent genet‐ ic testing and were found to have a BRCA mutation. They did not find clear evidence of a protective effect with oral contraceptive use in BRCA carriers. Given the low incidence of adverse effects, before more definitive investigations are available, the use of oral contracep‐ tives as a chemopreventive strategy would appear to be a reasonable approach for the pa‐ tient who declines prophylactic salpingo-oophorectomy and for whom prevention of pregnancy is acceptable. However, the conflicting data should be reviewed with the patient before initiation [1-5, 18-29].

was a 47% reduction in the risk of breast cancer in premenopausal women who had prophy‐ lactic salpingo-oophorectomy. The effectiveness of prophylactic salpingo-oophorectomy in reduction of ovarian cancer risk has also been demonstrated in prospective studies. Prophy‐ lactic salpingo-oophorectomy failures may be divided into groups of those patients who are found to harbor an occult malignancy at the time of surgery and those who go on to develop carcinoma at a later time. The existence of occult ovarian cancer in BRCA carriers with appa‐ rently healthy ovaries has been documented in small samples for a number of years. In a recent investigation that included 555 women who underwent prophylactic salpingo-oopho‐ rectomy, the rate of occult fallopian tube or ovarian cancer was 2.2%, consistent with prior reports. Although a low incidence, this risk should routinely be discussed with patients be‐ fore surgery and highlights the need for an extensive pathologic assessment of the entire ad‐ nexa, including the fallopian tubes [30-43]. Development of primary peritoneal carcinoma (PPC) represents the vast majority of failures after prophylactic salpingo-oophorectomy. In a multicenter investigation of 1,828 carriers, the cumulative risk of PPC was 4.3% at 20 years after prophylactic salpingo-oophorectomy.24 It is hypothesized that PPC arises from the peritoneal coelomic epithelium, derived from the same embryonic tissue that gives rise to the epithelial covering of the ovaries. Ovarian and peritoneal epithelium share common em‐ bryonal origin, originating both from the coelomic epithelium (mesodermal origin). Coelo‐ mic epithelium is thought to be of mesonephric origin. With the overall point being that normal ovarian and peritoneal tissue is derived from the mesonephros. On the contrary, fal‐ lopian tube epithelium, endometrium and endocervix are related to paramesonephros (Mül‐ lerian duct). Surprisingly, epithelial ovarian cancer and primary peritoneal cancer are histologically similar to the Mullerian epithelium; not their embryonal origin, the meso‐ nephros. Either a metaplasia has occurred or Mullerian remnants have been left behind in coelemic epithelium, which have turned oncogenic. Although the precise causes are not known, a link with certain variants of BRCA1/2 has been described. Furthermore, women with BRCA1/2 mutation have a 5% risk of developing primary peritoneal cancer even after prophylactic oophorectomy. Primary peritoneal carcinoma shows similar rates of tumor suppressor gene dysfunction (p53, BRCA, WT1) as ovarian cancer and can also show an in‐ creased expression of HER-2/neu. An association with vascular endothelial growth factor has been observed. Although the absolute risk of fallopian tube cancer is unknown in pa‐ tients with BRCA mutations, it is agreed that it is substantially elevated, with a relative risk of 120 in one study. It remains unknown if the 4.3% failure rate found by Finch et al consists entirely of PPC or if a proportion is in fact peritoneal recurrences of a fallopian tube carcino‐ ma missed at the time of prophylactic salpingo-oophorectomy. Regardless, it is widely ac‐ cepted that removal of the fallopian tubes is essential at the time of prophylactic surgery. There is an abundance of evidence supporting the efficacy of prophylactic salpingo-oopho‐ rectomy, but less information exists to counsel the clinician as to the optimal timing of pro‐ phylactic surgery. Reasonable guidelines can be inferred from existing data regarding the onset of ovarian cancer in BRCA carriers. The cumulative incidence of breast cancer is 11.6% by age 40 for women with BRCA1 mutations. In contrast, the rate is only 2.3% for ovarian cancer by age 40. By age 45, 6.5% of BRCA1 carriers will be diagnosed with ovarian cancer; 13.2% by age 50. As a result, for BRCA1 carriers, most physicians recommend prophylactic

The Genetics of Ovarian Cancer http://dx.doi.org/10.5772/54541 149

**Prophylactic salpingo-oophorectomy.** The use of oral contraceptives, having and breast‐ feeding children do not certainly offer enough protection for BRCA1/BRCA2 carriers. The removal of the "at-risk" tissue is the most important step to prevent Ovarian Cancer. Wom‐ en who have a high risk of ovarian cancer must be informed about the possibility of a pro‐ phylactic oophorectomy. This includes women who have inherited certain changes in the BRCA1 and BRCA2 genes or in the genes linked to hereditary nonpolyposis colon cancer (HNPCC). It is very important to have a cancer risk assessment and counseling before mak‐ ing this decision. These and other factors should be discussed: Early menopause: 90% reduc‐ tion in risk of ovarian cancer observed among women with a BRCA1 or BRCA2 mutation. BRCA1 or BRCA2 mutations occur in 0.1–0.8% of the general population and are inherited in an autosomal dominant manner. They are well recognized to have a higher incidence in certain ethnic groups, such as women of Ashkenazi Jewish descent. S Vaughan Given the newly appreciated importance of the fallopian tube in the genesis of high-grade serous ovar‐ ian cancer, it is recommended that the complete removal of the fallopian tube should be‐ come standard of care in any woman undergoing hysterectomy and/or removal of the ovaries (oophorectomy). Oophorectomy in premenopausal women induces early meno‐ pause. As a consequence, and with the changed view of the role of the fallopian tube in ovarian cancer, some clinicians have recommended that only the fallopian tubes should be removed (salpingectomy) in women with germline BRCA1 or BRCA2 mutations, or in wom‐ en with a strong family history of breast and/or ovarian cancer34. However, until compre‐ hensive comparative data are available, it is premature to recommend that only the fallopian tubes are removed in high-risk women [1-5, 30-43].

Women who have completed childbearing are candidates for surgery. For the majority of women, this surgery can be performed laparoscopically as an outpatient procedure. In con‐ trast to surveillance and chemoprevention, this intervention is very effective in reducing the risk of ovarian cancer. Bilateral tubal ligation and hysterectomy are associated with reduced ovarian cancer risk, including in BRCA1/BRCA2 mutation carriers. Ovarian cancer risk is re‐ duced more than 90% in women with documented BRCA1 or BRCA2 mutations who chose risk-reducing salpingo-oophorectomy. In this same population, prophylactic removal of the ovaries also resulted in a nearly 50% reduction in the risk of subsequent breast cancer. In a retrospective analysis of 551 patients, Rebbeck et al showed that women who had under‐ gone prophylactic salpingo-oophorectomy had an odds ratio of 0.04 for ovarian cancer, com‐ pared with carriers without prophylactic salpingo-oophorectomy. Over a median follow-up of 8.8 years, two primary peritoneal cancers were diagnosed in the 259 women who under‐ went prophylactic salpingo-oophorectomy compared with 58 ovarian/peritoneal cancers in the 292 women who did not have prophylactic salpingo-oophorectomy. An added benefit was a 47% reduction in the risk of breast cancer in premenopausal women who had prophy‐ lactic salpingo-oophorectomy. The effectiveness of prophylactic salpingo-oophorectomy in reduction of ovarian cancer risk has also been demonstrated in prospective studies. Prophy‐ lactic salpingo-oophorectomy failures may be divided into groups of those patients who are found to harbor an occult malignancy at the time of surgery and those who go on to develop carcinoma at a later time. The existence of occult ovarian cancer in BRCA carriers with appa‐ rently healthy ovaries has been documented in small samples for a number of years. In a recent investigation that included 555 women who underwent prophylactic salpingo-oopho‐ rectomy, the rate of occult fallopian tube or ovarian cancer was 2.2%, consistent with prior reports. Although a low incidence, this risk should routinely be discussed with patients be‐ fore surgery and highlights the need for an extensive pathologic assessment of the entire ad‐ nexa, including the fallopian tubes [30-43]. Development of primary peritoneal carcinoma (PPC) represents the vast majority of failures after prophylactic salpingo-oophorectomy. In a multicenter investigation of 1,828 carriers, the cumulative risk of PPC was 4.3% at 20 years after prophylactic salpingo-oophorectomy.24 It is hypothesized that PPC arises from the peritoneal coelomic epithelium, derived from the same embryonic tissue that gives rise to the epithelial covering of the ovaries. Ovarian and peritoneal epithelium share common em‐ bryonal origin, originating both from the coelomic epithelium (mesodermal origin). Coelo‐ mic epithelium is thought to be of mesonephric origin. With the overall point being that normal ovarian and peritoneal tissue is derived from the mesonephros. On the contrary, fal‐ lopian tube epithelium, endometrium and endocervix are related to paramesonephros (Mül‐ lerian duct). Surprisingly, epithelial ovarian cancer and primary peritoneal cancer are histologically similar to the Mullerian epithelium; not their embryonal origin, the meso‐ nephros. Either a metaplasia has occurred or Mullerian remnants have been left behind in coelemic epithelium, which have turned oncogenic. Although the precise causes are not known, a link with certain variants of BRCA1/2 has been described. Furthermore, women with BRCA1/2 mutation have a 5% risk of developing primary peritoneal cancer even after prophylactic oophorectomy. Primary peritoneal carcinoma shows similar rates of tumor suppressor gene dysfunction (p53, BRCA, WT1) as ovarian cancer and can also show an in‐ creased expression of HER-2/neu. An association with vascular endothelial growth factor has been observed. Although the absolute risk of fallopian tube cancer is unknown in pa‐ tients with BRCA mutations, it is agreed that it is substantially elevated, with a relative risk of 120 in one study. It remains unknown if the 4.3% failure rate found by Finch et al consists entirely of PPC or if a proportion is in fact peritoneal recurrences of a fallopian tube carcino‐ ma missed at the time of prophylactic salpingo-oophorectomy. Regardless, it is widely ac‐ cepted that removal of the fallopian tubes is essential at the time of prophylactic surgery. There is an abundance of evidence supporting the efficacy of prophylactic salpingo-oopho‐ rectomy, but less information exists to counsel the clinician as to the optimal timing of pro‐ phylactic surgery. Reasonable guidelines can be inferred from existing data regarding the onset of ovarian cancer in BRCA carriers. The cumulative incidence of breast cancer is 11.6% by age 40 for women with BRCA1 mutations. In contrast, the rate is only 2.3% for ovarian cancer by age 40. By age 45, 6.5% of BRCA1 carriers will be diagnosed with ovarian cancer; 13.2% by age 50. As a result, for BRCA1 carriers, most physicians recommend prophylactic

protective effect with oral contraceptive use in BRCA carriers. Given the low incidence of adverse effects, before more definitive investigations are available, the use of oral contracep‐ tives as a chemopreventive strategy would appear to be a reasonable approach for the pa‐ tient who declines prophylactic salpingo-oophorectomy and for whom prevention of pregnancy is acceptable. However, the conflicting data should be reviewed with the patient

**Prophylactic salpingo-oophorectomy.** The use of oral contraceptives, having and breast‐ feeding children do not certainly offer enough protection for BRCA1/BRCA2 carriers. The removal of the "at-risk" tissue is the most important step to prevent Ovarian Cancer. Wom‐ en who have a high risk of ovarian cancer must be informed about the possibility of a pro‐ phylactic oophorectomy. This includes women who have inherited certain changes in the BRCA1 and BRCA2 genes or in the genes linked to hereditary nonpolyposis colon cancer (HNPCC). It is very important to have a cancer risk assessment and counseling before mak‐ ing this decision. These and other factors should be discussed: Early menopause: 90% reduc‐ tion in risk of ovarian cancer observed among women with a BRCA1 or BRCA2 mutation. BRCA1 or BRCA2 mutations occur in 0.1–0.8% of the general population and are inherited in an autosomal dominant manner. They are well recognized to have a higher incidence in certain ethnic groups, such as women of Ashkenazi Jewish descent. S Vaughan Given the newly appreciated importance of the fallopian tube in the genesis of high-grade serous ovar‐ ian cancer, it is recommended that the complete removal of the fallopian tube should be‐ come standard of care in any woman undergoing hysterectomy and/or removal of the ovaries (oophorectomy). Oophorectomy in premenopausal women induces early meno‐ pause. As a consequence, and with the changed view of the role of the fallopian tube in ovarian cancer, some clinicians have recommended that only the fallopian tubes should be removed (salpingectomy) in women with germline BRCA1 or BRCA2 mutations, or in wom‐ en with a strong family history of breast and/or ovarian cancer34. However, until compre‐ hensive comparative data are available, it is premature to recommend that only the fallopian

Women who have completed childbearing are candidates for surgery. For the majority of women, this surgery can be performed laparoscopically as an outpatient procedure. In con‐ trast to surveillance and chemoprevention, this intervention is very effective in reducing the risk of ovarian cancer. Bilateral tubal ligation and hysterectomy are associated with reduced ovarian cancer risk, including in BRCA1/BRCA2 mutation carriers. Ovarian cancer risk is re‐ duced more than 90% in women with documented BRCA1 or BRCA2 mutations who chose risk-reducing salpingo-oophorectomy. In this same population, prophylactic removal of the ovaries also resulted in a nearly 50% reduction in the risk of subsequent breast cancer. In a retrospective analysis of 551 patients, Rebbeck et al showed that women who had under‐ gone prophylactic salpingo-oophorectomy had an odds ratio of 0.04 for ovarian cancer, com‐ pared with carriers without prophylactic salpingo-oophorectomy. Over a median follow-up of 8.8 years, two primary peritoneal cancers were diagnosed in the 259 women who under‐ went prophylactic salpingo-oophorectomy compared with 58 ovarian/peritoneal cancers in the 292 women who did not have prophylactic salpingo-oophorectomy. An added benefit

before initiation [1-5, 18-29].

148 Ovarian Cancer - A Clinical and Translational Update

tubes are removed in high-risk women [1-5, 30-43].

salpingo-oophorectomy between ages 35 to 40 years. However, performing prophylactic sal‐ pingo-oophorectomy before age 45 must be considered in the context of the potential mor‐ bidity of estrogen deprivation at an early age. Oophorectomy before age 45 has been associated with a hazard ratio of 1.96 for death from all causes (p 0.002). However, adminis‐ tration of estrogen replacement eliminated this risk. Many physicians consider estrogen therapy for women without a personal history of breast cancer who undergo prophylactic salpingo-oophorectomy before the age of 45.29 It should be noted that early prophylactic salpingo-oophorectomy is less important for BRCA2 carriers who are known to develop ovarian cancer at approximately the same age as patients with sporadic cancer. Only 1.2% of BRCA2 carriers will have ovarian cancer by the age of 50, so prophylactic salpingo-oopho‐ rectomy may safely be delayed until these patients are closer to menopause. The disadvan‐ tages of prophylactic salpingo-oophorectomy include the fact that it is an invasive surgical intervention, there is loss of ovarian tissue with accompanying hormone deprivation, and it is an irreversible decision. However, in contrast to surveillance and chemoprevention, pro‐ phylactic salpingo-oophorectomy has proven efficacy over an extended time period. Cost analyses comparing surveillance, oral contraceptives, and prophylactic salpingo-oophorec‐ tomy have shown that although any primary prevention strategy was cost effective, prophy‐ lactic salpingo-oophorectomy dominated all other strategies in women with BRCA mutations. Consequently, prophylactic salpingo-oophorectomy is recommended for all BRCA carriers, with timing dependent on the type of BRCA1 or BRCA2 mutation, childbear‐ ing status, and the age of onset of ovarian cancer within the family. The resultant physical and emotional outcomes of repeated gynecological screening or prophylactic oophorectomy must be discussed before and after genetic testing. A study of 315 women with documented HNPCC–associated germline mutations found no ovarian cancer among 47 women who had bilateral salpingo-oophorectomy and 12 cases (5%) among women with mutations who had not had surgery for a prevented fraction of 100% (95% CI, 62%–100%).

**6. Treatment**

lapses and improved overall survival [44].

Over the past ten years, the focus of management for BRCA1/2mutation carriers has been on cancer prevention and early cancer detection. However, despite prophylactic measures to reduce risk of EOC, many BRCA1/2 carriers have cancer at the time their mutation is diag‐ nosed and more will develop in the future. The treatment of patients with BRCA associated EOC is so far identical to those with sporadic disease. Data suggested that cancers associat‐ ed with BRCA mutations responded differently to chemotherapy. Tan et al. compared 22 BRCA-positive patients with EOC to 44 nonhereditary EOC controls in a matched case-con‐ trol study. They found that BRCA-positive patients have higher response rates to first line platinum-based treatment (81.8%versus 43.2%, P =.004) as well as to subsequent lines of platinum-based treatments (second line, 91.7% versus 40.9%, P =.004), third line, 100% v 14,3% (P<. 002) and time of first relapse (5v 1.6 years; P<. 001). They conclude that BRCApositive EOC patients have better outcomes than nonhereditary EOC cases. There exists a clinical syndrome of BRCAness that includes serous histology, high response rates to first and subsequent lines of platinum-based treatment, longer tumor free interval between re‐

The Genetics of Ovarian Cancer http://dx.doi.org/10.5772/54541 151

Over recent years the investigation of DNA repair in cancer cells has been a very active area of translational research. All cells have a number of overlapping pathways to protect the ge‐ nome from DNA damage, which occurs as a result of normal cell cycling, environmental in‐ sults, or cytotoxic chemotherapy. It is well recognized that when mutations occur within these DNA repair pathways there is an increased risk of malignant transformation and che‐ motherapy resistance. Much research has focused on protecting cells from DNA damage and/or restoring DNA repair function. However, emerging data suggest that the concept of "synthetic lethality," that is, exploiting the vulnerability of cancer cells, which have lost one mechanism of DNA repair by targeting a second pathway, may be a particularly attractive therapeutic approach.. Targeting the nuclear enzyme PARP-1 represents a new and novel approach to the treatment of EOC and appears to be particularly promising for those carry‐ ing mutations in the BRCA1 and 2 genes. Poly(ADPribose) polymerase (PARP) is an en‐ zyme, which plays an important role in the recognition and repair of single-strand DNA breaks via the base excision repair pathway. Over the last few years it has become apparent that in cells, which have lost BRCA1 or BRCA2, components of a second DNA repair path‐ way, homologous recombination, are particularly sensitive to PARP inhibition. These data suggest that PARP inhibitors may be particularly useful for the treatment of women with hereditary BRCA1/2-associated EOC. Targeted therapy using PARP inhibitors has become an important novel strategy for treating those with hereditary ovarian cancer. Furthermore the identification of other subpopulations of women with EOC who may benefit from this approach is an active area of research. There are currently 17 members of the PARP super‐ family identified. PARP-1 is the most studied enzyme. In the preclinical setting, PARP-1 in‐ hibitors enhance the cytotoxic effects of ionizing radiation and cytotoxic chemotherapy. Additionally, in the preclinical setting, the use of PARP-1 inhibitors as single agents did not cause any measurable toxicity, but the combination of PARP-1 inhibitor with temozolomide in the tumor bearing mice caused significant toxicity. There did not seem to be a correlation,

The degree of risk of ovarian cancer, potential morbidity and mortality of surgery, and the risks associated with early menopause, should be taken into account when consider‐ ing prophylactic oophorectomy for high-risk women. Adverse effects of bilateral oopho‐ rectomy and premature menopause include infertility, vasomotor symptoms, decline in sexual interest and activity, cardiovascular disease, and osteoporosis. Among women who have not taken hormone therapy, women undergoing bilateral oophorecotmy were twice as likely to have moderate or severe hot flashes than women who underwent nat‐ ural menopause (odds ratio [OR] = 2.44; 95% CI, 1.03–5.77). Women at increased heredi‐ tary risk of ovarian cancer who underwent oophorectomy without hormone therapy reported statistically significantly more vasomotor symptoms than women choosing screening or those using hormone replacement therapy (HRT). These women also report‐ ed lower sexual function scores but the difference was not statistically significant. A meta-analysis of early menopause as a risk factor for cardiovascular disease observed a pooled risk of 4.55 (95% CI, 2.56–8.01) among women with bilateral oophorectomy and early menopause (defined as younger than 50 years). Early menopause is also associated with an increased risk of fracture (OR = 1.5; 95% CI, 1.2–1.8).

#### **6. Treatment**

salpingo-oophorectomy between ages 35 to 40 years. However, performing prophylactic sal‐ pingo-oophorectomy before age 45 must be considered in the context of the potential mor‐ bidity of estrogen deprivation at an early age. Oophorectomy before age 45 has been associated with a hazard ratio of 1.96 for death from all causes (p 0.002). However, adminis‐ tration of estrogen replacement eliminated this risk. Many physicians consider estrogen therapy for women without a personal history of breast cancer who undergo prophylactic salpingo-oophorectomy before the age of 45.29 It should be noted that early prophylactic salpingo-oophorectomy is less important for BRCA2 carriers who are known to develop ovarian cancer at approximately the same age as patients with sporadic cancer. Only 1.2% of BRCA2 carriers will have ovarian cancer by the age of 50, so prophylactic salpingo-oopho‐ rectomy may safely be delayed until these patients are closer to menopause. The disadvan‐ tages of prophylactic salpingo-oophorectomy include the fact that it is an invasive surgical intervention, there is loss of ovarian tissue with accompanying hormone deprivation, and it is an irreversible decision. However, in contrast to surveillance and chemoprevention, pro‐ phylactic salpingo-oophorectomy has proven efficacy over an extended time period. Cost analyses comparing surveillance, oral contraceptives, and prophylactic salpingo-oophorec‐ tomy have shown that although any primary prevention strategy was cost effective, prophy‐ lactic salpingo-oophorectomy dominated all other strategies in women with BRCA mutations. Consequently, prophylactic salpingo-oophorectomy is recommended for all BRCA carriers, with timing dependent on the type of BRCA1 or BRCA2 mutation, childbear‐ ing status, and the age of onset of ovarian cancer within the family. The resultant physical and emotional outcomes of repeated gynecological screening or prophylactic oophorectomy must be discussed before and after genetic testing. A study of 315 women with documented HNPCC–associated germline mutations found no ovarian cancer among 47 women who had bilateral salpingo-oophorectomy and 12 cases (5%) among women with mutations who

150 Ovarian Cancer - A Clinical and Translational Update

had not had surgery for a prevented fraction of 100% (95% CI, 62%–100%).

with an increased risk of fracture (OR = 1.5; 95% CI, 1.2–1.8).

The degree of risk of ovarian cancer, potential morbidity and mortality of surgery, and the risks associated with early menopause, should be taken into account when consider‐ ing prophylactic oophorectomy for high-risk women. Adverse effects of bilateral oopho‐ rectomy and premature menopause include infertility, vasomotor symptoms, decline in sexual interest and activity, cardiovascular disease, and osteoporosis. Among women who have not taken hormone therapy, women undergoing bilateral oophorecotmy were twice as likely to have moderate or severe hot flashes than women who underwent nat‐ ural menopause (odds ratio [OR] = 2.44; 95% CI, 1.03–5.77). Women at increased heredi‐ tary risk of ovarian cancer who underwent oophorectomy without hormone therapy reported statistically significantly more vasomotor symptoms than women choosing screening or those using hormone replacement therapy (HRT). These women also report‐ ed lower sexual function scores but the difference was not statistically significant. A meta-analysis of early menopause as a risk factor for cardiovascular disease observed a pooled risk of 4.55 (95% CI, 2.56–8.01) among women with bilateral oophorectomy and early menopause (defined as younger than 50 years). Early menopause is also associated Over the past ten years, the focus of management for BRCA1/2mutation carriers has been on cancer prevention and early cancer detection. However, despite prophylactic measures to reduce risk of EOC, many BRCA1/2 carriers have cancer at the time their mutation is diag‐ nosed and more will develop in the future. The treatment of patients with BRCA associated EOC is so far identical to those with sporadic disease. Data suggested that cancers associat‐ ed with BRCA mutations responded differently to chemotherapy. Tan et al. compared 22 BRCA-positive patients with EOC to 44 nonhereditary EOC controls in a matched case-con‐ trol study. They found that BRCA-positive patients have higher response rates to first line platinum-based treatment (81.8%versus 43.2%, P =.004) as well as to subsequent lines of platinum-based treatments (second line, 91.7% versus 40.9%, P =.004), third line, 100% v 14,3% (P<. 002) and time of first relapse (5v 1.6 years; P<. 001). They conclude that BRCApositive EOC patients have better outcomes than nonhereditary EOC cases. There exists a clinical syndrome of BRCAness that includes serous histology, high response rates to first and subsequent lines of platinum-based treatment, longer tumor free interval between re‐ lapses and improved overall survival [44].

Over recent years the investigation of DNA repair in cancer cells has been a very active area of translational research. All cells have a number of overlapping pathways to protect the ge‐ nome from DNA damage, which occurs as a result of normal cell cycling, environmental in‐ sults, or cytotoxic chemotherapy. It is well recognized that when mutations occur within these DNA repair pathways there is an increased risk of malignant transformation and che‐ motherapy resistance. Much research has focused on protecting cells from DNA damage and/or restoring DNA repair function. However, emerging data suggest that the concept of "synthetic lethality," that is, exploiting the vulnerability of cancer cells, which have lost one mechanism of DNA repair by targeting a second pathway, may be a particularly attractive therapeutic approach.. Targeting the nuclear enzyme PARP-1 represents a new and novel approach to the treatment of EOC and appears to be particularly promising for those carry‐ ing mutations in the BRCA1 and 2 genes. Poly(ADPribose) polymerase (PARP) is an en‐ zyme, which plays an important role in the recognition and repair of single-strand DNA breaks via the base excision repair pathway. Over the last few years it has become apparent that in cells, which have lost BRCA1 or BRCA2, components of a second DNA repair path‐ way, homologous recombination, are particularly sensitive to PARP inhibition. These data suggest that PARP inhibitors may be particularly useful for the treatment of women with hereditary BRCA1/2-associated EOC. Targeted therapy using PARP inhibitors has become an important novel strategy for treating those with hereditary ovarian cancer. Furthermore the identification of other subpopulations of women with EOC who may benefit from this approach is an active area of research. There are currently 17 members of the PARP super‐ family identified. PARP-1 is the most studied enzyme. In the preclinical setting, PARP-1 in‐ hibitors enhance the cytotoxic effects of ionizing radiation and cytotoxic chemotherapy. Additionally, in the preclinical setting, the use of PARP-1 inhibitors as single agents did not cause any measurable toxicity, but the combination of PARP-1 inhibitor with temozolomide in the tumor bearing mice caused significant toxicity. There did not seem to be a correlation,

however, between the antitumor activity and the toxicity of the PARP inhibitor-temozolo‐ mide combinations, suggesting that toxicity and chemosensitization were by different mech‐ anisms. In 2005, two preclinical papers demonstrated the sensitivity of BRCA1- and BRCA2 deficient cell lines to PARP inhibition. The first paper by Bryant et al. demonstrated reduced survival of BRCA2-deficient cell lines with four PARP inhibitors. They concluded that BRCA2- deficient cells were sensitive to PARP inhibition, and that monotherapy with one of these agents could selectively kill cancer cells. In the same year, Farmer et al. demonstrated how both BRCA1- and BRCA2-deficient cells lines were sensitive to inhibition of PARP-1, and that BRCA2 deficient cells were more than 1000 times more sensitive to nanomolar con‐ centrations of PARP inhibitor. Both of these papers demonstrated how homozygotes (tumor cells) are sensitive to the mechanism of PARP inhibition, whereas heterozygotes (the rest of the patient's cells) are insensitive to this mechanism and should not exhibit toxicity. These findings from two independent groups using different chemical classes of PARP inhibitors on different BRCA deficient cell lines were the first to suggest the potent effect of PARP in‐ hibition. A number of PARP inhibitors have entered the clinic in both intravenous and oral formulations. The four, which are furthest along in terms of development, are AGO14699 (Pfizer), AZD2281 (AstraZeneca), ABT-888 (Abbott), and BSI-201 (BI Par), and all four of these compounds demonstrate profound inhibition of PARP-1.Olaparib (AZD2281, KU-0059436, AstraZeneca) is an oral small-molecule PARP inhibitor. Yap et al. presented the first clinical evidence demonstrating the sensitivity of BRCA-mutated cancers to PARP in‐ hibitor monotherapy in a study in 2007. This phase I trial included 44 patients, of which 11 patients had a BRCA mutation associated cancer. Dose escalation was guided by toxicity, pharmacokinetic and pharmacodynamic data. Based on the encouraging antitumor activity, many in whom had BRCA1/2 mutations, the trial was subsequently expanded to concentrate on cancers in patients with BRCA mutations. The drug was well tolerated in both BRCA mutated and normal populations. Most toxicities were grade 1-2 (≥95%), consisting of fati‐ gue (28%), nausea (28%), vomiting (18%), loss of taste (13%), and anorexia (12%). Grade 3-4 toxicities were rare, consisting of myelosuppression (≤5%), nausea and vomiting (2-3%), and dizziness or mood changes (2- 3%) [27]. Of the 60 patients that were enrolled and treated, 19 of 23 BRCA-positive carriers were evaluable. 12 of the 19 (63%) had a clinical benefit from olaparib, with radiologic or tumor marker responses, or stable disease for 4 months or more. Patient response was seen in those receiving a minimum of 100 mg twice daily up to 400 mg twice daily. Response was the greatest in patients with platinum-sensitive disease, although duration of response was the same regardless of the platinum-free interval. Recently data was presented from a phase II study of olaparib in women with advanced EOC with known mutations in BRCA1/2. Two patient cohorts received continuous oral olaparib in 28-day cy‐ cles; 33 patients received 400 mg orally twice daily, while 24 patients received 100 mg twice daily. The choice of dosing and schedule was based on the phase I trial above. The objective response rate measured by RECIST criteria was 33% at the 400 mg dose, and 12.5% at the 100 mg dose, suggesting that there may be a dose response effect. The toxicity profile was mainly mild, consisting of grade 1 or 2 nausea (44%) and fatigue (35%), with few grade 3 or 4 toxicities. Interestingly, although numbers were low, in this study there appeared to be a higher response rate in platinum resistant patients (38% versus 14%), which was opposite to

that observed in the earlier phase I study, where response was the greatest in platinum-sen‐ sitive patients. Laboratory studies have previously suggested that platinum resistant pa‐ tients may reacquire BRCA function thus potentially making them resistant to the effects of PARP inhibition. Taken together, the clinical data suggest that we still have a lot to learn with regard to target populations and the role of PARP inhibition. Furthermore, data from the phase II study appears to give an early indication that response (both RECIST and CA125) may be greater in those patients with BRCA2 mutations. This would be in line with the known mechanism of action of the two BRCA proteins as BRCA2 plays a key role in the repair pathway; whereas BRCA1 functions as a signaling molecule. This phase II study con‐ cluded that oral olaparib is well-tolerated and highly active in advanced, chemotherapy re‐ fractory BRCA-deficient EOC, with greater activity seen at a higher dose of 400 mg twice daily. The optimal patient group with respect to platinum sensitivity has not been defined. Reassuringly in the clinical studies there does not appear to be an increase in toxicity be‐ tween BRCA mutation carriers compared to noncarriers, supporting the theory that PARP inhibitors should not result in increased toxicity to heterozygote cells. These recent phase I and phase II trials are particularly promising for patients with BRCA-associated EOC. Fur‐ ther phase II trials are currently underway which will help further elucidate the role and po‐ tential for this new targeted therapy. Loss of BRCA1/2 function is not exclusive to inheriting a mutation in the BRCA1/2 genes. The results seen in known BRCA1 and 2 mutation carriers may also be relevant to the sporadic EOC patient population. Epigenetic gene inactivation is a well-recognized phenomenon with 31% of EOC exhibiting aberrant methylation of the BRCA1 promoter. Furthermore, genetic or epigenetic events occurring in other components of the HR pathway can be found in sporadic EOC. These tumors seem to be similar to BRCA1- or BRCA2-mutated tumors, even though they do not have mutations to either of these genes, a concept called "BRCAness." One molecular characterization study suggested that over 50% of patients with high-grade EOC had loss of BRCA function, either by genetic or epigenetic events [34]. Studies have shown that the loss of functional proteins in the HR pathway may lead these cells to be sensitive to PARP inhibition. Identification of "BRCAlike" EOC populations who may benefit from this new therapy through the identification and validation of biomarkers is an active area of ongoing research. Several PARP inhibitors are under investigation either as single agents and/or in combination with other agents or treatment modalities. Phase II studies in women with advanced EOC in both BRCA1/2 mu‐ tation carriers and high-grade EOC of unknown BRCA status are ongoing. Currently, ola‐ parib is being evaluated in a randomized phase II trial comparing this agent with pegylated liposomal doxorubicin in patients with BRCA-mutated EOC with a platinum-free interval of 0–12 months. More combination studies in women with both hereditary and sporadic EOC are expected in the future. Further defining the role of PARP inhibitors in the clinic is ongo‐ ing. Olaparib is being evaluated in a randomized placebo-controlled trial as a maintenance therapy in patients with sporadic EOC at high risk of early recurrence. Furthermore, some suggest that PARP inhibitors could be used to prevent cancers in patients who are BRCA mutation carriers. This approach, however, requires careful consideration and some caution with the potential for the development of drug resistance in long-term use of PARP inhibi‐ tors. Investigation of the PARP inhibitors in the nonhereditary EOC population is very ac‐

The Genetics of Ovarian Cancer http://dx.doi.org/10.5772/54541 153

that observed in the earlier phase I study, where response was the greatest in platinum-sen‐ sitive patients. Laboratory studies have previously suggested that platinum resistant pa‐ tients may reacquire BRCA function thus potentially making them resistant to the effects of PARP inhibition. Taken together, the clinical data suggest that we still have a lot to learn with regard to target populations and the role of PARP inhibition. Furthermore, data from the phase II study appears to give an early indication that response (both RECIST and CA125) may be greater in those patients with BRCA2 mutations. This would be in line with the known mechanism of action of the two BRCA proteins as BRCA2 plays a key role in the repair pathway; whereas BRCA1 functions as a signaling molecule. This phase II study con‐ cluded that oral olaparib is well-tolerated and highly active in advanced, chemotherapy re‐ fractory BRCA-deficient EOC, with greater activity seen at a higher dose of 400 mg twice daily. The optimal patient group with respect to platinum sensitivity has not been defined. Reassuringly in the clinical studies there does not appear to be an increase in toxicity be‐ tween BRCA mutation carriers compared to noncarriers, supporting the theory that PARP inhibitors should not result in increased toxicity to heterozygote cells. These recent phase I and phase II trials are particularly promising for patients with BRCA-associated EOC. Fur‐ ther phase II trials are currently underway which will help further elucidate the role and po‐ tential for this new targeted therapy. Loss of BRCA1/2 function is not exclusive to inheriting a mutation in the BRCA1/2 genes. The results seen in known BRCA1 and 2 mutation carriers may also be relevant to the sporadic EOC patient population. Epigenetic gene inactivation is a well-recognized phenomenon with 31% of EOC exhibiting aberrant methylation of the BRCA1 promoter. Furthermore, genetic or epigenetic events occurring in other components of the HR pathway can be found in sporadic EOC. These tumors seem to be similar to BRCA1- or BRCA2-mutated tumors, even though they do not have mutations to either of these genes, a concept called "BRCAness." One molecular characterization study suggested that over 50% of patients with high-grade EOC had loss of BRCA function, either by genetic or epigenetic events [34]. Studies have shown that the loss of functional proteins in the HR pathway may lead these cells to be sensitive to PARP inhibition. Identification of "BRCAlike" EOC populations who may benefit from this new therapy through the identification and validation of biomarkers is an active area of ongoing research. Several PARP inhibitors are under investigation either as single agents and/or in combination with other agents or treatment modalities. Phase II studies in women with advanced EOC in both BRCA1/2 mu‐ tation carriers and high-grade EOC of unknown BRCA status are ongoing. Currently, ola‐ parib is being evaluated in a randomized phase II trial comparing this agent with pegylated liposomal doxorubicin in patients with BRCA-mutated EOC with a platinum-free interval of 0–12 months. More combination studies in women with both hereditary and sporadic EOC are expected in the future. Further defining the role of PARP inhibitors in the clinic is ongo‐ ing. Olaparib is being evaluated in a randomized placebo-controlled trial as a maintenance therapy in patients with sporadic EOC at high risk of early recurrence. Furthermore, some suggest that PARP inhibitors could be used to prevent cancers in patients who are BRCA mutation carriers. This approach, however, requires careful consideration and some caution with the potential for the development of drug resistance in long-term use of PARP inhibi‐ tors. Investigation of the PARP inhibitors in the nonhereditary EOC population is very ac‐

however, between the antitumor activity and the toxicity of the PARP inhibitor-temozolo‐ mide combinations, suggesting that toxicity and chemosensitization were by different mech‐ anisms. In 2005, two preclinical papers demonstrated the sensitivity of BRCA1- and BRCA2 deficient cell lines to PARP inhibition. The first paper by Bryant et al. demonstrated reduced survival of BRCA2-deficient cell lines with four PARP inhibitors. They concluded that BRCA2- deficient cells were sensitive to PARP inhibition, and that monotherapy with one of these agents could selectively kill cancer cells. In the same year, Farmer et al. demonstrated how both BRCA1- and BRCA2-deficient cells lines were sensitive to inhibition of PARP-1, and that BRCA2 deficient cells were more than 1000 times more sensitive to nanomolar con‐ centrations of PARP inhibitor. Both of these papers demonstrated how homozygotes (tumor cells) are sensitive to the mechanism of PARP inhibition, whereas heterozygotes (the rest of the patient's cells) are insensitive to this mechanism and should not exhibit toxicity. These findings from two independent groups using different chemical classes of PARP inhibitors on different BRCA deficient cell lines were the first to suggest the potent effect of PARP in‐ hibition. A number of PARP inhibitors have entered the clinic in both intravenous and oral formulations. The four, which are furthest along in terms of development, are AGO14699 (Pfizer), AZD2281 (AstraZeneca), ABT-888 (Abbott), and BSI-201 (BI Par), and all four of these compounds demonstrate profound inhibition of PARP-1.Olaparib (AZD2281, KU-0059436, AstraZeneca) is an oral small-molecule PARP inhibitor. Yap et al. presented the first clinical evidence demonstrating the sensitivity of BRCA-mutated cancers to PARP in‐ hibitor monotherapy in a study in 2007. This phase I trial included 44 patients, of which 11 patients had a BRCA mutation associated cancer. Dose escalation was guided by toxicity, pharmacokinetic and pharmacodynamic data. Based on the encouraging antitumor activity, many in whom had BRCA1/2 mutations, the trial was subsequently expanded to concentrate on cancers in patients with BRCA mutations. The drug was well tolerated in both BRCA mutated and normal populations. Most toxicities were grade 1-2 (≥95%), consisting of fati‐ gue (28%), nausea (28%), vomiting (18%), loss of taste (13%), and anorexia (12%). Grade 3-4 toxicities were rare, consisting of myelosuppression (≤5%), nausea and vomiting (2-3%), and dizziness or mood changes (2- 3%) [27]. Of the 60 patients that were enrolled and treated, 19 of 23 BRCA-positive carriers were evaluable. 12 of the 19 (63%) had a clinical benefit from olaparib, with radiologic or tumor marker responses, or stable disease for 4 months or more. Patient response was seen in those receiving a minimum of 100 mg twice daily up to 400 mg twice daily. Response was the greatest in patients with platinum-sensitive disease, although duration of response was the same regardless of the platinum-free interval. Recently data was presented from a phase II study of olaparib in women with advanced EOC with known mutations in BRCA1/2. Two patient cohorts received continuous oral olaparib in 28-day cy‐ cles; 33 patients received 400 mg orally twice daily, while 24 patients received 100 mg twice daily. The choice of dosing and schedule was based on the phase I trial above. The objective response rate measured by RECIST criteria was 33% at the 400 mg dose, and 12.5% at the 100 mg dose, suggesting that there may be a dose response effect. The toxicity profile was mainly mild, consisting of grade 1 or 2 nausea (44%) and fatigue (35%), with few grade 3 or 4 toxicities. Interestingly, although numbers were low, in this study there appeared to be a higher response rate in platinum resistant patients (38% versus 14%), which was opposite to

152 Ovarian Cancer - A Clinical and Translational Update

tive with both the impact of treatment on patients without BRCA defects and the search for populations of women who have lost functional proteins in the HR pathway. Investigation of PARP inhibitor resistance and ways to overcome this resistance are emerging fields. The emerging data regarding the use of PARP inhibitors in patients with BRCA-associated EOC are encouraging. Identification of further patient groups who will benefit from this ap‐ proach is also indicated. Clinical trials underway will hopefully improve the prognosis of women with Epithelial Ovarian Cancer [45-53].

[3] National Cancer Institute: PDQ® Ovarian Cancer Screening. Bethesda, MD: National Cancer Institute. Date last modified 01/26/2012. Available at:http://cancer.gov/cancer‐

The Genetics of Ovarian Cancer http://dx.doi.org/10.5772/54541 155

[4] National Cancer Institute: PDQ® Ovarian Cancer Screening. Bethesda, MD: National Cancer Institute. Date last modified 09/30/2011. Available athttp://cancer.gov/cancer‐

[5] Genetics of Breast and Ovarian Cancer (PDQ) Health Professional Version Last modified 08/08/2012. Available at: http://cancertopics/pdq/genetics/breast-and-ovari‐

[6] American Cancer Society: Cancer Facts and Figures 2012. Atlanta, Ga: American Cancer Society, 2012. Available online http://www.cancer.gov/global/web/policies/

[7] Landen Jr, CN,Birrer MJ and Sood AK. Early Events in the Pathogenesis of Epithelial

[8] Vaughan S et al. Rethinking ovarian cancer: recommendations for improving out‐

[9] Spellman, et al. Integrated Genomic Analyses of Ovarian Carcinoma. Nature

[10] BRCA1 and BRCA2: Cancer Risk and Genetic Testing. National Cancer Institute.

[11] Dowdy SC and Hartmann LC. Management of Hereditary Ovarian Cancer by Ameri‐

[12] Greene MH, Kohlmann W, Popik WC and Offit K. Cancer Genetics in Practice: Cur‐

[13] Risch HA, McLaughlin JR, Cole DE, et al. Prevalence and penetrance of germline BRCA1 and BRCA2 mutations in a population series of 649 women with ovarian can‐

[14] Garber JE, Offit K. Hereditary cancer predisposition syndromes. J Clin Oncol 2005; 23

[15] King MC, Marks JH, Mandell JB. Breast and ovarian cancer risks due to inherited

[16] Reedy M, Gallion H, Fowler JM, et al. Contribution of BRCA1 and BRCA2 to familial ovarian cancer: A gynecologic oncology group study. Gynecol Oncol 2002; 85

[17] Guillem JG, Wood WC, Moley JF, et al. ASCO/SSO review of current role of risk-re‐ ducing surgery in common hereditary cancer syndromes. J Clin Oncol 2006; 24

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276-292.

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4642-4660.

#### **7. Conclusions**

Genetic testing can identify women with a hereditary increased risk to develop Ovarian Cancer. This information is extremely useful if the candidate for genetic testing is willing to accept prophylactic surgery. For patients who already have Ovarian Cancer Genetic testing will offer useful information for the relatives but it can also help plan their own treatment. Published data regarding the use of PARP inhibitors in patients with BRCA-associated EOC are encouraging. Studies in combination with chemotherapy are also producing encourag‐ ing results and there are several ongoing studies in patients with hereditary and sporadic cancer as well. These studies will clarify the mechanisms of DNA repair and how this can be exploited to improve treatment results. The development of diagnostic tests in order to se‐ lect patients likely to be sensitive to PARP inhibitors will also be very useful. The combina‐ tion of prevention, early diagnosis and more effective disease management will hopefully improve EOC prognosis in the near future.

#### **Author details**

#### Constantine Gennatas

2nd Department Of Surgery, Areteion Hospital, University of Athens, Athens, Greece

#### **References**


[3] National Cancer Institute: PDQ® Ovarian Cancer Screening. Bethesda, MD: National Cancer Institute. Date last modified 01/26/2012. Available at:http://cancer.gov/cancer‐ topics/pdq/screening/ovarian/HealthProfessional. Accessed 09/16/2012.

tive with both the impact of treatment on patients without BRCA defects and the search for populations of women who have lost functional proteins in the HR pathway. Investigation of PARP inhibitor resistance and ways to overcome this resistance are emerging fields. The emerging data regarding the use of PARP inhibitors in patients with BRCA-associated EOC are encouraging. Identification of further patient groups who will benefit from this ap‐ proach is also indicated. Clinical trials underway will hopefully improve the prognosis of

Genetic testing can identify women with a hereditary increased risk to develop Ovarian Cancer. This information is extremely useful if the candidate for genetic testing is willing to accept prophylactic surgery. For patients who already have Ovarian Cancer Genetic testing will offer useful information for the relatives but it can also help plan their own treatment. Published data regarding the use of PARP inhibitors in patients with BRCA-associated EOC are encouraging. Studies in combination with chemotherapy are also producing encourag‐ ing results and there are several ongoing studies in patients with hereditary and sporadic cancer as well. These studies will clarify the mechanisms of DNA repair and how this can be exploited to improve treatment results. The development of diagnostic tests in order to se‐ lect patients likely to be sensitive to PARP inhibitors will also be very useful. The combina‐ tion of prevention, early diagnosis and more effective disease management will hopefully

2nd Department Of Surgery, Areteion Hospital, University of Athens, Athens, Greece

[1] National Cancer Institute: PDQ® Ovarian Epithelial Cancer Treatment. Bethesda, MD: National Cancer Institute. Date last modified 06/22/2012. Available at: http:// cancer.gov/cancertopics/pdq/treatment/ovarianepithelial /Health Professional. Ac‐

[2] National Cancer Institute: PDQ® Ovarian Cancer Prevention. Bethesda, MD: Nation‐ al Cancer Institute. Date last modified 02/06/2012. Available at: http://cancer.gov/ cancertopics/pdq/prevention/ovarian/HealthProfessional. Accessed 08/08/2012.

women with Epithelial Ovarian Cancer [45-53].

154 Ovarian Cancer - A Clinical and Translational Update

improve EOC prognosis in the near future.

**7. Conclusions**

**Author details**

**References**

Constantine Gennatas

cessed 09/16/2012.


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156 Ovarian Cancer - A Clinical and Translational Update


[45] Roux R. Zwifel M,Gordon JS and Rustin GJS. Biologicals in the Up front Treatment of Ovarian Cancer: Focus on Bevacizumab and Poly(ADP-Ribose) Polymerase Inhibi‐ tors. Journal of Oncology 2010; Article ID 151750, 6 pages doi:10.1155/2010/151750

**Section 3**

**Ovarian Cancer Therapeutics**


**Ovarian Cancer Therapeutics**

[45] Roux R. Zwifel M,Gordon JS and Rustin GJS. Biologicals in the Up front Treatment of Ovarian Cancer: Focus on Bevacizumab and Poly(ADP-Ribose) Polymerase Inhibi‐ tors. Journal of Oncology 2010; Article ID 151750, 6 pages doi:10.1155/2010/151750

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[47] Huehls AM, Wagner JM, Huntoon CJ, Karnitz LM. Identification of DNA Repair Pathways that Affect the Survival of Ovarian Cancer Cells Treated with a PARP In‐ hibitor in a Novel Drug Combination. Mol Pharmacol 2012; July 25. [Epub ahead of

[48] Marchetti C, Imperiale L, Gasparri ML, Palaia I, Pignata S, Boni T, Bellati F, Benedetti Panici P. Olaparib, PARP1 inhibitor in ovarian cancer.Expert Opin Investig Drugs

[49] Maxwell KN, Domchek SM. Cancer treatment according to BRCA1 and BRCA2 mu‐ tations.Nat Rev Clin Oncol 2012; 14;9(9):520-8. doi: 10.1038/nrclinonc.2012.123. Epub

[50] Marchetti C, Imperiale L, Gasparri ML, Palaia I, Pignata S, Boni T, Bellati F, Benedetti Panici P. Olaparib, PARP1 inhibitor in ovarian cancer.Expert Opin Investig Drugs 2012; 21(10) 1575-84. Epub 2012 Jul 13.PMID22788971Ratner ES, Sartorelli AC, Lin ZP Poly (ADP-ribose) polymerase inhibitors: on the horizon of tailored and personalized therapies for epithelial ovarian cancer. Curr Opin Oncol 2012; 24(5) 564-71. PMID:

[51] Westin SN, Herzog TJ, Coleman RL. Investigational agents in development for the treatment of ovarian cancer.Invest New Drugs 2012; June 4. [Epub ahead of print]

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[53] Tinker AV, Gelmon K. The Role of PARP Inhibitors in the Treatment of Ovarian Car‐

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cinomas. Curr Pharm Des. 2012;18 (25):3770-4. PMID: 22591423

151750, 6 pages doi:10.1155/2010/151750

158 Ovarian Cancer - A Clinical and Translational Update

2012 Jul 24.PMID: 22825375

2012; 21(10):1575-84. Epub 2012 Jul 13.PMID:22788971

print]

22759740.

PMID: 22661305.

**Chapter 8**

**Surgical Treatment of Ovarian Cancer**

Javier Valero de Bernabé and Ivan Diaz-Padilla

Despite great efforts in developing novel screening, diagnosis and therapeutic strategies, the incidence and mortality of ovarian cancer have not significantly changed in the last 30 years. [1] It remains the leading cause of death from gynecologic malignancy with a lifetime proba‐ bility of developing the disease of 1 in 59.[1] Worldwide, approximately 200.000 women are annually diagnosed with ovarian cancer,[2] and almost 70% of them will be diagnosed at ad‐ vanced stage disease.[3] With current treatment modalities, the 5-year survival rate ranges from 80–95% for those with organ-confined or early stage disease (International Federation of Gynecology and Obstetrics (FIGO) stage I-II); to 30 – 40% for those women with advanced dis‐ ease, FIGO stage III-IV. Thus, ovarian cancer is a challenging and complex malignancy.[4]

Surgical management of ovarian cancer remains as the cornerstone treatment of this disease. [5] An adequate full surgical staging in women with early stage disease has demonstrated to improve oncologic outcome.[6] On the other hand, complete surgical cytoreduction is the only modifiable prognosis factor for patients with advanced disease. This chapter will de‐ scribe the rationale and surgical steps for an adequate surgical staging for women with early stage ovarian cancer, and for obtaining the maximal surgical cytoreduction in women affect‐

Approximately 25% of newly diagnosed ovarian cancer will be early stage disease. Progno‐ sis is good with survival rates ranging from 80 % to 95 % when recommended treatment is

and reproduction in any medium, provided the original work is properly cited.

© 2013 Minig et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

**2. Surgical treatment of early stage epithelial ovarian cancer**

Additional information is available at the end of the chapter

Lucas Minig, M. Guadalupe Patrono,

ed by advanced stage and relapsed disease.

Rafael Alvarez Gallego,

http://dx.doi.org/10.5772/53972

**1. Introduction**

### **Surgical Treatment of Ovarian Cancer**

Lucas Minig, M. Guadalupe Patrono, Rafael Alvarez Gallego, Javier Valero de Bernabé and Ivan Diaz-Padilla

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53972

#### **1. Introduction**

Despite great efforts in developing novel screening, diagnosis and therapeutic strategies, the incidence and mortality of ovarian cancer have not significantly changed in the last 30 years. [1] It remains the leading cause of death from gynecologic malignancy with a lifetime proba‐ bility of developing the disease of 1 in 59.[1] Worldwide, approximately 200.000 women are annually diagnosed with ovarian cancer,[2] and almost 70% of them will be diagnosed at ad‐ vanced stage disease.[3] With current treatment modalities, the 5-year survival rate ranges from 80–95% for those with organ-confined or early stage disease (International Federation of Gynecology and Obstetrics (FIGO) stage I-II); to 30 – 40% for those women with advanced dis‐ ease, FIGO stage III-IV. Thus, ovarian cancer is a challenging and complex malignancy.[4]

Surgical management of ovarian cancer remains as the cornerstone treatment of this disease. [5] An adequate full surgical staging in women with early stage disease has demonstrated to improve oncologic outcome.[6] On the other hand, complete surgical cytoreduction is the only modifiable prognosis factor for patients with advanced disease. This chapter will de‐ scribe the rationale and surgical steps for an adequate surgical staging for women with early stage ovarian cancer, and for obtaining the maximal surgical cytoreduction in women affect‐ ed by advanced stage and relapsed disease.

#### **2. Surgical treatment of early stage epithelial ovarian cancer**

Approximately 25% of newly diagnosed ovarian cancer will be early stage disease. Progno‐ sis is good with survival rates ranging from 80 % to 95 % when recommended treatment is

© 2013 Minig et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

followed.[5] These patients are initially managed by comprehensive surgical staging, which is relevant not only for identifying women with truly early stage disease, but also to select patients who will be candidates for adjuvant chemotherapy.

Peritoneal washing from paracolic gutters, pelvis and abdominal cavity should be done in the absence of ascites. It is estimated that over 30% of patients with stage I disease have tu‐ moral cells on cytological examination.[11] Careful inspection and palpation is preformed to detect extra-ovarian implants in a systematic way: starting by right paracolic space, advanc‐ ing the hand to the right kidney, suprahepatic space, the right diaphragm, right hepatic lobe, gallbladder, Morrison´s pouch, left hemi-diaphragm, left hepatic lobe, spleen, stomach, transverse colon, left kidney and left paracolic space. The lesser sac is entered on the left side of the gastrocolic ligament. Both surfaces of the mesentery should be examined and retro‐ peritoneal vascular areas should be palpated as well. The result of this comprehensive pro‐

Surgical Treatment of Ovarian Cancer http://dx.doi.org/10.5772/53972 163

The ovaries need to be examined for capsule rupture or external excrescences. The affected ovary must then be removed for frozen section. Although the influence on the prognosis of the intraoperative rupture of malignant ovarian tumors is controversial,[12] adnexal masses should be removed intact. If malignancy is confirmed in the frozen section, full surgical staging, as previously described, must be performed by the extension of the incision up to xifoid appendix. Contralateral oophorectomy and total hysterectomy is completed due to

Even though controversial, random peritoneal biopsies are indicated in early-stage disease. A retrospective study demonstrated that less than 4% of patients with ovarian cancer were upstaged due to positive peritoneal biopsies. No patient, however, had a change in treat‐ ment recommendations based on these biopsies.[13] Infracolic omentectomy should be per‐ formed from the hepatic to splenic flexure. During dissection, the lesser sac is developed dissecting the posterior and anterior layer of the transverse mesocolon, while preserving the middle colic artery. The omentum is removed and the pedicles are sequentially sutured –

The incidence of lymph-node involvement in patients with disease confined to the ovary is 5% in only pelvic nodes, 9% in aortic nodes and 6% in both pelvic and aortic nodes.[14] Sys‐ tematic lymphadenectomy as part of surgical staging of apparent early stage ovarian cancer is associated with a statistically significant increase in median operative time, median blood loss, and the proportion of patients undergoing blood transfusions.[15] Systematic lympha‐ denectomy, however, significantly improves progression-free survival (PFS) rates, without a statistically significant impact on overall survival (OS). [14, 15] Lymphatic drainage of the ovaries is known to follow the gonadal blood supply that reaches the renal vein, on the left side, and the inferior vena cava, on the right side. Pelvic lymphadenectomy should include removal of nodes from paravesical and pararectal spaces, including bilateral common iliac

nodes. Aortic nodes should be removed from aortic bifurcation to the renal veins.[14]

ligated. Appendectomy is only reserved for mucinous histology.

**5. Retropetitoneal lymph node dissection**

cedure should be properly described.

the possibility of synchronous cancer.

#### **3. Rationale for surgical staging**

Adequate surgical staging procedures include: exploration of abdomen/pelvis, peritoneal washings, bilateral salpingo-oophorectomy, hysterectomy, peritoneal biopsies of Cul-de-sac, pelvic walls, paracolic gutters, diaphragm, suspicious areas, omentectomy, appendectomy, as well as pelvic and para-aortic node dissection up to the renal veins. (TABLE 1)[7],[8] These procedures are needed to find hidden disease in nearly 18% of women[8], which has implications in the prognosis and subsequent patient treatment.[9] Surgeon expertise is cru‐ cial given that it was correlated with under-staged ovarian cancer. Several studies demon‐ strated that over 30% of patients operated by general gynecologists or general surgeons were upstaged by gynecologist oncologists by finding disease on pelvic-aortic lymph nodes, diaphragm biopsies and omentum.[6, 10] Moreover, as it has been demonstrated, inade‐ quate initial surgical staging leads to a higher risk of developing recurrent disease despite receiving adjuvant chemotherapy.[6] Thus, if the operative risk is not too high, all patients should be routinely re-staged before starting chemotherapy.


**Table 1.** Surgical staging procedures for early stage ovarian cancer

#### **4. Surgical staging procedures**

Midline vertical incision is the recommended surgical approach for initial management of suspected early stage ovarian cancer. The incision is firstly made from the pubis to the um‐ bilicus and then progressed to xifoid appendix, if surgical staging is indicated following the frozen section diagnosis. The abdominal-pelvic cavity is opened and visualized. If free fluid is present, a minimum sample of 100 cc[3] should be obtained for cytological examination.

<sup>•</sup> Peritoneal cytology/ascites drainage

<sup>•</sup> Careful and systematic abdominal exploration – inspect and palpate all peritoneal surface

Peritoneal washing from paracolic gutters, pelvis and abdominal cavity should be done in the absence of ascites. It is estimated that over 30% of patients with stage I disease have tu‐ moral cells on cytological examination.[11] Careful inspection and palpation is preformed to detect extra-ovarian implants in a systematic way: starting by right paracolic space, advanc‐ ing the hand to the right kidney, suprahepatic space, the right diaphragm, right hepatic lobe, gallbladder, Morrison´s pouch, left hemi-diaphragm, left hepatic lobe, spleen, stomach, transverse colon, left kidney and left paracolic space. The lesser sac is entered on the left side of the gastrocolic ligament. Both surfaces of the mesentery should be examined and retro‐ peritoneal vascular areas should be palpated as well. The result of this comprehensive pro‐ cedure should be properly described.

The ovaries need to be examined for capsule rupture or external excrescences. The affected ovary must then be removed for frozen section. Although the influence on the prognosis of the intraoperative rupture of malignant ovarian tumors is controversial,[12] adnexal masses should be removed intact. If malignancy is confirmed in the frozen section, full surgical staging, as previously described, must be performed by the extension of the incision up to xifoid appendix. Contralateral oophorectomy and total hysterectomy is completed due to the possibility of synchronous cancer.

Even though controversial, random peritoneal biopsies are indicated in early-stage disease. A retrospective study demonstrated that less than 4% of patients with ovarian cancer were upstaged due to positive peritoneal biopsies. No patient, however, had a change in treat‐ ment recommendations based on these biopsies.[13] Infracolic omentectomy should be per‐ formed from the hepatic to splenic flexure. During dissection, the lesser sac is developed dissecting the posterior and anterior layer of the transverse mesocolon, while preserving the middle colic artery. The omentum is removed and the pedicles are sequentially sutured – ligated. Appendectomy is only reserved for mucinous histology.

#### **5. Retropetitoneal lymph node dissection**

followed.[5] These patients are initially managed by comprehensive surgical staging, which is relevant not only for identifying women with truly early stage disease, but also to select

Adequate surgical staging procedures include: exploration of abdomen/pelvis, peritoneal washings, bilateral salpingo-oophorectomy, hysterectomy, peritoneal biopsies of Cul-de-sac, pelvic walls, paracolic gutters, diaphragm, suspicious areas, omentectomy, appendectomy, as well as pelvic and para-aortic node dissection up to the renal veins. (TABLE 1)[7],[8] These procedures are needed to find hidden disease in nearly 18% of women[8], which has implications in the prognosis and subsequent patient treatment.[9] Surgeon expertise is cru‐ cial given that it was correlated with under-staged ovarian cancer. Several studies demon‐ strated that over 30% of patients operated by general gynecologists or general surgeons were upstaged by gynecologist oncologists by finding disease on pelvic-aortic lymph nodes, diaphragm biopsies and omentum.[6, 10] Moreover, as it has been demonstrated, inade‐ quate initial surgical staging leads to a higher risk of developing recurrent disease despite receiving adjuvant chemotherapy.[6] Thus, if the operative risk is not too high, all patients

patients who will be candidates for adjuvant chemotherapy.

should be routinely re-staged before starting chemotherapy.

• Total abdominal hysterectomy and bilateral salpingo-oophorectomy

• Biopsy or scrapings from the undersurface of both diaphragms

**Table 1.** Surgical staging procedures for early stage ovarian cancer

• Careful and systematic abdominal exploration – inspect and palpate all peritoneal surface

• Random and directed peritoneal biopsies – posterior cul-de-sac, bladder reflection, both pelvic sidewalls and both

Midline vertical incision is the recommended surgical approach for initial management of suspected early stage ovarian cancer. The incision is firstly made from the pubis to the um‐ bilicus and then progressed to xifoid appendix, if surgical staging is indicated following the frozen section diagnosis. The abdominal-pelvic cavity is opened and visualized. If free fluid is present, a minimum sample of 100 cc[3] should be obtained for cytological examination.

**3. Rationale for surgical staging**

162 Ovarian Cancer - A Clinical and Translational Update

• Peritoneal cytology/ascites drainage

• Pelvic and aortic lymphadenectomy

• Appendectomy (for mucinous histology)

**4. Surgical staging procedures**

• Infracolicomentectomy

paracolic spaces

The incidence of lymph-node involvement in patients with disease confined to the ovary is 5% in only pelvic nodes, 9% in aortic nodes and 6% in both pelvic and aortic nodes.[14] Sys‐ tematic lymphadenectomy as part of surgical staging of apparent early stage ovarian cancer is associated with a statistically significant increase in median operative time, median blood loss, and the proportion of patients undergoing blood transfusions.[15] Systematic lympha‐ denectomy, however, significantly improves progression-free survival (PFS) rates, without a statistically significant impact on overall survival (OS). [14, 15] Lymphatic drainage of the ovaries is known to follow the gonadal blood supply that reaches the renal vein, on the left side, and the inferior vena cava, on the right side. Pelvic lymphadenectomy should include removal of nodes from paravesical and pararectal spaces, including bilateral common iliac nodes. Aortic nodes should be removed from aortic bifurcation to the renal veins.[14]

#### **6. Minimally invasive surgery for surgical staging ovarian cancer**

Over the last years, laparoscopy has gained an important role for the management of suspect‐ ed adnexal masses. High-volume centers have reported their experience in performing a com‐ prehensive surgical staging by using minimally invasive surgery.[16],[17] Nezhat et al. [16] reported a case series of 36 patients with early stage invasive ovarian carcinoma managed by laparoscopy. They showed 100% OS rate with a mean duration of follow-up of 55.9 months. Chi et al. [17] conducted a case control study by staging 20 patients with early ovarian cancer with laparoscopy compared with 30 patients staged with laparotomy. There were no differen‐ ces in the omental specimen size or number of lymph nodes removed. Blood loss and hospital stay were lower for the laparoscopy group, with longer operating time. There were no conver‐ sions to laparotomy or other intraoperative complications in the laparoscopy group.

worldwide recommendation consists of primary maximal surgical cytoreduction followed

served for selected patients and it includes surgical cytoreduction in between chemotherapy courses, usually after three or four cycles. This strategy is called neoadjuvant chemotherapy followed by interval debulking surgery. (fig 1) The appropriate selection of patients for each

Prognostic factors in women with advanced stage EOC was described in literature based on

three prospective randomized phase III multicenter trials, which enrolled 3388 patients with advanced EOC between 1995 and 2002. Univariate and multivariate analysis revealed nonmodifiable significant prognostic factors for OS and PFS such as: age, performance status (ECOG 2 versus 0-1), FIGO stage (IIIC-IV versus IIB-IIIB), subtype histology (Mucinous ver‐ sus serous), histology grade (grade 2-3 versus 1), presence of large volume ascites (> 500 mL). The only significant modifiable prognosis factor was postoperative residual tumor (0

23] did a combined exploratory analysis of

5,7] An alternative strategy is re‐

Surgical Treatment of Ovarian Cancer http://dx.doi.org/10.5772/53972 165

by 6 cycles of intravenous carboplatin plus paclitaxel. [

modality of treatment will be described below.

**Figure 1.** Treatment options for advanced stage ovarian cancer

retrospective data.[22] Recently, Du Buois et al., [

**8. Prognostic factors**

Despite laparoscopic staging of early ovarian cancer seems to be a safe and feasible proce‐ dure performed by expert surgeons, the possibility of cyst rupture or port-site metastases re‐ main controversial. The immediate effect of tumor rupture is that a patient with a potentially curable disease will require additional adjuvant chemotherapy. Preoperative evaluation is essential, as well as the surgical experience and the quality of laparoscopic in‐ struments.[18] Even though there are no specific recommendations, adnexal masses up to 5-6 cm could be reasonably managed by laparoscopy.

The etiology of port-site metastases is uncertain. Several hypotheses include tumor cell en‐ trapment, direct spread from the trocar in which instruments are exchanged, and the ''chim‐ ney effect,'' which suggests that tumor cells travel along the sheath of the trocars with the leaking gas. Port-site metastases have been reported in 1% to 2% of patients with ovarian cancer. However, <5% of port metastases are clinically detected and these sites are likely to respond to chemotherapy.[9]

Robotic surgery has emerged as an innovative minimally invasive approach in the field of gynecology. The da Vinci Surgical System (Intuitive Surgical, Inc, Sunnyvale, California, USA) offers several advantages over conventional laparoscopy including three-dimensional view, greater dexterity, and tremor filtration. Most of the data regarding the application of robotic technology for ovarian cancer staging are included in the literature used in the as‐ sessment for its implementation in other gynecologic malignancies, such as cervical and en‐ dometrial cancer.[19] Data are still scarce but promising.

#### **7. Treatment of advanced stage disease: Surgical cytoreduction**

Advanced-stage disease means that the disease is extended to pelvic/ aortic lymph nodes, peritoneum, intra-abdominal organs or disease outside the abdominal cavity.[20] In 1975, a landmark study quantified residual disease and demonstrated for the first time an inverse relationship between residual tumor and oncologic outcome. [ 21] The goal of surgery is to resect as much tumor as possible obtaining, ideally, a complete resection. The standard worldwide recommendation consists of primary maximal surgical cytoreduction followed by 6 cycles of intravenous carboplatin plus paclitaxel. [ 5,7] An alternative strategy is re‐ served for selected patients and it includes surgical cytoreduction in between chemotherapy courses, usually after three or four cycles. This strategy is called neoadjuvant chemotherapy followed by interval debulking surgery. (fig 1) The appropriate selection of patients for each modality of treatment will be described below.

**Figure 1.** Treatment options for advanced stage ovarian cancer

#### **8. Prognostic factors**

**6. Minimally invasive surgery for surgical staging ovarian cancer**

sions to laparotomy or other intraoperative complications in the laparoscopy group.

5-6 cm could be reasonably managed by laparoscopy.

dometrial cancer.[19] Data are still scarce but promising.

relationship between residual tumor and oncologic outcome. [

respond to chemotherapy.[9]

164 Ovarian Cancer - A Clinical and Translational Update

Despite laparoscopic staging of early ovarian cancer seems to be a safe and feasible proce‐ dure performed by expert surgeons, the possibility of cyst rupture or port-site metastases re‐ main controversial. The immediate effect of tumor rupture is that a patient with a potentially curable disease will require additional adjuvant chemotherapy. Preoperative evaluation is essential, as well as the surgical experience and the quality of laparoscopic in‐ struments.[18] Even though there are no specific recommendations, adnexal masses up to

The etiology of port-site metastases is uncertain. Several hypotheses include tumor cell en‐ trapment, direct spread from the trocar in which instruments are exchanged, and the ''chim‐ ney effect,'' which suggests that tumor cells travel along the sheath of the trocars with the leaking gas. Port-site metastases have been reported in 1% to 2% of patients with ovarian cancer. However, <5% of port metastases are clinically detected and these sites are likely to

Robotic surgery has emerged as an innovative minimally invasive approach in the field of gynecology. The da Vinci Surgical System (Intuitive Surgical, Inc, Sunnyvale, California, USA) offers several advantages over conventional laparoscopy including three-dimensional view, greater dexterity, and tremor filtration. Most of the data regarding the application of robotic technology for ovarian cancer staging are included in the literature used in the as‐ sessment for its implementation in other gynecologic malignancies, such as cervical and en‐

Advanced-stage disease means that the disease is extended to pelvic/ aortic lymph nodes, peritoneum, intra-abdominal organs or disease outside the abdominal cavity.[20] In 1975, a landmark study quantified residual disease and demonstrated for the first time an inverse

resect as much tumor as possible obtaining, ideally, a complete resection. The standard

21] The goal of surgery is to

**7. Treatment of advanced stage disease: Surgical cytoreduction**

Over the last years, laparoscopy has gained an important role for the management of suspect‐ ed adnexal masses. High-volume centers have reported their experience in performing a com‐ prehensive surgical staging by using minimally invasive surgery.[16],[17] Nezhat et al. [16] reported a case series of 36 patients with early stage invasive ovarian carcinoma managed by laparoscopy. They showed 100% OS rate with a mean duration of follow-up of 55.9 months. Chi et al. [17] conducted a case control study by staging 20 patients with early ovarian cancer with laparoscopy compared with 30 patients staged with laparotomy. There were no differen‐ ces in the omental specimen size or number of lymph nodes removed. Blood loss and hospital stay were lower for the laparoscopy group, with longer operating time. There were no conver‐

> Prognostic factors in women with advanced stage EOC was described in literature based on retrospective data.[22] Recently, Du Buois et al., [ 23] did a combined exploratory analysis of three prospective randomized phase III multicenter trials, which enrolled 3388 patients with advanced EOC between 1995 and 2002. Univariate and multivariate analysis revealed nonmodifiable significant prognostic factors for OS and PFS such as: age, performance status (ECOG 2 versus 0-1), FIGO stage (IIIC-IV versus IIB-IIIB), subtype histology (Mucinous ver‐ sus serous), histology grade (grade 2-3 versus 1), presence of large volume ascites (> 500 mL). The only significant modifiable prognosis factor was postoperative residual tumor (0

ovarian cancer

versus >1 mm). (Table 2) This study highlighted the importance of an adequate surgical management of women affected by ovarian cancer as the key-point for improving oncologic outcomes given that the quality of surgical cytoreduction was the only modifiable prognosis factor for survival.

are essentially resistant to chemotherapy.[29] Primary surgical cytoreduction may stim‐ ulate G0 residual tumor cells to re-enter in the normal cell cycle, increasing the chemo‐

Surgical Treatment of Ovarian Cancer http://dx.doi.org/10.5772/53972 167

Residual tumor disease is commonly described as the diameter, in millimeters, of the biggest nodule left after surgical debulking. Griffiths *et al*., first described the importance of residual disease after surgery in women with ovarian cancer.[21] They demonstrated an inverse rela‐ tionship between residual disease and patient survival. In 1994, the Gynecology Oncology Group (GOG) published a sub-analysis of two retrospective series (GOG protocol 52 & 97) of patients affected by advanced stage EOC who underwent primary cytoreduction fol‐ lowed by chemotherapy. The study showed significant differences in OS in women with mi‐ croscopic disease or less than 2 cm in comparison with of residual disease of more than 2 cm diameter. The maximum diameter of residual disease was firstly found to be an independ‐ ent predictor of OS after controlling other variables. Thus, surgery with residual disease of less than 2 cm was defined as "optimal" cytoreduction; while more than 2 cm was called

In 2002, a meta-analysis of 6885 patients with stage III or IV ovarian cancer was reported. [22]The study analyzed 81 cohorts of patients treated in the platinum era to evaluate the ef‐ fect of maximal cytoreductive surgery and other prognostic factors on survival. The investi‐ gators demonstrated that each 10% increase in the proportion of patients undergoing maximal cytoreduction was associated with a concomitant 5.5% increase in median cohort survival time. The mean weighted median survival time was 29 months. Thus, for all clinical trials that followed, the GOG established ≤ 1 cm residual disease as the criterion for optimal

Winter III et al. [31] reported the GOG collective experience analyzing the data of seven tri‐ als (GOG 11, 114, 132, 152, 158, 162 and 172) that studied the efficacy of chemotherapy in 1895 stage III and 360 stage IV ovarian cancer patients. All patients underwent primary de‐ bulking surgery followed by 6 courses of cisplatin and paclitaxel. Residual disease after sur‐ gery was an independent prognostic factor. The median OS reported was 79.1, 42.4 and 35 months in patients with microscopic, 1-10 mm and > 10 mm of residual disease, respectively. The authors suggested a modification of the term "optimal residual disease" from < 1 cm to

These results were confirmed when 3 large phase III randomized trials conducted by the AGO (AGO-OVAR 3, 5 and 7) of patients with stage IIB-IV ovarian cancer receiving platinum/ taxanes chemotherapy following primary cytoreduction surgery were analyzed. [23] Patients with microscopic residual disease had significantly longer median OS than those with any re‐ sidual disease, 99.1 months versus less than 36 months, respectively. Thus the current goal of

the surgery in ovarian cancer is to obtain a complete cytoreduction. (Fig 2)

**10. Residual tumor disease: Definition and relevance**

therapy efficacy.[29]

"suboptimal".[30]

cytoreduction.

microscopic.


#### **9. Rationale for primary surgical cytoreduction**


are essentially resistant to chemotherapy.[29] Primary surgical cytoreduction may stim‐ ulate G0 residual tumor cells to re-enter in the normal cell cycle, increasing the chemo‐ therapy efficacy.[29]

#### **10. Residual tumor disease: Definition and relevance**

versus >1 mm). (Table 2) This study highlighted the importance of an adequate surgical management of women affected by ovarian cancer as the key-point for improving oncologic outcomes given that the quality of surgical cytoreduction was the only modifiable prognosis

**Table 2.** Prognosis factors of overall survival and progression free survival in patients with advanced stage epithelial

**1. Improvement of oncologic outcomes:** a large body of retrospective and non-random‐ ized prospective studies consistently show an inverse correlation between survival and the amount of postoperative residual disease [22]. Results of two meta-analysis[22],[24] evaluated women affected by advanced stage EOC that were treated with primary sur‐ gical cytoreduction and platinum-based neoadjuvant chemotherapy and demonstrated

**2. Surgical reduction of tumor burden prior to chemotherapy:** it has been postulated that the proportion of tumor cells destroyed with each cycle of chemotherapy is constant. Thus, in cases of tumor cells not resistant to chemotherapy, fewer cycles would be nec‐ essary to eradicate them if the absolute number were less.[25] In addition, tumor size is correlated with an increased spontaneous mutation rate of malignant cells.[26] Animal models have also demonstrated that drug exposure allows the resistant cells to outgrow the sensitive tumor cells population.[27] Primary surgical cytoreduction, thus, reduces

the number of cancer cells decreasing the chance of inducing drug resistance.

**3. Improved drug diffusion:** large bulky tumors may have hypoperfused areas where concentration of chemotherapy agents can be suboptimal, increasing the possibility of

**4. Increased tumor cells growth rate:** During initial tumor growth, cancer cell division is almost exponential. But then, cell growth reaches a plateau. Thus, the great majority of cells in large tumoral masses are not dividing, being in G0 phase of the cell cycle, which

a mean weighted median survival of 29 and 24 months respectively.

**9. Rationale for primary surgical cytoreduction**

factor for survival.

166 Ovarian Cancer - A Clinical and Translational Update

• Patient performance status

• Post-surgical residual tumor

drug resistance.[28]

**Non-modifiable**

• Hystology subtype • Hystology grade • Large volume of ascites

• FIGO stage

**Modifiable**

ovarian cancer

Residual tumor disease is commonly described as the diameter, in millimeters, of the biggest nodule left after surgical debulking. Griffiths *et al*., first described the importance of residual disease after surgery in women with ovarian cancer.[21] They demonstrated an inverse rela‐ tionship between residual disease and patient survival. In 1994, the Gynecology Oncology Group (GOG) published a sub-analysis of two retrospective series (GOG protocol 52 & 97) of patients affected by advanced stage EOC who underwent primary cytoreduction fol‐ lowed by chemotherapy. The study showed significant differences in OS in women with mi‐ croscopic disease or less than 2 cm in comparison with of residual disease of more than 2 cm diameter. The maximum diameter of residual disease was firstly found to be an independ‐ ent predictor of OS after controlling other variables. Thus, surgery with residual disease of less than 2 cm was defined as "optimal" cytoreduction; while more than 2 cm was called "suboptimal".[30]

In 2002, a meta-analysis of 6885 patients with stage III or IV ovarian cancer was reported. [22]The study analyzed 81 cohorts of patients treated in the platinum era to evaluate the ef‐ fect of maximal cytoreductive surgery and other prognostic factors on survival. The investi‐ gators demonstrated that each 10% increase in the proportion of patients undergoing maximal cytoreduction was associated with a concomitant 5.5% increase in median cohort survival time. The mean weighted median survival time was 29 months. Thus, for all clinical trials that followed, the GOG established ≤ 1 cm residual disease as the criterion for optimal cytoreduction.

Winter III et al. [31] reported the GOG collective experience analyzing the data of seven tri‐ als (GOG 11, 114, 132, 152, 158, 162 and 172) that studied the efficacy of chemotherapy in 1895 stage III and 360 stage IV ovarian cancer patients. All patients underwent primary de‐ bulking surgery followed by 6 courses of cisplatin and paclitaxel. Residual disease after sur‐ gery was an independent prognostic factor. The median OS reported was 79.1, 42.4 and 35 months in patients with microscopic, 1-10 mm and > 10 mm of residual disease, respectively. The authors suggested a modification of the term "optimal residual disease" from < 1 cm to microscopic.

These results were confirmed when 3 large phase III randomized trials conducted by the AGO (AGO-OVAR 3, 5 and 7) of patients with stage IIB-IV ovarian cancer receiving platinum/ taxanes chemotherapy following primary cytoreduction surgery were analyzed. [23] Patients with microscopic residual disease had significantly longer median OS than those with any re‐ sidual disease, 99.1 months versus less than 36 months, respectively. Thus the current goal of the surgery in ovarian cancer is to obtain a complete cytoreduction. (Fig 2)

## The goal of the surgery in ovarian cancer is to obtain a complete cytoreduction

Stratified residual tumor on expert series / International trials

**Figure 3.** Stratified residual tumor on expert series/international traits

**Figure 4.** Stratified residual tumor on less experienced centers

¢ȱ¢ȱǯǽŚŗǾȬǽŚřǾ

**)LJXUH**\$GGFDSWLRQ

2YDULDQ&DQFHU

**Stratified residual tumor on less experienced centers** 

**ŗŘǯȱȱ¢ȱ ȱ¢ȱȱȱ¢**

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Surgical Treatment of Ovarian Cancer http://dx.doi.org/10.5772/53972 169

#### **Figure 2.** Goal of the surgery in ovarian cancer

Chang and Bristow in 2012, reported a single institution series and cooperative group trials since 2003 of patients who underwent primary debulking surgery followed by adjuvant che‐ motherapy. Over 14000 patients in 15 studies were analyzed.[32] A marked inverse correla‐ tion between the maximal diameter of residual tumor and OS was noted. The weighted median OS for 3593 patients with no gross residual disease was 77.8 months compared to 39.0 months for the 4780 patients with 0.1–1 cm residual disease and 31.1 months for the 3518 patients with residual tumor >1 cm in maximal diameter. The magnitude of the incre‐ mental improvement in OS strongly suggests that complete resection should be the surgical objective whenever feasible.

#### **11. Feasibility of complete primary cytoreduction**

In the presence of a preoperative suspected adnexal mass whit ascites and peritoneal carci‐ nomatosis are present, the feasibility of complete cytoreduction should be determined by ex‐ clusion of multiple liver or pulmonary metastases by imaging studies such as computed tomography (CT). In the absence of extra-peritoneal lesions and surgical contraindications, patients should undergo primary debulking surgery. The feasibility of optimal cytoreduc‐ tion depends on the disease distribution, the patient´s overall medical condition and the sur‐ geon's expertise. However, obtaining an optimal cytoreduction ≤ 1 cm of residual disease is not an easy task. In highly specialized centers, the rate with optimal primary cytoreduction is over 75 %. (Fig 3) But this rate falls down to 25% when low-volume ovarian cancer surger‐ ies centers are included in the analysis. (Fig 4) Nevertheless, as it was previously mentioned, according with collecting data of the latter,[23],[31] primary debulking surgery is beneficial if complete cytoreduction is achieved. According with the literature, this is achievable in on‐ ly 30% of patients when a gynecologist oncologist performs the surgery, a higher rate when compared with general gynecologists or general surgeons.[33]-[38]

#### **12. Neoadjuvant chemotherapy followed by interval debulking surgery**

Despite upfront primary debulking surgery (PDS) for newly diagnosed patients with ad‐ vanced stage ovarian cancer is considered the standard of care,[5] limitations to this strategy have been postulated.[39],[40] For instance, patients with incomplete primary cytoreduction seem to have no meaningful impact on OS.[23],[31] Furthermore, only experienced surgeons

**Figure 3.** Stratified residual tumor on expert series/international traits

**Stratified residual tumor on less experienced centers** 

The goal of the surgery in ovarian cancer

is to obtain a complete cytoreduction

Chang and Bristow in 2012, reported a single institution series and cooperative group trials since 2003 of patients who underwent primary debulking surgery followed by adjuvant che‐ motherapy. Over 14000 patients in 15 studies were analyzed.[32] A marked inverse correla‐ tion between the maximal diameter of residual tumor and OS was noted. The weighted median OS for 3593 patients with no gross residual disease was 77.8 months compared to 39.0 months for the 4780 patients with 0.1–1 cm residual disease and 31.1 months for the 3518 patients with residual tumor >1 cm in maximal diameter. The magnitude of the incre‐ mental improvement in OS strongly suggests that complete resection should be the surgical

In the presence of a preoperative suspected adnexal mass whit ascites and peritoneal carci‐ nomatosis are present, the feasibility of complete cytoreduction should be determined by ex‐ clusion of multiple liver or pulmonary metastases by imaging studies such as computed tomography (CT). In the absence of extra-peritoneal lesions and surgical contraindications, patients should undergo primary debulking surgery. The feasibility of optimal cytoreduc‐ tion depends on the disease distribution, the patient´s overall medical condition and the sur‐ geon's expertise. However, obtaining an optimal cytoreduction ≤ 1 cm of residual disease is not an easy task. In highly specialized centers, the rate with optimal primary cytoreduction is over 75 %. (Fig 3) But this rate falls down to 25% when low-volume ovarian cancer surger‐ ies centers are included in the analysis. (Fig 4) Nevertheless, as it was previously mentioned, according with collecting data of the latter,[23],[31] primary debulking surgery is beneficial if complete cytoreduction is achieved. According with the literature, this is achievable in on‐ ly 30% of patients when a gynecologist oncologist performs the surgery, a higher rate when

**12. Neoadjuvant chemotherapy followed by interval debulking surgery**

Despite upfront primary debulking surgery (PDS) for newly diagnosed patients with ad‐ vanced stage ovarian cancer is considered the standard of care,[5] limitations to this strategy have been postulated.[39],[40] For instance, patients with incomplete primary cytoreduction seem to have no meaningful impact on OS.[23],[31] Furthermore, only experienced surgeons

**Figure 2.** Goal of the surgery in ovarian cancer

168 Ovarian Cancer - A Clinical and Translational Update

objective whenever feasible.

**11. Feasibility of complete primary cytoreduction**

compared with general gynecologists or general surgeons.[33]-[38]

**ŗŘǯȱȱ¢ȱ ȱ¢ȱȱȱ¢**

ȱ ȱ ¢ȱ ȱ ¢ȱ ǻǼȱ ȱ ¢ȱ ȱ ȱ ȱ Ȭ ȱȱȱȱȱȱȱȱȱǰǽśǾȱȱȱȱ¢ ȱȱǯǽřşǾǰǽŚŖǾȱȱǰȱȱ ȱȱ¢ȱ¢ ȱȱȱȱȱȱȱǯǽŘřǾǰǽřŗǾȱǰȱ¢ȱ¡ȱ ȱ ¡ȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ

¢ǰȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ȱ ǻǼȱ ȱȱ¢ȱȱǯǽřşǾǰǽŚŖǾȱȱ¢ȱȱȱȱȱȱȬ ȱȱȱȱřȱȱȱȬ¡ȱ¢ȱ ȱ¢ȱȱȱȬ ȱ ¢ȱ ǻǼȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ǯȱǽŚŚǾȱȱȱȱȱ¢ȱȱȱȱȱ¡ȱȱȱȱǰȱ¢ ȱȱȱȱȱǰȱȱȱȱȱ¢ȱȱȬ

ȱȱȱȱȱȱǰȱ ȱȱȱȱȱȱǯ

ȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ȱȱ ȱȱȱȬȱȱȱȱȱȱ ȱ¢ǯȱȱȱǰȱ ǰȱ ȱȱȱȱ

**Figure 4.** Stratified residual tumor on less experienced centers

**)LJXUH**\$GGFDSWLRQ

¢ȱ¢ȱǯǽŚŗǾȬǽŚřǾ

with extended formal training in cytoreductive techniques obtain an acceptable complete primary cytoreduction rate.[41]-[43]

IV ovarian cancer and complete resection of all gross lesions remains the objective of the cy‐ toreductive surgery whether performed as primary or after neoadjuvant chemotherapy. However, optimal cytoreduction (<1 cm residual disease) was achieved in only 41.6% of pa‐ tients in the PDS arm, a substantially lower rate than the published by expert series.[31],[42], [43] The PFS and OS for patients randomized to the PDS arm were substantially lower than those reported in previous studies, including prospective trials of the Gynecologic Oncology

Surgical Treatment of Ovarian Cancer http://dx.doi.org/10.5772/53972 171

A recent report from the Memorial Sloan-Kettering Cancer Center contradicts the findings of the EORTC study and suggests that the strategy of neoadjuvant chemotherapy requires fur‐ ther investigation. A total of 316 stage IIIC–IV ovarian cancer patients were treated at the institution during the same period in which the EORTC-NCIC trial were evaluated, using identical inclusion criteria.[53] The optimal cytoreduction rate was 71% and the median OS time was 50 months. This study suggested that primary cytoreductive surgery should be considered as t he preferred initial management strategy for patients with this disease.

It seems, therefore, that neoadjuvant chemotherapy should not be performed routinely in patients with advanced ovarian cancer and be done in selected patients who are at risk of morbidity associated with primary surgery and less likely to have a complete cytoreduction.

Group (GOG) as well.[30],[31],[43]

**Figure 5.** Initial approach of suspected advanced ovarian cáncer

(Fig. 5)

Consequently, an alternative approach such as neoadjuvant chemotherapy (NACT) has been proposed by several authors.[39],[40] This strategy of treatment consists in the admin‐ istration of at least 3 courses of platinum-taxanes chemotherapy followed by an interval de‐ bulking surgery (IDS) and further adjuvant treatment in patients responsive to chemotherapy. [44] (Figure 1) The goal of this modality is to reduce the extension of the dis‐ ease and, by performing a less radical surgical procedure, to improve the complete cytore‐ duction rate reducing the surgical time and complication rate, while improving the PFS and OS rate.

Objective indications for neoadjuvant chemotherapy are patients with poor performance status and with significant medical co-morbidities making them unsuitable for an aggressive debulking surgery. These indications include, however, the smallest proportion of patients who underwent neoadjuvant chemotherapy in the series published in the literature. [39], [40],[45],[46] The majority of women receive either NACT or PDS based on tumor extension and on estimated tumor resectability.[47] The latter is a subjective and highly surgeon-de‐ pendent indication. [24] Although several criteria have been tested for predicting the surgi‐ cal resectability of ovarian tumors, its accuracy and clinical applicability is still controversial. [48] Some of these criteria include ascites volume, serum CA 125 values[48] and computer tomography scan parameters.[49] For example, terms like "dense adhesion between bowel and omentum", "large diaphragm disease", and "large tumor nodules adherent to abdomi‐ nal structures" have been postulated by some authors as criteria of unresectability.[50] These terms show how subjective is the definition of a patient as debulkable or not. These criteria are mostly based on CT scan findings but, sometimes, a direct laparoscopic assess‐ ment of is recommended.[51] (Fig. 5)

On the other hand it is a common belief to associate NACT with less complex surgical proce‐ dures, shorter surgical time, and lower incidence of complications after IDS.[44],[46] How‐ ever, this strategy does not exclude the necessity of performing complex surgical procedures at the time of IDS in order to obtain an optimal cytoreduction. Thus, referring these patients to a specialized gynecologist is mandatory as well.

Recently, the results of a randomized, controlled, prospective trial conducted by the Europe‐ an Organization for Research and Treatment of Cancer (EORTC) were published.[52] Six hundred and seventy patients with stage IIIC and IV ovarian cancer were randomly as‐ signed to primary cytoreductive surgery group or neoadjuvant chemotherapy group. There were no significant differences in OS (29 months for primary cytoreductive surgery group versus 30 months for neoadjuvant chemotherapy group) between the two groups. Complete cytoreduction with no gross residual disease was possible in 20% of patients who under‐ went primary cytoreduction and 52% of those who had neoadjuvant chemotherapy. On multivariate analysis, the strongest independent predictor factor of prolonged survival was the absence of residual tumor after surgery (p < 0.001). The authors concluded that neoadju‐ vant chemotherapy followed by interval debulking surgery has similar efficacy compared with primary debulking surgery followed by chemotherapy for patients with stage IIIC or IV ovarian cancer and complete resection of all gross lesions remains the objective of the cy‐ toreductive surgery whether performed as primary or after neoadjuvant chemotherapy. However, optimal cytoreduction (<1 cm residual disease) was achieved in only 41.6% of pa‐ tients in the PDS arm, a substantially lower rate than the published by expert series.[31],[42], [43] The PFS and OS for patients randomized to the PDS arm were substantially lower than those reported in previous studies, including prospective trials of the Gynecologic Oncology Group (GOG) as well.[30],[31],[43]

with extended formal training in cytoreductive techniques obtain an acceptable complete

Consequently, an alternative approach such as neoadjuvant chemotherapy (NACT) has been proposed by several authors.[39],[40] This strategy of treatment consists in the admin‐ istration of at least 3 courses of platinum-taxanes chemotherapy followed by an interval de‐ bulking surgery (IDS) and further adjuvant treatment in patients responsive to chemotherapy. [44] (Figure 1) The goal of this modality is to reduce the extension of the dis‐ ease and, by performing a less radical surgical procedure, to improve the complete cytore‐ duction rate reducing the surgical time and complication rate, while improving the PFS and

Objective indications for neoadjuvant chemotherapy are patients with poor performance status and with significant medical co-morbidities making them unsuitable for an aggressive debulking surgery. These indications include, however, the smallest proportion of patients who underwent neoadjuvant chemotherapy in the series published in the literature. [39], [40],[45],[46] The majority of women receive either NACT or PDS based on tumor extension and on estimated tumor resectability.[47] The latter is a subjective and highly surgeon-de‐ pendent indication. [24] Although several criteria have been tested for predicting the surgi‐ cal resectability of ovarian tumors, its accuracy and clinical applicability is still controversial. [48] Some of these criteria include ascites volume, serum CA 125 values[48] and computer tomography scan parameters.[49] For example, terms like "dense adhesion between bowel and omentum", "large diaphragm disease", and "large tumor nodules adherent to abdomi‐ nal structures" have been postulated by some authors as criteria of unresectability.[50] These terms show how subjective is the definition of a patient as debulkable or not. These criteria are mostly based on CT scan findings but, sometimes, a direct laparoscopic assess‐

On the other hand it is a common belief to associate NACT with less complex surgical proce‐ dures, shorter surgical time, and lower incidence of complications after IDS.[44],[46] How‐ ever, this strategy does not exclude the necessity of performing complex surgical procedures at the time of IDS in order to obtain an optimal cytoreduction. Thus, referring these patients

Recently, the results of a randomized, controlled, prospective trial conducted by the Europe‐ an Organization for Research and Treatment of Cancer (EORTC) were published.[52] Six hundred and seventy patients with stage IIIC and IV ovarian cancer were randomly as‐ signed to primary cytoreductive surgery group or neoadjuvant chemotherapy group. There were no significant differences in OS (29 months for primary cytoreductive surgery group versus 30 months for neoadjuvant chemotherapy group) between the two groups. Complete cytoreduction with no gross residual disease was possible in 20% of patients who under‐ went primary cytoreduction and 52% of those who had neoadjuvant chemotherapy. On multivariate analysis, the strongest independent predictor factor of prolonged survival was the absence of residual tumor after surgery (p < 0.001). The authors concluded that neoadju‐ vant chemotherapy followed by interval debulking surgery has similar efficacy compared with primary debulking surgery followed by chemotherapy for patients with stage IIIC or

primary cytoreduction rate.[41]-[43]

170 Ovarian Cancer - A Clinical and Translational Update

ment of is recommended.[51] (Fig. 5)

to a specialized gynecologist is mandatory as well.

OS rate.

A recent report from the Memorial Sloan-Kettering Cancer Center contradicts the findings of the EORTC study and suggests that the strategy of neoadjuvant chemotherapy requires fur‐ ther investigation. A total of 316 stage IIIC–IV ovarian cancer patients were treated at the institution during the same period in which the EORTC-NCIC trial were evaluated, using identical inclusion criteria.[53] The optimal cytoreduction rate was 71% and the median OS time was 50 months. This study suggested that primary cytoreductive surgery should be considered as t he preferred initial management strategy for patients with this disease.

It seems, therefore, that neoadjuvant chemotherapy should not be performed routinely in patients with advanced ovarian cancer and be done in selected patients who are at risk of morbidity associated with primary surgery and less likely to have a complete cytoreduction. (Fig. 5)

**Figure 5.** Initial approach of suspected advanced ovarian cáncer

#### **13. Surgical cytoreduction technique**

Women should be placed on supine position with legs spread apart. Vertical midline inci‐ sion is recommended in order to access to the entire abdominal cavity. Ascites is evacuated and sent for cytological evaluation. As described above, a careful inspection and palpation of the entire peritoneal cavity and retroperitoneum is carried out in order to assess the ex‐ tent of the primary and metastatic disease. The localization and diameter of the primary tu‐ mor and its extension into surrounding organs is described as the diameter of the larger metastases. Sometimes, there are regions that cannot be accessed before larger tumor masses are removed. This careful inspection and palpation is essential in order to establish the feasi‐ bility and extension of surgical cytoreduction. Complete cytoreduction may by difficult in cases of bulky suprarenal nodes, extensive disease in the liver parenchyma, along the root of the small bowel mesentery and in the bowel serosa, close to the origin of the superior mes‐ enteric artery, or in the porta hepatis. If complete surgical cytoreduction is not feasible, neo‐ adjuvant chemotherapy is preferred. (Figure 5)

lemma for physicians. To date, there is no consensus for optimal treatment strategies. Three essential options are proposed: surgical resection followed by chemotherapy, chemotherapy only or enrollment into clinical trials. This dilemma will be fundamentally responded by the localization of the disease, by the disease free interval (DFI) between the end of standard front-line chemotherapy (platinum/taxanes-based) and the date of documented disease re‐ currence. This period will divide patients in three groups: *platinum sensible* with a DFI more than 6 months; *platinum resistant:* patients with a DFI less than 6 months; and the group of *platinum refractory:* patients who will never respond to front line therapy or who will experi‐ ence progression of disease. The latter represents 20-30% of the patients with FIGO stage III-IV who underwent surgical cytoreduction followed by carboplatin /paclitaxel.[55],[56] DFI has been established as the most important predictor factor for response to treatment of the

Surgical Treatment of Ovarian Cancer http://dx.doi.org/10.5772/53972 173

Surgical resection for ovarian cancer recurrence means secondary cytoreduction. Although primary cytoreductive surgery is well accepted as the cornerstone of initial management, the use of cytoreductive surgery in the setting of recurrent disease is defined less clearly. Benefits of secondary cytoreduction are encountered in several studies.[59] No randomized studies exist regarding the benefits of surgical resection over chemotherapy in patients with recurrent disease. The available data is controversial and biased by the decision whether or not to expose patients to a surgical treatment. In general, studies included patients with more favorable characteristics such as younger age, fewer medical comorbidity, scarce num‐ ber of lesions, better performance status, absence of ascites at recurrence, early stage at diag‐ nosis, DFI more than 12 months, and optimal primary cytoreduction.[60]-[64] All of the previous characteristics are favorable prognostic factors and constitute the standard indica‐ tions for secondary surgical resection.[60] A recent meta-analysis studied 2.019 patients en‐ rolled in 40 retrospective and prospective trials who underwent secondary cytoreduction due to recurrent ovarian cancer. The mean weighted median OS time after recurrence was 30.3 months. Complete cytoreduction was identified as an independent factor for the im‐ proving OS after secondary cytoreduction. In addition, the multivariate analysis showed that the survival time is increased 3.0 months each 10 % increase in the proportion of pa‐

The objective of secondary cytoreduction should be to achieve complete debulking. In pa‐ tients who are able to tolerate a major surgical procedure, secondary cytoreduction should be offered to those with a single site disease regardless of DFI, as well as to all patients with a DFI of greater than 30 months regardless the amount of disease sites. Patients with carci‐ nomatosis and a DFI of less than 12 months should not be considered for secondary cytore‐ duction. The decisions must be, however, individualized based on each patient's goals,

performance status, operative risk, and available therapeutic options.[66] (Table 3)

relapsed disease.[55],[57],[58]

**15. Secondary cytoreduction**

tients undergoing complete cytoreductive surgery.[65]

Radical omentectomy use to be the first surgical step because it is the first tumor encoun‐ tered upon entering the peritoneal cavity. The infracolic omentum is separated from the transverse colon and resected. If the omental metastases involve the gastrocolic omentum, it is resected as well. The next step is to remove the primary tumor in the pelvis with the other adnexa and the uterus in the usual fashion if no extension to other pelvic organs is present. However, advanced ovarian cancer often involves the uterus, rectosigmoid, cecum, ileum and bladder. Metastases of the pelvic peritoneum sometimes completely obliterate the ante‐ rior and posterior cul-de-sac. In this case, the retroperitoneal approach is the most reasona‐ ble way for removing *in block* the entire tumor. This procedure is accompanied by performing a rectosigmoid resection with an end-to end mechanical anastomosis.[54] Tumor spread to the hilum of the spleen may be carefully inspected as well. Splenectomy may be sometimes indicated to achieve maximal tumor debulking. Any peritoneal implants should be removed, particularly if there are large, isolated masses and their removal will render the patient optimally cytoreduced. Diaphragm peritoneum should be visualized and resected if the disease is present. Sometimes, it can involve muscle resection that can be sutured with non-reabsorbed monofilament continuous suture. Pelvic and /or aortic lymph node involve‐ ment is seen is approximately 60% of patient with advanced stage disease. Despite contro‐ versial, pelvic and aortic lymphadenectomy should be completed starting form aortic bifurcation up to the renal veins. The incidence of complications and morbidity of this ap‐ proach should be also taken into consideration for patient selection. The most common com‐ plications include: infections, cardiac morbidity, pulmonary thromboembolism, coagulopathy, gastrointestinal, renal failure, re-laparotomy and death.

#### **14. Surgical treatment of relapsed ovarian cancer: Secondary cytoreduction**

Once recurrence is confirmed, the next step is to determine the best treatment approach for each individual case. Recurrent epithelial ovarian carcinoma is, however, a therapeutic di‐ lemma for physicians. To date, there is no consensus for optimal treatment strategies. Three essential options are proposed: surgical resection followed by chemotherapy, chemotherapy only or enrollment into clinical trials. This dilemma will be fundamentally responded by the localization of the disease, by the disease free interval (DFI) between the end of standard front-line chemotherapy (platinum/taxanes-based) and the date of documented disease re‐ currence. This period will divide patients in three groups: *platinum sensible* with a DFI more than 6 months; *platinum resistant:* patients with a DFI less than 6 months; and the group of *platinum refractory:* patients who will never respond to front line therapy or who will experi‐ ence progression of disease. The latter represents 20-30% of the patients with FIGO stage III-IV who underwent surgical cytoreduction followed by carboplatin /paclitaxel.[55],[56] DFI has been established as the most important predictor factor for response to treatment of the relapsed disease.[55],[57],[58]

#### **15. Secondary cytoreduction**

**13. Surgical cytoreduction technique**

172 Ovarian Cancer - A Clinical and Translational Update

adjuvant chemotherapy is preferred. (Figure 5)

Women should be placed on supine position with legs spread apart. Vertical midline inci‐ sion is recommended in order to access to the entire abdominal cavity. Ascites is evacuated and sent for cytological evaluation. As described above, a careful inspection and palpation of the entire peritoneal cavity and retroperitoneum is carried out in order to assess the ex‐ tent of the primary and metastatic disease. The localization and diameter of the primary tu‐ mor and its extension into surrounding organs is described as the diameter of the larger metastases. Sometimes, there are regions that cannot be accessed before larger tumor masses are removed. This careful inspection and palpation is essential in order to establish the feasi‐ bility and extension of surgical cytoreduction. Complete cytoreduction may by difficult in cases of bulky suprarenal nodes, extensive disease in the liver parenchyma, along the root of the small bowel mesentery and in the bowel serosa, close to the origin of the superior mes‐ enteric artery, or in the porta hepatis. If complete surgical cytoreduction is not feasible, neo‐

Radical omentectomy use to be the first surgical step because it is the first tumor encoun‐ tered upon entering the peritoneal cavity. The infracolic omentum is separated from the transverse colon and resected. If the omental metastases involve the gastrocolic omentum, it is resected as well. The next step is to remove the primary tumor in the pelvis with the other adnexa and the uterus in the usual fashion if no extension to other pelvic organs is present. However, advanced ovarian cancer often involves the uterus, rectosigmoid, cecum, ileum and bladder. Metastases of the pelvic peritoneum sometimes completely obliterate the ante‐ rior and posterior cul-de-sac. In this case, the retroperitoneal approach is the most reasona‐ ble way for removing *in block* the entire tumor. This procedure is accompanied by performing a rectosigmoid resection with an end-to end mechanical anastomosis.[54] Tumor spread to the hilum of the spleen may be carefully inspected as well. Splenectomy may be sometimes indicated to achieve maximal tumor debulking. Any peritoneal implants should be removed, particularly if there are large, isolated masses and their removal will render the patient optimally cytoreduced. Diaphragm peritoneum should be visualized and resected if the disease is present. Sometimes, it can involve muscle resection that can be sutured with non-reabsorbed monofilament continuous suture. Pelvic and /or aortic lymph node involve‐ ment is seen is approximately 60% of patient with advanced stage disease. Despite contro‐ versial, pelvic and aortic lymphadenectomy should be completed starting form aortic bifurcation up to the renal veins. The incidence of complications and morbidity of this ap‐ proach should be also taken into consideration for patient selection. The most common com‐ plications include: infections, cardiac morbidity, pulmonary thromboembolism,

coagulopathy, gastrointestinal, renal failure, re-laparotomy and death.

**cytoreduction**

**14. Surgical treatment of relapsed ovarian cancer: Secondary**

Once recurrence is confirmed, the next step is to determine the best treatment approach for each individual case. Recurrent epithelial ovarian carcinoma is, however, a therapeutic di‐ Surgical resection for ovarian cancer recurrence means secondary cytoreduction. Although primary cytoreductive surgery is well accepted as the cornerstone of initial management, the use of cytoreductive surgery in the setting of recurrent disease is defined less clearly. Benefits of secondary cytoreduction are encountered in several studies.[59] No randomized studies exist regarding the benefits of surgical resection over chemotherapy in patients with recurrent disease. The available data is controversial and biased by the decision whether or not to expose patients to a surgical treatment. In general, studies included patients with more favorable characteristics such as younger age, fewer medical comorbidity, scarce num‐ ber of lesions, better performance status, absence of ascites at recurrence, early stage at diag‐ nosis, DFI more than 12 months, and optimal primary cytoreduction.[60]-[64] All of the previous characteristics are favorable prognostic factors and constitute the standard indica‐ tions for secondary surgical resection.[60] A recent meta-analysis studied 2.019 patients en‐ rolled in 40 retrospective and prospective trials who underwent secondary cytoreduction due to recurrent ovarian cancer. The mean weighted median OS time after recurrence was 30.3 months. Complete cytoreduction was identified as an independent factor for the im‐ proving OS after secondary cytoreduction. In addition, the multivariate analysis showed that the survival time is increased 3.0 months each 10 % increase in the proportion of pa‐ tients undergoing complete cytoreductive surgery.[65]

The objective of secondary cytoreduction should be to achieve complete debulking. In pa‐ tients who are able to tolerate a major surgical procedure, secondary cytoreduction should be offered to those with a single site disease regardless of DFI, as well as to all patients with a DFI of greater than 30 months regardless the amount of disease sites. Patients with carci‐ nomatosis and a DFI of less than 12 months should not be considered for secondary cytore‐ duction. The decisions must be, however, individualized based on each patient's goals, performance status, operative risk, and available therapeutic options.[66] (Table 3)


years after first treatment.[69],[72],[73],[75],[79] In absolute numbers, this translates in an ex‐

Despite the consensus and the advantages explained above, population-based studies indi‐ cate that access to specialist care in gynecologic oncology for women with suspected ovarian cancer has been less than universal.[35],[36],[70] Reports from countries such as USA,[80] and UK[35],[81] have consistently shown that the majority of patients were treated in lowvolume hospitals by low-volume surgeons. For example, the accessibility of patients with ovarian cancer to a specialized center was reported in 18% of patients in The Nether‐

In summary, the configuration of health-care delivery systems to facilitate quick and consis‐ tent centralized referral will be necessary to ensure widespread access for women with sus‐ pected ovarian cancer to such health-care providers. Only through such efforts will contemporary patterns of surgical practice conform to the definition of high-quality cancer

, Rafael Alvarez Gallego2

1 Gynecologic Oncology Division, Department of Obstetrics and Gynecology. HM Universi‐

2 Medical Oncology Division, Gynecologic Oncology Program, Medical Oncology Division, HM Universitario Sanchinarro, Centro Integral Oncológico Clara Campal, Madrid, Spain

[1] Jema A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun M. Cancer Statistics, 2008.

[2] Siegel R, Desantis C, Virgo K, Stein K, Mariotto A, Smith T, Cooper D, Gansler T, Ler‐ ro C, Fedewa S, Lin C, Leach C, Cannady RS, Cho H, Scoppa S, Hachey M, Kirch R, Jemal A, Ward E.Cancer treatment and survivorship statistics, 2012.CA Cancer J Clin.

[3] Pecorelli S, Creasman WT, Petterson F, Benedet JL, Shepard JH. FIGO annual report on the results of treatment in gynaecological cancer. J Epidemiol Biostat. 1998;3:75–

,

Surgical Treatment of Ovarian Cancer http://dx.doi.org/10.5772/53972 175

tension of survival of more than 10 months.[79]

care.[83]

**Author details**

**References**

102.

Javier Valero de Bernabé1

Lucas Minig1\*, M. Guadalupe Patrono1

lands[74], 35% in Canada[72] and 40% in Maryland, USA.[82]

and Ivan Diaz-Padilla2

tario Sanchinarro, Centro Integral Oncológico Clara Campal, Madrid, Spain

\*Address all correspondence to: lucasminig@yahoo.com

CA Cancer J Clin 2008;58;71-96.

2012 Jul;62(4):220-41.

**Table 3.** Recommendations for secondary cytoreduction (SC)

#### **16. Specialized gynecologists**

Surgical evaluation of a pelvic mass is one of the most common indications for gynecologic surgery and, therefore, it is unlikely that all patients with adnexal masses will be referred to a gynecologic oncologist. To assist in the referral process, the Society of Gynecologic Oncolo‐ gy established a guideline for patient referral with suspected ovarian cancer.[67]

It has been demonstrated that patients operated on by gynecologic oncologists are more likely to undergo an adequate staging procedure in early stage disease[34],[36],[37],[68] and a better percentage of optimal primary cytoreduction in advanced stage disease can be ach‐ ieved in comparison to general gynecologists or general surgeons.[33]-[38] Moreover, many studies from several countries around the world have shown over 10 months increased OS when ovarian cancer patients were initially operated by a gynecological oncologist rather than general gynecologist [33],[34],[69]-[71] or general surgeons.[68],[72] Thus, optimal pri‐ mary cytoreductive surgery performed by a surgeon with extended formal training in cytor‐ eductive techniques followed by an appropriate chemotherapy combination is among the most powerful clinician-driven determinants of survival for women with ovarian cancer.[24]

#### **17. Multidisciplinary team and centralization of treatment**

Ovarian cancer is a challenging, complex and multidisciplinary disease. It is not only impor‐ tant how well trained physicians are, but also how many physicians of different specialties are involved in the management of this malignancy. The holistic conception of patient care and the intrinsic complexity of ovarian cancer require the involvement of different special‐ ties to optimize the quality of care. The concept of multidisciplinary team approach in ovari‐ an cancer is not restricted to the operating room settings. Multidisciplinary approach is crucial from the diagnosis to the demise of disease.

Results of different studies consistently show that patients with ovarian cancer treated at re‐ ferral teaching high-volume hospitals receive better quality of care as accomplished by bet‐ ter surgical staging, better optimal cytoreduction[35],[39],[69],[73]-[75] and better chemotherapy administration rate and schemes.[69],[75]-[78] Treating patients at referral hospitals was independently associated with 10%-20% increased probability of survival at 5 years after first treatment.[69],[72],[73],[75],[79] In absolute numbers, this translates in an ex‐ tension of survival of more than 10 months.[79]

Despite the consensus and the advantages explained above, population-based studies indi‐ cate that access to specialist care in gynecologic oncology for women with suspected ovarian cancer has been less than universal.[35],[36],[70] Reports from countries such as USA,[80] and UK[35],[81] have consistently shown that the majority of patients were treated in lowvolume hospitals by low-volume surgeons. For example, the accessibility of patients with ovarian cancer to a specialized center was reported in 18% of patients in The Nether‐ lands[74], 35% in Canada[72] and 40% in Maryland, USA.[82]

In summary, the configuration of health-care delivery systems to facilitate quick and consis‐ tent centralized referral will be necessary to ensure widespread access for women with sus‐ pected ovarian cancer to such health-care providers. Only through such efforts will contemporary patterns of surgical practice conform to the definition of high-quality cancer care.[83]

#### **Author details**

**Disease-Free Interval Single site of**

174 Ovarian Cancer - A Clinical and Translational Update

**16. Specialized gynecologists**

**recurrence**

**Table 3.** Recommendations for secondary cytoreduction (SC)

**Multiple site od recurrence – but no carcinomatosis**

6 – 12 months Offer SC Consider SC No SC 12 – 30 months Offer SC Offer SC Consider SC > 30 months Offer SC Offer SC Offer SC

Surgical evaluation of a pelvic mass is one of the most common indications for gynecologic surgery and, therefore, it is unlikely that all patients with adnexal masses will be referred to a gynecologic oncologist. To assist in the referral process, the Society of Gynecologic Oncolo‐

It has been demonstrated that patients operated on by gynecologic oncologists are more likely to undergo an adequate staging procedure in early stage disease[34],[36],[37],[68] and a better percentage of optimal primary cytoreduction in advanced stage disease can be ach‐ ieved in comparison to general gynecologists or general surgeons.[33]-[38] Moreover, many studies from several countries around the world have shown over 10 months increased OS when ovarian cancer patients were initially operated by a gynecological oncologist rather than general gynecologist [33],[34],[69]-[71] or general surgeons.[68],[72] Thus, optimal pri‐ mary cytoreductive surgery performed by a surgeon with extended formal training in cytor‐ eductive techniques followed by an appropriate chemotherapy combination is among the most powerful clinician-driven determinants of survival for women with ovarian cancer.[24]

Ovarian cancer is a challenging, complex and multidisciplinary disease. It is not only impor‐ tant how well trained physicians are, but also how many physicians of different specialties are involved in the management of this malignancy. The holistic conception of patient care and the intrinsic complexity of ovarian cancer require the involvement of different special‐ ties to optimize the quality of care. The concept of multidisciplinary team approach in ovari‐ an cancer is not restricted to the operating room settings. Multidisciplinary approach is

Results of different studies consistently show that patients with ovarian cancer treated at re‐ ferral teaching high-volume hospitals receive better quality of care as accomplished by bet‐ ter surgical staging, better optimal cytoreduction[35],[39],[69],[73]-[75] and better chemotherapy administration rate and schemes.[69],[75]-[78] Treating patients at referral hospitals was independently associated with 10%-20% increased probability of survival at 5

gy established a guideline for patient referral with suspected ovarian cancer.[67]

**17. Multidisciplinary team and centralization of treatment**

crucial from the diagnosis to the demise of disease.

**Carcinomatosis**

Lucas Minig1\*, M. Guadalupe Patrono1 , Rafael Alvarez Gallego2 , Javier Valero de Bernabé1 and Ivan Diaz-Padilla2

\*Address all correspondence to: lucasminig@yahoo.com

1 Gynecologic Oncology Division, Department of Obstetrics and Gynecology. HM Universi‐ tario Sanchinarro, Centro Integral Oncológico Clara Campal, Madrid, Spain

2 Medical Oncology Division, Gynecologic Oncology Program, Medical Oncology Division, HM Universitario Sanchinarro, Centro Integral Oncológico Clara Campal, Madrid, Spain

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**Chapter 9**

**The Role of Chemotherapy in Recurrent Ovarian Cancer**

Miguel Angel Alonso Bermejo, Ana Fernandez Montes, Eva Perez Lopez, Miguel Angel Nuñez Viejo, Jesus Garcia Gomez and Jesus Garcia Mata

Epithelial ovarian cancer causes more deaths than any other cancer of the female reproductive system and it is the leading cause of death from gynecologic cancer. There is no universally accepted consensus on the surveillance of ovarian cancer, but if we review the main clinical guidelines, we can find similar recommendations for follow-up for patients with ovarian

Approximately 60% of patients will experience a relapse after the standard first-line treatment including cytoreductive surgery and adjuvant chemotherapy [1]. At this time, when relapse occurs, the chance of cure decreases drastically and treatment is solely palliative. This makes the increase in overall survival and the quality of life the primary endpoints. Surgery is not sufficiently validated due to the lack of phase III clinical trials, and there are no approved targeted therapies in relapsed ovarian cancer. Therefore, chemotherapy is the only option to achieve these objectives. We will review the role of chemotherapy in recurrent ovarian cancer

In stages I, II, III and IV complete responders, American guidelines recommend that, after completing primary surgery and adjuvant chemotherapy, follow-up visits should include a physical examination with a pelvic exam every 2 to 4 months for the first two years, then every 3 to 6 months until the fifth year, and then annually after the fifth year. Periodic monitoring of CA 125 and other tumor markers (e.g., CA 19.9, CEA) are also recommended if the markers were elevated previously. The rest of the examination, which ranges from performing

and reproduction in any medium, provided the original work is properly cited.

© 2013 Bermejo et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54171

cancer after chemotherapy treatment.

**2. Diagnosis of epithelial ovarian cancer relapse**

**1. Introduction**

in this chapter.


### **The Role of Chemotherapy in Recurrent Ovarian Cancer**

Miguel Angel Alonso Bermejo, Ana Fernandez Montes, Eva Perez Lopez, Miguel Angel Nuñez Viejo, Jesus Garcia Gomez and Jesus Garcia Mata

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54171

#### **1. Introduction**

[81] Jolly K, Parry J, Rouse A, Stevens A. Volumes of cancer surgery for breast, colorectal and ovarian cancer 1992– 97: is there evidence of increasing sub-specialization by

[82] Diaz-Montes TP, Zahurak ML, Giuntoli II RL, Gardner GJ, Gordon TA, Armstrong DK, et al. Surgical care of elderly women with ovarian cancer: a population-based

[83] Bristow RE, Berek JS. Surgery for ovarian cancer: how to improve survival. Lancet.

surgeons? Br J Cancer 2001;84:1308– 13.

2006;367:1558-60.

182 Ovarian Cancer - A Clinical and Translational Update

perspective. Gynecol Oncol 2005;99(2):352–7.

Epithelial ovarian cancer causes more deaths than any other cancer of the female reproductive system and it is the leading cause of death from gynecologic cancer. There is no universally accepted consensus on the surveillance of ovarian cancer, but if we review the main clinical guidelines, we can find similar recommendations for follow-up for patients with ovarian cancer after chemotherapy treatment.

Approximately 60% of patients will experience a relapse after the standard first-line treatment including cytoreductive surgery and adjuvant chemotherapy [1]. At this time, when relapse occurs, the chance of cure decreases drastically and treatment is solely palliative. This makes the increase in overall survival and the quality of life the primary endpoints. Surgery is not sufficiently validated due to the lack of phase III clinical trials, and there are no approved targeted therapies in relapsed ovarian cancer. Therefore, chemotherapy is the only option to achieve these objectives. We will review the role of chemotherapy in recurrent ovarian cancer in this chapter.

#### **2. Diagnosis of epithelial ovarian cancer relapse**

In stages I, II, III and IV complete responders, American guidelines recommend that, after completing primary surgery and adjuvant chemotherapy, follow-up visits should include a physical examination with a pelvic exam every 2 to 4 months for the first two years, then every 3 to 6 months until the fifth year, and then annually after the fifth year. Periodic monitoring of CA 125 and other tumor markers (e.g., CA 19.9, CEA) are also recommended if the markers were elevated previously. The rest of the examination, which ranges from performing

Computerized Tomography (CT), Magnetic Resonance Imaging (MRI) or Positron Emission Tomography/Computerized Tomography (PET/CT), will be performed as clinically indicated such as weight loss, fatigue, bloating, pelvic pain or bowel occlusion [2].

In 1993, Thigpen defined two subgroups of patients with relapsed ovarian carcinoma based on the volume of relapse and the time to relapse after the end of treatment with platinum. Patients with small-volume disease confined to the peritoneal cavity have a far better chance of achieving a response to second-line chemotherapy with subsequent prolonged survival than those with bulky disease or disease outside the abdomen. Thus, we can classify the patients into those who are still "clinically sensitive" to the platinum-based regimens (initial response to platinum-based therapy and relapse more than 6 months after cessation of treatment) and those with "clinically resistant" disease (defined as progression disease during or within 6 months of first-line treatment platinum-based therapy). We should choose a platinumcontaining regimen for relapse for those patients classified as clinically sensitive and an alternative treatment without platinum salts for those with clinically resistant disease [13].

**Interval**

No response Any Primary Platinum-resistant Response < 6 months Potentially platinum-sensitive Response > 6 months Potentially platinum-sensitive

No response Any Platinum-resistant Response < 6 months Platinum-resistant Response > 6 months Platinum-sensitive

Progression ---- Platinum-refractory

No response Any Platinum-refractory Response < 4 months Platinum-refractory

Response > 12 months Platinum-sensitive

No response Any Platinum-refractory

Response < 6 months Platinum-resistant

Response > 12 months Platinum-sensitive

Response > 6 - 12 months Partially platinum-sensitive

Response > 4 - 12 months Intermediate platinum-sensitive

Markman [11,12] Progression ----- Primary Platinum-resistant

Thigpen [13] Progression ---- Platinum-resistant

NICE 2005 [15] Progression ---- Platinum-refractory

**Classification**

The Role of Chemotherapy in Recurrent Ovarian Cancer

http://dx.doi.org/10.5772/54171

185

Until recently, this was the most utilized and simplest classification.

**Author Best response to platinum Platinum Free**

1998 International Workshop Consensus

2010 GCIG Consensus

**Table 1.** Classification of relapsed ovarian cancer

[14]

[16]

European clinical guidelines recommend a physical exam and routine measurement of CA 125 every 3 months for 2 years, every 4 months during the third year and every 6 months during years 4 and 5. CT scan will be performed if the CA 125 is elevated or if there is clinical evidence of relapse [3].

A physical examination to detect recurrent ovarian cancer has limited value and detects abnormalities that indicate a recurrence only in 3.8 to 4.6% of patients [4, 5]. CT has a sensitivi‐ ty of 40 - 93%, depending on the presence of peritoneal disease, tumor location and the pres‐ ence of ascites. The sensitivity of MRI ranges from 62 to 91%, depending on the location of the tumor and tumor size. MRI facilitates the detection of disease on the peritoneal and intestinal surface [6].

We can define the relapse of ovarian cancer with the RECIST (Response Evaluation Criteria in Solid Tumors) criteria. However, relapse can also be defined as a doubling from the upper limit of normal value of CA 125 (30 U/mL) in patients who normalized their value after finishing their treatment, or doubling this value from the nadir (minimum value) in patients who never had normalized values [7-9]. It is estimated that this rise in the CA 125 level precedes the clinical detection of recurrence by about three months [10], and this may have implications at the beginning of the second-line treatment.

#### **3. Classification of relapse**

There are several classifications of patients with relapsed ovarian cancer based on the plati‐ num-free interval (Table 1).

Markman suggested that the probability of response in the re-treatment with platinum-based chemotherapy depends on the platinum-free interval. In a retrospective analysis conducted at the Memorial Sloan-Kettering Cancer Center (New York, United States of America), these authors found a subgroup of patients with a higher likelihood of response to platinum salts. They selected 82 patients who received initial chemotherapeutic treatment with a cisplatinbased regimen and second-line treatment with a cisplatin- or carboplatin-based regimen, with a platinum-free interval of more than 4 months. The response rate to second-line treatment in the three groups according to the platinum-free interval at 5 to 12 months, 13 to 24 months and more than 24 months, was 27%, 33% and 59%, respectively [11]. They proposed to classify patients into different groups according to their previous response to platinum-based treat‐ ment and platinum-free interval: primary platinum-resistant (patients who progressed before the completion of the planned treatment), secondary platinum-resistant (patients who responded to a platinum regimen and did not respond to a second platinum-based treatment), and potentially platinum-sensitive (all patients who respond to a platinum-based treatment, subdivided into patients with platinum-free intervals of less than 6, 6 to 12 months and more than 12 months) [12].

In 1993, Thigpen defined two subgroups of patients with relapsed ovarian carcinoma based on the volume of relapse and the time to relapse after the end of treatment with platinum. Patients with small-volume disease confined to the peritoneal cavity have a far better chance of achieving a response to second-line chemotherapy with subsequent prolonged survival than those with bulky disease or disease outside the abdomen. Thus, we can classify the patients into those who are still "clinically sensitive" to the platinum-based regimens (initial response to platinum-based therapy and relapse more than 6 months after cessation of treatment) and those with "clinically resistant" disease (defined as progression disease during or within 6 months of first-line treatment platinum-based therapy). We should choose a platinumcontaining regimen for relapse for those patients classified as clinically sensitive and an alternative treatment without platinum salts for those with clinically resistant disease [13]. Until recently, this was the most utilized and simplest classification.


**Table 1.** Classification of relapsed ovarian cancer

Computerized Tomography (CT), Magnetic Resonance Imaging (MRI) or Positron Emission Tomography/Computerized Tomography (PET/CT), will be performed as clinically indicated

European clinical guidelines recommend a physical exam and routine measurement of CA 125 every 3 months for 2 years, every 4 months during the third year and every 6 months during years 4 and 5. CT scan will be performed if the CA 125 is elevated or if there is clinical evidence

A physical examination to detect recurrent ovarian cancer has limited value and detects abnormalities that indicate a recurrence only in 3.8 to 4.6% of patients [4, 5]. CT has a sensitivi‐ ty of 40 - 93%, depending on the presence of peritoneal disease, tumor location and the pres‐ ence of ascites. The sensitivity of MRI ranges from 62 to 91%, depending on the location of the tumor and tumor size. MRI facilitates the detection of disease on the peritoneal and intestinal

We can define the relapse of ovarian cancer with the RECIST (Response Evaluation Criteria in Solid Tumors) criteria. However, relapse can also be defined as a doubling from the upper limit of normal value of CA 125 (30 U/mL) in patients who normalized their value after finishing their treatment, or doubling this value from the nadir (minimum value) in patients who never had normalized values [7-9]. It is estimated that this rise in the CA 125 level precedes the clinical detection of recurrence by about three months [10], and this may have implications

There are several classifications of patients with relapsed ovarian cancer based on the plati‐

Markman suggested that the probability of response in the re-treatment with platinum-based chemotherapy depends on the platinum-free interval. In a retrospective analysis conducted at the Memorial Sloan-Kettering Cancer Center (New York, United States of America), these authors found a subgroup of patients with a higher likelihood of response to platinum salts. They selected 82 patients who received initial chemotherapeutic treatment with a cisplatinbased regimen and second-line treatment with a cisplatin- or carboplatin-based regimen, with a platinum-free interval of more than 4 months. The response rate to second-line treatment in the three groups according to the platinum-free interval at 5 to 12 months, 13 to 24 months and more than 24 months, was 27%, 33% and 59%, respectively [11]. They proposed to classify patients into different groups according to their previous response to platinum-based treat‐ ment and platinum-free interval: primary platinum-resistant (patients who progressed before the completion of the planned treatment), secondary platinum-resistant (patients who responded to a platinum regimen and did not respond to a second platinum-based treatment), and potentially platinum-sensitive (all patients who respond to a platinum-based treatment, subdivided into patients with platinum-free intervals of less than 6, 6 to 12 months and more

such as weight loss, fatigue, bloating, pelvic pain or bowel occlusion [2].

of relapse [3].

184 Ovarian Cancer - A Clinical and Translational Update

surface [6].

at the beginning of the second-line treatment.

**3. Classification of relapse**

num-free interval (Table 1).

than 12 months) [12].

The International Workshop Consensus established a different classification in 1998 and stratified patients into platinum-refractory (progression during or within 4 months), inter‐ mediate platinum-sensitive (initial response but relapse 4 -12 months) and platinum-sensitive (relapse after 12 months) [14].

chemotherapy (57% versus 50%; Hazard Ratio (HR): 0.82; 95% CI 0.69 - 0.97; p = 0.02). For PFS, there was a HR: 0.76 (95% CI 0.66 - 0.89; p = 0.0004) in favor of paclitaxel plus platinum-based chemotherapy, which translates into an absolute difference in median PFS of 3 months in favor of the combination regimen (9 versus 12 months). The response rate (RR) seemed to be higher in the combination arm (66%) compared to the conventional chemotherapy arm (p = 0.06). There were no differences between the quality of life measures in both groups. The results showed no difference between different subgroups (randomization group, time to relapse, number of previous lines of chemotherapy, type of prior chemotherapy, age and performance

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Paclitaxel plus platinum-based chemotherapy was generally more toxic than conventional platinum-based chemotherapy, causing more alopecia and neurotoxicity (20% of patients), while conventional platinum-based chemotherapy was associated with more hematological

The ICON4/AGO-OVAR 2.2 trial was the first large clinical trial that showed the superiority

Similar results were found in a Spanish randomized phase II clinical trial conducted by GEICO (Grupo Español de Investigación en Cáncer de Ovario) [18]. In this trial, 81 patients with platinum-sensitive recurrent ovarian carcinoma were randomized to carboplatin or carbopla‐ tin plus paclitaxel. The primary endpoint was objective response and secondary endpoints were time to progression, overall survival, tolerability and quality of life. The platinum-free interval was greater than 12 months in 57.7% of patients. In the intent-to-treat analysis, they reported a higher response rate in the group treated with carboplatin plus paclitaxel than in the carboplatin group (75.6% versus 50%; p = 0.017). The median time to progression (49.1 versus 33.7 weeks; p = 0.021) and overall survival (not reached versus 72.7 weeks; p = 0.0021) were also better in the group treated with the combination therapy. There were no differences in the quality of life. As in the ICON4/AGO-OVAR 2.2 trial, alopecia (86.8%) and neurotoxicity (23.7%) were more frequent in patients treated with paclitaxel. Stomatitis (18.4%) and myal‐ gias/arthralgias (36.8%) were also more frequent in this group. In the ICON4/AGO OVAR 2.2 trial, only 40% of patients received paclitaxel as part of a previous treatment, which could affect the superiority of the paclitaxel arm following the relapse. In the Spanish trial, 87.2% of patients received paclitaxel previously, so it was suggested that carboplatin plus paclitaxel could be

of polychemotherapy versus monotherapy in platinum-sensitive ovarian cancer.

administered at relapse in patients who received this treatment as first-line therapy.

Neurotoxicity is the main drawback for the re-treatment with carboplatin plus paclitaxel because, among other factors, co-administration of paclitaxel and platinum compounds can increase the development of neurotoxicity [19]. Neurotoxicity is a cumulative dosedependent toxicity; 715 mg/m2 is the mean cumulative dose to onset of grade 2 or greater

In the ICON4-AGO OVAR 2.2 study, moderate or severe neurological effects were observed in 20% of patients in the combination arm, and the majority of patients experienced grades 1

**4.2. Carboplatin versus carboplatin/gemcitabine (AGO-OVAR 2.5)**

toxic effects than paclitaxel plus platinum chemotherapy.

status).

neurotoxicity [20].

More recently, the National Institute for Health and Clinical Excellence (NICE) in 2005 [15] and the Gynecologic Cancer InterGroup (GCIG) in 2010 [16] have developed new classifica‐ tions, including partially platinum-sensitive patients (those who relapse between 6 and 12 months after completion of initial platinum-based chemotherapy).

#### **4. Treatment of platinum-sensitive disease**

Until the early 2000s, monotherapy with platinum salts was the standard treatment for patients with platinum-sensitive disease because clinical trials attempting to prove the superiority of polychemotherapy were negative.

More recent clinical trials have demonstrated the superiority of polychemotherapy versus monotherapy, making this strategy the standard treatment in patients with platinum-sensitive disease. We discuss the main previous studies in this section.

#### **4.1. Carboplatin versus carboplatin/paclitaxel (ICON4/AGO-OVAR 2.2)**

In parallel, two pragmatic clinical trials were designed to determine whether the combination of carboplatin and paclitaxel should be used at first relapse after platinum-based chemother‐ apy [the International Collaborative Ovarian Neoplasm 4 (ICON4), coordinated by the Instituto Mario Negri, Milan, Italy (IRFMN) and the Medical Research Council's Clinical Trials Unit, London, United Kingdom (MRC CTU), and Arbeitsgemeinschaft Gynaekologische Onkologie (AGO) OVAR 2.2 coordinated by AGO, Karlsruhe, Germany] [17].

They randomized 802 patients with relapsed epithelial ovarian cancer who previously received platinum-based chemotherapy and had a platinum-free interval of more than 6 months (more than 12 months in the ICON4 group) to receive a conventional platinum-based chemotherapy (the majority of patients (71%) received carboplatin alone) or a combined treatment with paclitaxel 175 mg/m2 plus cisplatin 50 mg/m2 or carboplatin AUC 5 – 6 every 3 weeks for at least 6 cycles. The primary endpoint was overall survival (OS), and secondary endpoints were progression-free survival (PFS) and quality of life. The platinum-free interval was greater than 12 months in 75% of patients.

Patients in the AGO protocol must have previously received cisplatin or carboplatin plus paclitaxel, patients in the MRC CTU protocol trial were permitted to have had more than one line of previous chemotherapy and patients randomized into the Italian protocol required measurable disease.

With a median follow-up of 42 months, OS was increased by 5 months (24 versus 29 months), with an absolute difference in 2-year survival of 7% in favor of paclitaxel plus platinum-based chemotherapy (57% versus 50%; Hazard Ratio (HR): 0.82; 95% CI 0.69 - 0.97; p = 0.02). For PFS, there was a HR: 0.76 (95% CI 0.66 - 0.89; p = 0.0004) in favor of paclitaxel plus platinum-based chemotherapy, which translates into an absolute difference in median PFS of 3 months in favor of the combination regimen (9 versus 12 months). The response rate (RR) seemed to be higher in the combination arm (66%) compared to the conventional chemotherapy arm (p = 0.06). There were no differences between the quality of life measures in both groups. The results showed no difference between different subgroups (randomization group, time to relapse, number of previous lines of chemotherapy, type of prior chemotherapy, age and performance status).

The International Workshop Consensus established a different classification in 1998 and stratified patients into platinum-refractory (progression during or within 4 months), inter‐ mediate platinum-sensitive (initial response but relapse 4 -12 months) and platinum-sensitive

More recently, the National Institute for Health and Clinical Excellence (NICE) in 2005 [15] and the Gynecologic Cancer InterGroup (GCIG) in 2010 [16] have developed new classifica‐ tions, including partially platinum-sensitive patients (those who relapse between 6 and 12

Until the early 2000s, monotherapy with platinum salts was the standard treatment for patients with platinum-sensitive disease because clinical trials attempting to prove the superiority of

More recent clinical trials have demonstrated the superiority of polychemotherapy versus monotherapy, making this strategy the standard treatment in patients with platinum-sensitive

In parallel, two pragmatic clinical trials were designed to determine whether the combination of carboplatin and paclitaxel should be used at first relapse after platinum-based chemother‐ apy [the International Collaborative Ovarian Neoplasm 4 (ICON4), coordinated by the Instituto Mario Negri, Milan, Italy (IRFMN) and the Medical Research Council's Clinical Trials Unit, London, United Kingdom (MRC CTU), and Arbeitsgemeinschaft Gynaekologische

They randomized 802 patients with relapsed epithelial ovarian cancer who previously received platinum-based chemotherapy and had a platinum-free interval of more than 6 months (more than 12 months in the ICON4 group) to receive a conventional platinum-based chemotherapy (the majority of patients (71%) received carboplatin alone) or a combined treatment with

least 6 cycles. The primary endpoint was overall survival (OS), and secondary endpoints were progression-free survival (PFS) and quality of life. The platinum-free interval was greater than

Patients in the AGO protocol must have previously received cisplatin or carboplatin plus paclitaxel, patients in the MRC CTU protocol trial were permitted to have had more than one line of previous chemotherapy and patients randomized into the Italian protocol required

With a median follow-up of 42 months, OS was increased by 5 months (24 versus 29 months), with an absolute difference in 2-year survival of 7% in favor of paclitaxel plus platinum-based

or carboplatin AUC 5 – 6 every 3 weeks for at

months after completion of initial platinum-based chemotherapy).

**4. Treatment of platinum-sensitive disease**

disease. We discuss the main previous studies in this section.

**4.1. Carboplatin versus carboplatin/paclitaxel (ICON4/AGO-OVAR 2.2)**

Onkologie (AGO) OVAR 2.2 coordinated by AGO, Karlsruhe, Germany] [17].

plus cisplatin 50 mg/m2

(relapse after 12 months) [14].

186 Ovarian Cancer - A Clinical and Translational Update

polychemotherapy were negative.

paclitaxel 175 mg/m2

measurable disease.

12 months in 75% of patients.

Paclitaxel plus platinum-based chemotherapy was generally more toxic than conventional platinum-based chemotherapy, causing more alopecia and neurotoxicity (20% of patients), while conventional platinum-based chemotherapy was associated with more hematological toxic effects than paclitaxel plus platinum chemotherapy.

The ICON4/AGO-OVAR 2.2 trial was the first large clinical trial that showed the superiority of polychemotherapy versus monotherapy in platinum-sensitive ovarian cancer.

Similar results were found in a Spanish randomized phase II clinical trial conducted by GEICO (Grupo Español de Investigación en Cáncer de Ovario) [18]. In this trial, 81 patients with platinum-sensitive recurrent ovarian carcinoma were randomized to carboplatin or carbopla‐ tin plus paclitaxel. The primary endpoint was objective response and secondary endpoints were time to progression, overall survival, tolerability and quality of life. The platinum-free interval was greater than 12 months in 57.7% of patients. In the intent-to-treat analysis, they reported a higher response rate in the group treated with carboplatin plus paclitaxel than in the carboplatin group (75.6% versus 50%; p = 0.017). The median time to progression (49.1 versus 33.7 weeks; p = 0.021) and overall survival (not reached versus 72.7 weeks; p = 0.0021) were also better in the group treated with the combination therapy. There were no differences in the quality of life. As in the ICON4/AGO-OVAR 2.2 trial, alopecia (86.8%) and neurotoxicity (23.7%) were more frequent in patients treated with paclitaxel. Stomatitis (18.4%) and myal‐ gias/arthralgias (36.8%) were also more frequent in this group. In the ICON4/AGO OVAR 2.2 trial, only 40% of patients received paclitaxel as part of a previous treatment, which could affect the superiority of the paclitaxel arm following the relapse. In the Spanish trial, 87.2% of patients received paclitaxel previously, so it was suggested that carboplatin plus paclitaxel could be administered at relapse in patients who received this treatment as first-line therapy.

#### **4.2. Carboplatin versus carboplatin/gemcitabine (AGO-OVAR 2.5)**

Neurotoxicity is the main drawback for the re-treatment with carboplatin plus paclitaxel because, among other factors, co-administration of paclitaxel and platinum compounds can increase the development of neurotoxicity [19]. Neurotoxicity is a cumulative dosedependent toxicity; 715 mg/m2 is the mean cumulative dose to onset of grade 2 or greater neurotoxicity [20].

In the ICON4-AGO OVAR 2.2 study, moderate or severe neurological effects were observed in 20% of patients in the combination arm, and the majority of patients experienced grades 1 to 4 neurotoxicity (75% to 83%) with the combination of carboplatin–paclitaxel and cisplatinpaclitaxel.

In this trial, a total of 976 patients with histologically confirmed ovarian cancer with recurrence more than 6 months after first- or second-line platinum- and taxane-based therapies were randomly assigned to receive carboplatin AUC 5 on day 1 plus pegylated liposomal doxorubicin (PLD) 30 mg/m2 on day 1, every 28 days, or carboplatin AUC 5 on day 1 plus paclitaxel 175 mg/m2 on day 1 at 3-week intervals for at least 6 cycles (in case of stabilization of disease or if partial response was achieved after 6 courses, the patients were allowed to receive therapy until progression). The platinum-free interval was greater

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The study was designed as a non-inferiority trial. The primary endpoint was progression-free

With a median follow-up of 22 months, PFS was statistically superior for patients treated with carboplatin/PLD than patients in the carboplatin/paclitaxel arm (11.3 versus 9.4 months with

Severe non-hematologic toxicity (grades 3 to 4) was more frequent in patients in the carboplatin/paclitaxel arm than in patients treated with carboplatin/PLD (36.8% versus 28.4%; p = 0.001). Grade 2 or greater palmar-plantar erythrodisestesia (12% versus 2.2%), nausea (35.2% versus 24.2%), vomiting (22.5% versus 15.6%) and mucositis (13.9% versus 7%) occurred more commonly in the carboplatin/PLD arm. Grade 2 or greater neurotoxic‐ ity (4.9% versus 26.9%), complete hair loss (7% versus 83.6%) and allergic/hypersensitivi‐ ty reactions (5.6% versus 18.8%) were more frequent in patients treated with carboplatin and paclitaxel. The allergic/hypersensitivity reactions were mainly secondary to carbopla‐ tin administration and was the reason for significantly lower rates of early discontinua‐ tion of one or both drugs in the paclitaxel arm compared with the PLD arm (1% versus 6%; p > 0.001). Fewer patients discontinued treatment early for toxicity in the carboplatin/

Regarding hematologic toxicities, they were generally similar between the treatment groups, although grades 3 to 4 neutropenia was more frequent in patients treated with carboplatin/ paclitaxel (35.2% versus 45.7%) and grades 3 to 4 thrombocytopenia was more frequent in patients treated with carboplatin/PLD (15.9% versus 6.2%). There were no differences in febrile neutropenia or the use of supportive treatment (e.g., transfusion, granulocyte colony-stimu‐

Recently, data on the final OS were reported. With a median follow-up of 49 months, no statistically significant difference in OS was observed between the two arms (HR: 0.99; 95% CI: 0.85 - 1.16; p = 0.94). The median OS was 30.7 months in patients treated with carbopla‐ tin and PLD and 33.0 months for patients treated with carboplatin and paclitaxel. The authors rationalize this fact with an imbalanced post-study cross-over between arms, with a greater proportion of patients randomized to carboplatin/paclitaxel receiving post-study PLD (68%) than patients in the carboplatin/PLD arm receiving post-study paclitaxel (43%;

The improved disease-related outcomes achieved with carboplatin/PLD treatment were not

survival, and secondary endpoints were toxicity, quality of life, and overall survival.

than 12 months in 63.9% of patients.

HR: 0.821; 95% CI, 0.72 to 0.94; p = 0.005).

PLD arm (6% versus 15%; p < 0.001).

lating factor).

p < 0.001) [23].

at the expense of quality of life [24].

For these reasons, an alternative combination with carboplatin and gemcitabine was designed to avoid toxic effects, such as neurotoxicity, derived from the combination of carboplatin or cisplatin and paclitaxel.

In the AGO-OVAR 2.5 [21] clinical trial, the AGO-OVAR investigators, in collaboration with the National Cancer Institute of Canada Clinical Trials Group (NCIC CTG) and the European Organization for Research and Treatment of Cancer Gynecological Cancer Group (EORTC GCG), randomized 356 patients with platinum-sensitive recurrent ovarian cancer to receive either carboplatin alone (AUC 5) every 21 days or carboplatin AUC 4 on day 1 plus gemcitabine 1000 mg/m2 on days 1 and 8, every 21 days. Patients could receive 6 to 10 cycles in both arms. The primary objective was progression-free survival, and secondary objectives included the response rate, duration of response, overall survival, quality of life and toxicity. Both groups were well balanced: 70.8% of patients had received platinum-based plus taxane as first-line therapy, and 59.8% of patients had a platinum-free interval greater than 12 months. The study was not powered to detect differences in OS.

With a median follow-up of 17 months, the median PFS in the combination arm and the single-agent arm were 8.6 months (95% CI, 7.9 - 9.7) and 5.8 months (95% CI, 5.2 - 7.1), respectively, with a 28% reduction in the progression-free event rate (HR: 0.72; 95% CI, 0.58 - 0.90; p = 0.0031). On the other hand, the RR was significantly higher in the gemcita‐ bine plus carboplatin arm than in the carboplatin arm (47.2% versus 30.9%; p = 0.0016). The HR for overall survival was 0.96 (95% CI, 0.75 - 1.23; p = 0.7349). There was no difference in OS, which was 18 months for patients treated with carboplatin and gemcitabine versus 17.3 for patients treated with carboplatin alone. Furthermore, there was no difference in the quality of life between treatment arms.

A significant increase in serious (grade 3 to 4) hematologic adverse events was documented in both arms, including neutropenia (71% versus 12%), thrombocytopenia (35% versus 11%) and anemia (27% versus 8%). These adverse events appeared more commonly in the combi‐ nation arm. The use of granulocyte colony-stimulating factor was more frequent in patients treated with carboplatin and gemcitabine (24% versus 10%).

The results of the AGO-OVAR 2.5 trial confirmed the superiority of platinum-based polyche‐ motherapy over platinum salts in monotherapy.

The results of this clinical trial provide a treatment alternative to carboplatin/paclitaxel, with a different profile of toxicity, including less alopecia and neurotoxicity, which can affect the quality of life for women with ovarian cancer.

#### **4.3. Carboplatin/paclitaxel versus carboplatin/Pegylated Liposomal Doxorubicin (PLD) (CALYPSO)**

In an attempt to establish a new second-line treatment with improved tolerance and equal or greater efficacy than the standard treatment with carboplatin and paclitaxel, the CALYPSO clinical trial was designed [22].

In this trial, a total of 976 patients with histologically confirmed ovarian cancer with recurrence more than 6 months after first- or second-line platinum- and taxane-based therapies were randomly assigned to receive carboplatin AUC 5 on day 1 plus pegylated liposomal doxorubicin (PLD) 30 mg/m2 on day 1, every 28 days, or carboplatin AUC 5 on day 1 plus paclitaxel 175 mg/m2 on day 1 at 3-week intervals for at least 6 cycles (in case of stabilization of disease or if partial response was achieved after 6 courses, the patients were allowed to receive therapy until progression). The platinum-free interval was greater than 12 months in 63.9% of patients.

to 4 neurotoxicity (75% to 83%) with the combination of carboplatin–paclitaxel and cisplatin-

For these reasons, an alternative combination with carboplatin and gemcitabine was designed to avoid toxic effects, such as neurotoxicity, derived from the combination of carboplatin or

In the AGO-OVAR 2.5 [21] clinical trial, the AGO-OVAR investigators, in collaboration with the National Cancer Institute of Canada Clinical Trials Group (NCIC CTG) and the European Organization for Research and Treatment of Cancer Gynecological Cancer Group (EORTC GCG), randomized 356 patients with platinum-sensitive recurrent ovarian cancer to receive either carboplatin alone (AUC 5) every 21 days or carboplatin AUC 4 on day 1 plus gemcitabine 1000 mg/m2 on days 1 and 8, every 21 days. Patients could receive 6 to 10 cycles in both arms. The primary objective was progression-free survival, and secondary objectives included the response rate, duration of response, overall survival, quality of life and toxicity. Both groups were well balanced: 70.8% of patients had received platinum-based plus taxane as first-line therapy, and 59.8% of patients had a platinum-free interval greater than 12 months. The study

With a median follow-up of 17 months, the median PFS in the combination arm and the single-agent arm were 8.6 months (95% CI, 7.9 - 9.7) and 5.8 months (95% CI, 5.2 - 7.1), respectively, with a 28% reduction in the progression-free event rate (HR: 0.72; 95% CI, 0.58 - 0.90; p = 0.0031). On the other hand, the RR was significantly higher in the gemcita‐ bine plus carboplatin arm than in the carboplatin arm (47.2% versus 30.9%; p = 0.0016). The HR for overall survival was 0.96 (95% CI, 0.75 - 1.23; p = 0.7349). There was no difference in OS, which was 18 months for patients treated with carboplatin and gemcitabine versus 17.3 for patients treated with carboplatin alone. Furthermore, there was no difference in

A significant increase in serious (grade 3 to 4) hematologic adverse events was documented in both arms, including neutropenia (71% versus 12%), thrombocytopenia (35% versus 11%) and anemia (27% versus 8%). These adverse events appeared more commonly in the combi‐ nation arm. The use of granulocyte colony-stimulating factor was more frequent in patients

The results of the AGO-OVAR 2.5 trial confirmed the superiority of platinum-based polyche‐

The results of this clinical trial provide a treatment alternative to carboplatin/paclitaxel, with a different profile of toxicity, including less alopecia and neurotoxicity, which can affect the

**4.3. Carboplatin/paclitaxel versus carboplatin/Pegylated Liposomal Doxorubicin (PLD)**

In an attempt to establish a new second-line treatment with improved tolerance and equal or greater efficacy than the standard treatment with carboplatin and paclitaxel, the CALYPSO

paclitaxel.

cisplatin and paclitaxel.

188 Ovarian Cancer - A Clinical and Translational Update

was not powered to detect differences in OS.

the quality of life between treatment arms.

motherapy over platinum salts in monotherapy.

quality of life for women with ovarian cancer.

**(CALYPSO)**

clinical trial was designed [22].

treated with carboplatin and gemcitabine (24% versus 10%).

The study was designed as a non-inferiority trial. The primary endpoint was progression-free survival, and secondary endpoints were toxicity, quality of life, and overall survival.

With a median follow-up of 22 months, PFS was statistically superior for patients treated with carboplatin/PLD than patients in the carboplatin/paclitaxel arm (11.3 versus 9.4 months with HR: 0.821; 95% CI, 0.72 to 0.94; p = 0.005).

Severe non-hematologic toxicity (grades 3 to 4) was more frequent in patients in the carboplatin/paclitaxel arm than in patients treated with carboplatin/PLD (36.8% versus 28.4%; p = 0.001). Grade 2 or greater palmar-plantar erythrodisestesia (12% versus 2.2%), nausea (35.2% versus 24.2%), vomiting (22.5% versus 15.6%) and mucositis (13.9% versus 7%) occurred more commonly in the carboplatin/PLD arm. Grade 2 or greater neurotoxic‐ ity (4.9% versus 26.9%), complete hair loss (7% versus 83.6%) and allergic/hypersensitivi‐ ty reactions (5.6% versus 18.8%) were more frequent in patients treated with carboplatin and paclitaxel. The allergic/hypersensitivity reactions were mainly secondary to carbopla‐ tin administration and was the reason for significantly lower rates of early discontinua‐ tion of one or both drugs in the paclitaxel arm compared with the PLD arm (1% versus 6%; p > 0.001). Fewer patients discontinued treatment early for toxicity in the carboplatin/ PLD arm (6% versus 15%; p < 0.001).

Regarding hematologic toxicities, they were generally similar between the treatment groups, although grades 3 to 4 neutropenia was more frequent in patients treated with carboplatin/ paclitaxel (35.2% versus 45.7%) and grades 3 to 4 thrombocytopenia was more frequent in patients treated with carboplatin/PLD (15.9% versus 6.2%). There were no differences in febrile neutropenia or the use of supportive treatment (e.g., transfusion, granulocyte colony-stimu‐ lating factor).

Recently, data on the final OS were reported. With a median follow-up of 49 months, no statistically significant difference in OS was observed between the two arms (HR: 0.99; 95% CI: 0.85 - 1.16; p = 0.94). The median OS was 30.7 months in patients treated with carbopla‐ tin and PLD and 33.0 months for patients treated with carboplatin and paclitaxel. The authors rationalize this fact with an imbalanced post-study cross-over between arms, with a greater proportion of patients randomized to carboplatin/paclitaxel receiving post-study PLD (68%) than patients in the carboplatin/PLD arm receiving post-study paclitaxel (43%; p < 0.001) [23].

The improved disease-related outcomes achieved with carboplatin/PLD treatment were not at the expense of quality of life [24].

This study provides an optional scheme of treatment for patients with platinum-sensitive ovarian cancer, with a reduction in severe toxicities, including carboplatin hypersensitivity reactions and peripheral neurotoxicity, both of which can be a reason for limiting the dose. Carboplatin/PLD also induced far less alopecia, one of the most feared adverse effects of chemotherapy for the majority of women.

dary endpoints included the overall response rate, duration of treatment response, overall survival, safety and tolerability, and health-related quality of life. Ninety-seven patients were randomly assigned (1:1:1 ratio) to receive olaparib 200 mg twice per day, 400 mg twice per day

status and platinum sensitivity (sensitive or resistant). There was no statistically significant difference in PFS between the olaparib 200 mg, olaparib 400 mg and PLD groups (6.5 months, 8.8 months and 7.1 months, respectively). The overall response rate was 25%, 31% and 18%, respectively, with no statistically significant difference. A similar duration of response was also observed (6.0, 6.8 and 5.5 months). There was no difference among groups in the OS or the health-related quality of life. Nausea, vomiting, fatigue and anemia were the most common adverse events related to olaparib; the adverse events related to PLD were stomatitis and

In a second randomized phase II study, olaparib was evaluated in the maintenance treatment for patients with platinum-sensitive relapsed high-grade (grades 2 or 3) ovarian cancer who responded to their most recent platinum-based chemotherapy [28]. A total of 265 patients were randomized to receive olaparib 400 mg twice daily or placebo after completion of their last dose of platinum-based chemotherapy. The primary endpoint was progression-free survival; it was significantly longer in the olaparib group (8.4 months) than in the placebo group (4.8 months), with a hazard ratio for progression or death of 0.35 (95% CI 0.25 to 0.49; p < 0.001). Secondary efficacy endpoints were time to progression, objective response rate and overall survival. The time to progression was also significantly longer in patients treated with olaparib (8.3 versus 3.7 months; HR: 0.35; 95% CI 0.25 to 0.47; p < 0.001). According to the RECIST criteria, there was no difference in the response rate (12% versus 4%; p = 0.12) or in the overall survival in the interim analysis at 38% maturity (29.7 versus 29.9 months; p = 0.75). Nausea, vomiting, fatigue and anemia were the adverse events, with an incidence of at least 10% or

The results of these two trials underline the necessity of further exploring the role of olaparib and other PARP inhibitors in the treatment of women with recurrent ovarian cancer. It may well be that their use has to be restricted to BRCA mutated patients, but a better definition of

Patients with platinum-resistant disease have a worse prognosis than patients with platinumsensitive disease and a poorer response rate to cytostatic treatment. Although there is no clear recommendation for the standard treatment in these patients, there is a long list of drugs that have shown activity in phase II clinical trials in this situation: pegylated liposomal doxorubicin, topotecan, gemcitabine, paclitaxel, docetaxel, trabectedin, vinorelbine, ifosfamide, etoposide,

higher in the olaparib group; the majority of them were grade 1 or 2.

BRCAness should then be standardized. [29]

and pemetrexed (Table 2).

**5. Treatment of platinum-resistant disease**

every 28 days. Patients were stratified by BRCA1 or BRCA2

The Role of Chemotherapy in Recurrent Ovarian Cancer

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191

continuously or PLD 50 mg/m2

palmo-plantar erythrodysesthesia.

#### **4.4. Carboplatin/gemcitabine versus carboplatin/gemcitabine/bevacizumab (OCEANS)**

In ovarian cancer, as in other tumors, the addition of new treatments is required for improved outcomes.

In a phase III clinical trial, 484 patients with relapsed platinum-sensitive ovarian cancer were randomly assigned to receive Carboplatin AUC 4 on day 1 and Gemcitabine 1000 mg/m2 on days 1 and 8, every 21 days with placebo, or bevacizumab 15 mg/kg on day 1, every 21 days [25]. After 6 to 10 cycles of chemotherapy, bevacizumab or placebo were continued until toxicity or progression. The primary endpoint was progression-free survival, and secondary endpoints were overall response rate, overall survival and the duration of response.

With a median follow-up of 24 months, the analysis showed an increase in PFS (12.4 versus 8.4 months with a HR of 0.484; 95% CI 0.388 to 0.605; p < 0.0001) and in the RR (78.5% versus 57.4%, p < 0.0001) for bevacizumab. The duration of response was also significant‐ ly increased with the addition of bevacizumab (10.4 versus 7.4 months; HR: 0.534; 95% CI: 0.408 - 0.698). With the number of events for the final analysis not yet reached, the OS was 35.2 months in the placebo arm versus 33.3 months in the bevacizumab arm. This could be related to subsequent therapy, including patients receiving bevacizumab in the place‐ bo arm (31%).

The bevacizumab arm had a higher incidence of grade 3 or higher hypertension (17.4% versus 1%) and proteinuria (8.5% versus < 1%). There was no gastrointestinal perforation in any group.

This is the first positive phase III trial evaluating the addition of a targeting therapy to a standard platinum-based chemotherapy regimen for recurrent ovarian cancer.

#### **4.5. New perspectives in the treatment of platinum-sensitive disease**

The poly (adenosine diphosphate [ADP]-ribose) polymerases (PARPs) are a family of enzymes that play a role in the repair of DNA damage by repairing base excisions. The tumor-suppressor proteins BRCA1 and BRCA2 are components of the DNA repair pathway, and it is known that a germ-line mutation in BRCA1 or BRCA2 is associated with a high risk of the development of some cancers, including breast, prostate and ovarian cancer. Olaparib (AZD2281) is an oral PARP inhibitor that has shown activity in cancers associated with BRCA1 or BRCA2 mutations with an acceptable side-effect profile [26].

A randomized phase II clinical trial was designed to compare the efficacy of olaparib and PLD in patients with confirmed germ-line BRCA1 or BRCA2 mutations and recurrent or progressed ovarian cancer within 12 months of the most recent platinum-based chemotherapy regimen [27]. The primary endpoint was the progression-free survival by RECIST criteria, and secon‐ dary endpoints included the overall response rate, duration of treatment response, overall survival, safety and tolerability, and health-related quality of life. Ninety-seven patients were randomly assigned (1:1:1 ratio) to receive olaparib 200 mg twice per day, 400 mg twice per day continuously or PLD 50 mg/m2 every 28 days. Patients were stratified by BRCA1 or BRCA2 status and platinum sensitivity (sensitive or resistant). There was no statistically significant difference in PFS between the olaparib 200 mg, olaparib 400 mg and PLD groups (6.5 months, 8.8 months and 7.1 months, respectively). The overall response rate was 25%, 31% and 18%, respectively, with no statistically significant difference. A similar duration of response was also observed (6.0, 6.8 and 5.5 months). There was no difference among groups in the OS or the health-related quality of life. Nausea, vomiting, fatigue and anemia were the most common adverse events related to olaparib; the adverse events related to PLD were stomatitis and palmo-plantar erythrodysesthesia.

In a second randomized phase II study, olaparib was evaluated in the maintenance treatment for patients with platinum-sensitive relapsed high-grade (grades 2 or 3) ovarian cancer who responded to their most recent platinum-based chemotherapy [28]. A total of 265 patients were randomized to receive olaparib 400 mg twice daily or placebo after completion of their last dose of platinum-based chemotherapy. The primary endpoint was progression-free survival; it was significantly longer in the olaparib group (8.4 months) than in the placebo group (4.8 months), with a hazard ratio for progression or death of 0.35 (95% CI 0.25 to 0.49; p < 0.001). Secondary efficacy endpoints were time to progression, objective response rate and overall survival. The time to progression was also significantly longer in patients treated with olaparib (8.3 versus 3.7 months; HR: 0.35; 95% CI 0.25 to 0.47; p < 0.001). According to the RECIST criteria, there was no difference in the response rate (12% versus 4%; p = 0.12) or in the overall survival in the interim analysis at 38% maturity (29.7 versus 29.9 months; p = 0.75). Nausea, vomiting, fatigue and anemia were the adverse events, with an incidence of at least 10% or higher in the olaparib group; the majority of them were grade 1 or 2.

The results of these two trials underline the necessity of further exploring the role of olaparib and other PARP inhibitors in the treatment of women with recurrent ovarian cancer. It may well be that their use has to be restricted to BRCA mutated patients, but a better definition of BRCAness should then be standardized. [29]

#### **5. Treatment of platinum-resistant disease**

This study provides an optional scheme of treatment for patients with platinum-sensitive ovarian cancer, with a reduction in severe toxicities, including carboplatin hypersensitivity reactions and peripheral neurotoxicity, both of which can be a reason for limiting the dose. Carboplatin/PLD also induced far less alopecia, one of the most feared adverse effects of

**4.4. Carboplatin/gemcitabine versus carboplatin/gemcitabine/bevacizumab (OCEANS)**

In ovarian cancer, as in other tumors, the addition of new treatments is required for improved

In a phase III clinical trial, 484 patients with relapsed platinum-sensitive ovarian cancer were randomly assigned to receive Carboplatin AUC 4 on day 1 and Gemcitabine 1000 mg/m2 on days 1 and 8, every 21 days with placebo, or bevacizumab 15 mg/kg on day 1, every 21 days [25]. After 6 to 10 cycles of chemotherapy, bevacizumab or placebo were continued until toxicity or progression. The primary endpoint was progression-free survival, and secondary

With a median follow-up of 24 months, the analysis showed an increase in PFS (12.4 versus 8.4 months with a HR of 0.484; 95% CI 0.388 to 0.605; p < 0.0001) and in the RR (78.5% versus 57.4%, p < 0.0001) for bevacizumab. The duration of response was also significant‐ ly increased with the addition of bevacizumab (10.4 versus 7.4 months; HR: 0.534; 95% CI: 0.408 - 0.698). With the number of events for the final analysis not yet reached, the OS was 35.2 months in the placebo arm versus 33.3 months in the bevacizumab arm. This could be related to subsequent therapy, including patients receiving bevacizumab in the place‐

The bevacizumab arm had a higher incidence of grade 3 or higher hypertension (17.4% versus 1%) and proteinuria (8.5% versus < 1%). There was no gastrointestinal perforation in any group.

This is the first positive phase III trial evaluating the addition of a targeting therapy to a

The poly (adenosine diphosphate [ADP]-ribose) polymerases (PARPs) are a family of enzymes that play a role in the repair of DNA damage by repairing base excisions. The tumor-suppressor proteins BRCA1 and BRCA2 are components of the DNA repair pathway, and it is known that a germ-line mutation in BRCA1 or BRCA2 is associated with a high risk of the development of some cancers, including breast, prostate and ovarian cancer. Olaparib (AZD2281) is an oral PARP inhibitor that has shown activity in cancers associated with BRCA1 or BRCA2 mutations

A randomized phase II clinical trial was designed to compare the efficacy of olaparib and PLD in patients with confirmed germ-line BRCA1 or BRCA2 mutations and recurrent or progressed ovarian cancer within 12 months of the most recent platinum-based chemotherapy regimen [27]. The primary endpoint was the progression-free survival by RECIST criteria, and secon‐

standard platinum-based chemotherapy regimen for recurrent ovarian cancer.

**4.5. New perspectives in the treatment of platinum-sensitive disease**

with an acceptable side-effect profile [26].

endpoints were overall response rate, overall survival and the duration of response.

chemotherapy for the majority of women.

190 Ovarian Cancer - A Clinical and Translational Update

outcomes.

bo arm (31%).

Patients with platinum-resistant disease have a worse prognosis than patients with platinumsensitive disease and a poorer response rate to cytostatic treatment. Although there is no clear recommendation for the standard treatment in these patients, there is a long list of drugs that have shown activity in phase II clinical trials in this situation: pegylated liposomal doxorubicin, topotecan, gemcitabine, paclitaxel, docetaxel, trabectedin, vinorelbine, ifosfamide, etoposide, and pemetrexed (Table 2).


**5.1. Topotecan versus paclitaxel**

topotecan group (9 weeks; p = 0.041).

response rates.

myalgia and neurotoxicity.

Topotecan and paclitaxel are active in platinum-resistant relapsed ovarian cancer. To compare the activity of these two drugs in this setting, a phase III clinical trial was conducted in patients who had progressed during or after platinum-based therapy [41, 42]. A total of 226 patients

duration of treatment was dependent on response. Patients with a complete or partial response continued treatment until progression or for 6 months past the maximal response. Patients who progressed were removed from the study and patients with stable disease after six courses were removed from the study or switched to the alternate regimen (the study allowed crossover of the arms). None of the patients had previously received topotecan or paclitaxel (not included in standard first-line therapy as of now). Patients were stratified as platinumresistant or as early, interim and late relapse groups. In the study, 53% of the patients did not respond to platinum-based treatment or had progression within 6 months; they had platinum-

The primary efficacy parameters were the response rate, duration of response and time to progression. The secondary criteria for efficacy were the time to response and survival.

In the whole group of patients in the study, no differences in the response rates (topotecan 20.5% versus paclitaxel 13.2%; p = 0.138) or in the median survival (63 weeks for topotecan versus 53 weeks for paclitaxel, p = 0.44) were achieved. The duration of response was 32.1 weeks in patients treated with topotecan and 19.7 weeks in patients treated with paclitaxel (p = 0.222). There was no statistically significant difference in the time to progression after therapy (18.9 weeks for topotecan versus 14.7 weeks for paclitaxel; p = 0.08). The median time to documented radiologic response was inferior in the paclitaxel group (6 weeks) than in the

Among platinum-resistant patients, the response rates were superior in the topotecan group than in the paclitaxel group (13.1 versus 6.7%, p = 0.303), and the median overall survival was

Patients who had no ascites, better performance status and a smaller tumor burden had higher

The results of questionnaires on the quality of life, including pain, anorexia, diarrhea, fatigue, nausea and vomiting, dyspnea, constipation and insomnia, were similar in both groups.

Different toxicities were observed in the two groups. Hematologic toxicity was more frequent in the topotecan group, including grade 4 neutropenia (79% versus 23% in paclitaxel group; p < 0.01) and grade 4 thrombocytopenia (25% versus 2% in paclitaxel group; p < 0.01). Other toxicities more frequent in patients treated with topotecan were fatigue, nausea and vomiting (generally grades 1 – 2). Patients in the paclitaxel group experienced more alopecia, arthralgia,

Patients who received topotecan after paclitaxel in their third-line treatment had an overall response rate of 13%, compared to 10% (p = 0.638) in patients who received paclitaxel after

28.4 weeks in the topotecan group and 39.7 weeks in patients treated with paclitaxel.

/24 h on 5 consecutive

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, every 21 days (114 patients). The

The Role of Chemotherapy in Recurrent Ovarian Cancer

were randomized to receive chemotherapy with topotecan 1.5 mg/m2

resistant disease (55% in the topotecan group and 52% in the paclitaxel group).

days, every 21 days (112 patients), or paclitaxel 175 mg/m2

#### **Table 2.** Response rate and toxicity for platinum-resistant disease

The comparisons between some of these drugs in phase III clinical trials do not yield superior results for any of the drugs in terms of overall or progression-free survival.

As explained, the response rate to platinum compounds is too low in patients with platinumresistant disease, so monotherapy with a non-platinum drug is usually preferred because studies with doublets have not demonstrated superiority in platinum-resistant patients or either have presented greater toxicity [30 - 37].

Despite its frequent use in clinical practice, endocrine treatment (e.g., Tamoxifen, Letrozole) is not approved, and there is no good evidence supporting its use. Data on tamoxifen were obtained from observational studies, not comparative ones, and do not allow us to make any evidence-based recommendations [38]. In a phase II trial with letrozol carried out in 44 patients (half of them with platinum-resistant disease) who had primary tumors that expressed the estrogen receptor, a 9% overall response and 42% stabilization at 12 weeks was obtained in 33 patients with radiologically measurable disease, with a minimal toxicity [39]. In any case, there are worse data in the literature on the impact of endocrine therapy versus chemotherapy on progression-free survival [40]. Unfortunately, there are no phase III trials to make any recommendations about the use of hormone treatment in relapsed ovarian cancer.

The main phase III clinical trials comparing different agents in platinum-resistant relapsed ovarian cancer are shown below.

#### **5.1. Topotecan versus paclitaxel**

**Drug Response rate Main toxicity**

192 Ovarian Cancer - A Clinical and Translational Update

Gemcitabine 9 - 16% Hematologic

Docetaxel 23% Hematologic

Trabectedin 6% Hematologic

Vinorelbine 3 - 21% Neutropenia

Etoposide 27% Neutropenia

**Table 2.** Response rate and toxicity for platinum-resistant disease

either have presented greater toxicity [30 - 37].

ovarian cancer are shown below.

Pemetrexed 9 - 21% Neutropenia, asthenia

results for any of the drugs in terms of overall or progression-free survival.

Pegylated liposomal doxorubicin 20% Hand-foot syndrome, mucositis

Topotecan 6 - 20% Hematologic, alopecia

Paclitaxel 13 - 17% Neurotoxicity, alopecia

Ifosfamide 12% Hematologic, central nervous toxicity

The comparisons between some of these drugs in phase III clinical trials do not yield superior

As explained, the response rate to platinum compounds is too low in patients with platinumresistant disease, so monotherapy with a non-platinum drug is usually preferred because studies with doublets have not demonstrated superiority in platinum-resistant patients or

Despite its frequent use in clinical practice, endocrine treatment (e.g., Tamoxifen, Letrozole) is not approved, and there is no good evidence supporting its use. Data on tamoxifen were obtained from observational studies, not comparative ones, and do not allow us to make any evidence-based recommendations [38]. In a phase II trial with letrozol carried out in 44 patients (half of them with platinum-resistant disease) who had primary tumors that expressed the estrogen receptor, a 9% overall response and 42% stabilization at 12 weeks was obtained in 33 patients with radiologically measurable disease, with a minimal toxicity [39]. In any case, there are worse data in the literature on the impact of endocrine therapy versus chemotherapy on progression-free survival [40]. Unfortunately, there are no phase III trials to make any

recommendations about the use of hormone treatment in relapsed ovarian cancer.

The main phase III clinical trials comparing different agents in platinum-resistant relapsed

Topotecan and paclitaxel are active in platinum-resistant relapsed ovarian cancer. To compare the activity of these two drugs in this setting, a phase III clinical trial was conducted in patients who had progressed during or after platinum-based therapy [41, 42]. A total of 226 patients were randomized to receive chemotherapy with topotecan 1.5 mg/m2 /24 h on 5 consecutive days, every 21 days (112 patients), or paclitaxel 175 mg/m2 , every 21 days (114 patients). The duration of treatment was dependent on response. Patients with a complete or partial response continued treatment until progression or for 6 months past the maximal response. Patients who progressed were removed from the study and patients with stable disease after six courses were removed from the study or switched to the alternate regimen (the study allowed crossover of the arms). None of the patients had previously received topotecan or paclitaxel (not included in standard first-line therapy as of now). Patients were stratified as platinumresistant or as early, interim and late relapse groups. In the study, 53% of the patients did not respond to platinum-based treatment or had progression within 6 months; they had platinumresistant disease (55% in the topotecan group and 52% in the paclitaxel group).

The primary efficacy parameters were the response rate, duration of response and time to progression. The secondary criteria for efficacy were the time to response and survival.

In the whole group of patients in the study, no differences in the response rates (topotecan 20.5% versus paclitaxel 13.2%; p = 0.138) or in the median survival (63 weeks for topotecan versus 53 weeks for paclitaxel, p = 0.44) were achieved. The duration of response was 32.1 weeks in patients treated with topotecan and 19.7 weeks in patients treated with paclitaxel (p = 0.222). There was no statistically significant difference in the time to progression after therapy (18.9 weeks for topotecan versus 14.7 weeks for paclitaxel; p = 0.08). The median time to documented radiologic response was inferior in the paclitaxel group (6 weeks) than in the topotecan group (9 weeks; p = 0.041).

Among platinum-resistant patients, the response rates were superior in the topotecan group than in the paclitaxel group (13.1 versus 6.7%, p = 0.303), and the median overall survival was 28.4 weeks in the topotecan group and 39.7 weeks in patients treated with paclitaxel.

Patients who had no ascites, better performance status and a smaller tumor burden had higher response rates.

The results of questionnaires on the quality of life, including pain, anorexia, diarrhea, fatigue, nausea and vomiting, dyspnea, constipation and insomnia, were similar in both groups.

Different toxicities were observed in the two groups. Hematologic toxicity was more frequent in the topotecan group, including grade 4 neutropenia (79% versus 23% in paclitaxel group; p < 0.01) and grade 4 thrombocytopenia (25% versus 2% in paclitaxel group; p < 0.01). Other toxicities more frequent in patients treated with topotecan were fatigue, nausea and vomiting (generally grades 1 – 2). Patients in the paclitaxel group experienced more alopecia, arthralgia, myalgia and neurotoxicity.

Patients who received topotecan after paclitaxel in their third-line treatment had an overall response rate of 13%, compared to 10% (p = 0.638) in patients who received paclitaxel after topotecan. The data analysis for those patients receiving the other drug (paclitaxel or topote‐ can) in the third-line therapy showed that there was a degree of non-cross-resistance between them [43]. Therefore, the use of paclitaxel in first-line therapy does not prevent the adminis‐ tration of topotecan in relapsed epithelial ovarian cancer.

In the subgroup of platinum-resistant patients, (54% of the population of the study; 255 patients) there were no statistically significant differences in the response rate (12.3% for PLD and 6.5% for topotecan, p = 0.118), the PFS (9.1 weeks in patients who received PLD compared to 13.6 weeks in the topotecan group, p = 0.733), or OS (35.6 weeks for the PLD group and 41.3 for the topotecan group, p = 0.455, with a HR = 1.069, 95% CI 0.823 to 1.387, p = 0.618).

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The toxicity profiles of the two drugs were different. The main toxicities in patients treated with PLD were hand-foot syndrome (49%) and stomatitis (40%). The main toxicities in patients treated with topotecan were hematological toxicity, so they were more likely to receive granulocyte colony-stimulating factor (29.1%), erythropoietin (23.1%) and transfusions (57.8%). Moreover, the toxicity caused by PLD was usually mild to moderate, while the toxicity caused by topotecan was more severe. Despite this difference, there was no difference in the

Two randomized phase III trials compared gemcitabine with PLD in patients with platinum-

The first trial [47] was carried out in 195 patients with platinum-resistant ovarian cancer who were randomly assigned to receive gemcitabine 1000 mg/m2 on days 1 and 8, every 21 days,

over treatment was administered at progression. The primary endpoint was progression-free survival, and secondary endpoints were response rate, time to treatment failure, survival and

The response rate was similar in both groups (9.2% for gemcitabine versus 11.7% for PLD, p = 0.772). There was no difference in the progression-free survival between patients treated with gemcitabine and patients treated with PLD (3.6 months versus 3.1 months, p = 0.870). The overall survival was similar in patients treated with gemcitabine followed by PLD and patients

The toxicity profiles were different, with more hand-foot syndrome and stomatitis in the PLD arm and increased constipation, nausea and vomiting, fatigue and neutropenia in the gemci‐ tabine arm. During the cross-over treatment, toxicity was similar to those observed during the

In a second study [48], 153 patients previously treated with platinum/paclitaxel who had relapsed or progressed within 12 months (53% within 6 months) were randomized to receive

There were no differences in the response rate (29% for gemcitabine versus 16% for PLD, p = 0.066) or time to progression (20 weeks in gemcitabine group versus 16 weeks in PLD group, p = 0.411). Although the overall survival was higher in the PLD arm (51 weeks versus 56 weeks, p = 0.048), this difference was not detected in the platinum-resistant subgroup (relapse or progression < 6 months). The toxicity profile was similar to the previous study. Health-related

on days 1, 8 and 15, every 28 days, or PLD 40 mg/m2

every 28 days.

who received the inverse sequence (12.7 months versus 13.5 months, p = 0.997).

every 28 days until the progression of disease or unacceptable toxicity. Cross-

health-related quality of life questionnaire at 12 weeks.

resistant disease.

or PLD 50 mg/m2

quality of life.

initial treatment phase.

gemcitabine 1000 mg/m2

quality of life favored the PLD arm.

**5.4. Gemcitabine versus pegylated liposomal doxorubicin**

#### **5.2. Paclitaxel versus pegylated liposomal doxorubicin**

One study compared PLD 50 mg/m2 every 4 weeks versus paclitaxel 175 mg/m2 every 3 weeks in 214 patients with relapsed epithelial ovarian cancer [44].

There were no differences in the response rates among patients who received pegylated liposomal doxorubicin and patients who received paclitaxel (17.8% versus 22.4%; p = 0.034). There was also no difference in the PFS (21.7 weeks versus 22.4 weeks; p = 0.15) or OS (45.7 weeks versus 56.1 weeks; p = 0.44).

There were no observed differences in the PFS or OS in platinum-resistant or platinumsensitive patients.

In the PLD group, hand-foot syndrome, stomatitis, nausea, and vomiting were more frequent. Conversely, alopecia, myalgia, arthralgia, and paresthesia were more frequent in the paclitaxel group.

#### **5.3. Pegylated liposomal doxorubicin versus topotecan**

To compare the efficacy and safety of PLD and topotecan in relapsed ovarian cancer after chemotherapy with platinum and taxanes, a phase III clinical trial was carried out in 474 patients [45, 46].

Patients were randomized to receive treatment with PLD 50 mg/m2 every 28 days (239 patients), or topotecan 1.5 mg/m2 /24 h on 5 consecutive days, every 21 days (235 patients). The primary endpoint was time to progression, and the secondary endpoints included overall survival, response rate, time to response, duration of response and toxicity. The trial included 54% of the platinum-resistant patients in the PLD group and 53% of such patients in the topotecan group.

There was no difference in the rate of response between the two groups (19.7% in patients treated with PLD versus 17% in patients treated with topotecan; p = 0.390). A reduction in the risk of death by 18% was achieved in the group of patients treated with PLD compared to topotecan (HR = 1.216: 95% CI 1.000 to 1.478, p = 0.050). The median survival was 62.7 weeks in the PLD group versus 59.7 weeks in the topotecan group.

In the platinum-sensitive population, there were benefits in survival among patients treated with PLD, with a reduced risk of death by 30% (HR 1.432, 95% CI 1.066 to 1.923, p = 0.017) and a median survival of 107.9 weeks in the PLD group compared to 70.1 weeks in the topotecan group. The progression-free survival was 28.9 weeks for the PLD group and 23.3 weeks for the topotecan group (p =0.037), although the response rate was similar between the two groups (28.4% in the PLD group versus 28.8% in the topotecan group, p = 0.964).

In the subgroup of platinum-resistant patients, (54% of the population of the study; 255 patients) there were no statistically significant differences in the response rate (12.3% for PLD and 6.5% for topotecan, p = 0.118), the PFS (9.1 weeks in patients who received PLD compared to 13.6 weeks in the topotecan group, p = 0.733), or OS (35.6 weeks for the PLD group and 41.3 for the topotecan group, p = 0.455, with a HR = 1.069, 95% CI 0.823 to 1.387, p = 0.618).

The toxicity profiles of the two drugs were different. The main toxicities in patients treated with PLD were hand-foot syndrome (49%) and stomatitis (40%). The main toxicities in patients treated with topotecan were hematological toxicity, so they were more likely to receive granulocyte colony-stimulating factor (29.1%), erythropoietin (23.1%) and transfusions (57.8%). Moreover, the toxicity caused by PLD was usually mild to moderate, while the toxicity caused by topotecan was more severe. Despite this difference, there was no difference in the health-related quality of life questionnaire at 12 weeks.

#### **5.4. Gemcitabine versus pegylated liposomal doxorubicin**

topotecan. The data analysis for those patients receiving the other drug (paclitaxel or topote‐ can) in the third-line therapy showed that there was a degree of non-cross-resistance between them [43]. Therefore, the use of paclitaxel in first-line therapy does not prevent the adminis‐

There were no differences in the response rates among patients who received pegylated liposomal doxorubicin and patients who received paclitaxel (17.8% versus 22.4%; p = 0.034). There was also no difference in the PFS (21.7 weeks versus 22.4 weeks; p = 0.15) or OS (45.7

There were no observed differences in the PFS or OS in platinum-resistant or platinum-

In the PLD group, hand-foot syndrome, stomatitis, nausea, and vomiting were more frequent. Conversely, alopecia, myalgia, arthralgia, and paresthesia were more frequent in the paclitaxel

To compare the efficacy and safety of PLD and topotecan in relapsed ovarian cancer after chemotherapy with platinum and taxanes, a phase III clinical trial was carried out in 474

Patients were randomized to receive treatment with PLD 50 mg/m2 every 28 days (239

primary endpoint was time to progression, and the secondary endpoints included overall survival, response rate, time to response, duration of response and toxicity. The trial included 54% of the platinum-resistant patients in the PLD group and 53% of such patients in the

There was no difference in the rate of response between the two groups (19.7% in patients treated with PLD versus 17% in patients treated with topotecan; p = 0.390). A reduction in the risk of death by 18% was achieved in the group of patients treated with PLD compared to topotecan (HR = 1.216: 95% CI 1.000 to 1.478, p = 0.050). The median survival was 62.7 weeks

In the platinum-sensitive population, there were benefits in survival among patients treated with PLD, with a reduced risk of death by 30% (HR 1.432, 95% CI 1.066 to 1.923, p = 0.017) and a median survival of 107.9 weeks in the PLD group compared to 70.1 weeks in the topotecan group. The progression-free survival was 28.9 weeks for the PLD group and 23.3 weeks for the topotecan group (p =0.037), although the response rate was similar between the two groups

/24 h on 5 consecutive days, every 21 days (235 patients). The

every 4 weeks versus paclitaxel 175 mg/m2

every 3 weeks

tration of topotecan in relapsed epithelial ovarian cancer.

**5.2. Paclitaxel versus pegylated liposomal doxorubicin**

in 214 patients with relapsed epithelial ovarian cancer [44].

**5.3. Pegylated liposomal doxorubicin versus topotecan**

in the PLD group versus 59.7 weeks in the topotecan group.

(28.4% in the PLD group versus 28.8% in the topotecan group, p = 0.964).

One study compared PLD 50 mg/m2

194 Ovarian Cancer - A Clinical and Translational Update

weeks versus 56.1 weeks; p = 0.44).

sensitive patients.

patients [45, 46].

topotecan group.

patients), or topotecan 1.5 mg/m2

group.

Two randomized phase III trials compared gemcitabine with PLD in patients with platinumresistant disease.

The first trial [47] was carried out in 195 patients with platinum-resistant ovarian cancer who were randomly assigned to receive gemcitabine 1000 mg/m2 on days 1 and 8, every 21 days, or PLD 50 mg/m2 every 28 days until the progression of disease or unacceptable toxicity. Crossover treatment was administered at progression. The primary endpoint was progression-free survival, and secondary endpoints were response rate, time to treatment failure, survival and quality of life.

The response rate was similar in both groups (9.2% for gemcitabine versus 11.7% for PLD, p = 0.772). There was no difference in the progression-free survival between patients treated with gemcitabine and patients treated with PLD (3.6 months versus 3.1 months, p = 0.870). The overall survival was similar in patients treated with gemcitabine followed by PLD and patients who received the inverse sequence (12.7 months versus 13.5 months, p = 0.997).

The toxicity profiles were different, with more hand-foot syndrome and stomatitis in the PLD arm and increased constipation, nausea and vomiting, fatigue and neutropenia in the gemci‐ tabine arm. During the cross-over treatment, toxicity was similar to those observed during the initial treatment phase.

In a second study [48], 153 patients previously treated with platinum/paclitaxel who had relapsed or progressed within 12 months (53% within 6 months) were randomized to receive gemcitabine 1000 mg/m2 on days 1, 8 and 15, every 28 days, or PLD 40 mg/m2 every 28 days.

There were no differences in the response rate (29% for gemcitabine versus 16% for PLD, p = 0.066) or time to progression (20 weeks in gemcitabine group versus 16 weeks in PLD group, p = 0.411). Although the overall survival was higher in the PLD arm (51 weeks versus 56 weeks, p = 0.048), this difference was not detected in the platinum-resistant subgroup (relapse or progression < 6 months). The toxicity profile was similar to the previous study. Health-related quality of life favored the PLD arm.

#### **5.5. Canfosfamide versus pegylated liposomal doxorubicin or topotecan**

A phase III clinical trial (ASSIST-1) was designed to attempt to demonstrate superiority in the overall survival (primary endpoint) and progression-free survival (secondary end‐ point) with canfosfamide versus PLD or topotecan in patients who progressed despite second-line treatment with either topotecan or PLD in platinum-refractory or -resistant patients [49].

in the platinum-free interval. In this population, the median PFS was 7.4 months in the PLD/ trabectedin group versus 5.5 months in the PLD group (HR = 0.65; p = 0.0152) [52]. The median OS was 22.4 months in the PLD/trabectedin group versus 16.4 months in the monotherapy arm

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In the OVA-301 study, similar proportions of patients received subsequent therapy in each arm (77% and 76%), with 56% and 57% receiving platinum-based therapies in the 6 - 12 months subgroup. In this subgroup, the time from randomization to subsequent platinum-based therapy was significantly longer for patients treated with PLD/trabectedin (9.8 versus 7.9 months; p = 0.0167). Patients randomized to the combination group experienced significantly longer survival after the initiation of subsequent platinum-based therapy (13.3 versus 9.8 months; HR = 0.63, p = 0.0357) [52]. These data support the hypothesis that the enhanced survival benefits may be due to an artificial extension of the platinum-free interval. In any case,

When the data on patients who received platinum-based therapy as the first subsequent treatment after PLD/trabectedin or PLD in the 6 - 12 months subset were analyzed, platinum was delayed 4 months (11.5 versus 7.5 months; HR: 0.61, p = 0.0203) and the overall survival from the first platinum treatment was significantly extended by a median of 8.7 months (18.6

The delay in platinum re-treatment could promote the recovery from toxicities, such as

As previously shown, a longer platinum-free interval is the most important factor associated with a higher likelihood of response and prolongation of progression-free survival. Therefore, patients who relapse after six months of completion of chemotherapy and are responders are

The considerations in the choice of a second and subsequent line of chemotherapy in recurrent ovarian cancer may also include assessment of efficacy, cumulative toxicities and the optimal

Currently, in patients with platinum-sensitive disease, it is preferred to administer a combi‐ nation regimen including a platinum compound and a second active drug (Table 3). The platinum compound most commonly used is carboplatin, due to its better toxicity profile. These treatments provide a high response rate and significant improvements in the quality of life and progression-free survival compared to platinum monotherapy. However, the ideal platinum combination is unknown, and several regimens are available. Recently, schemes without platinum, such as PLD/trabectedin, have been developed and can be useful. Never‐ theless, there are no data comparing these regimens to platinum-based schemes, so we must

this hypothesis should be confirmed in prospective randomized trials.

versus 9.9 months; HR = 0.54, p = 0.0169) [53].

candidates for re-treatment with platinum salts.

polyneuropathy or alopecia.

sequencing of available agents.

**7. Discussion**

be prudent.

(HR = 0.64; p = 0.0027) [36].

The study included 461 patients randomized to an active control arm (PLD 50 mg/m2 every 28 days or topotecan 1.5 mg/m2 on days 1 – 5, every 21 days, based on the prior therapy) or canfosfamide 1000 mg/m2 every 21 days.

The median overall survival was 8.5 months with canfosfamide and 13.5 months in the control arm (p < 0.01). The median OS was similar between PLD and topotecan (14.2 versus 10.8 months; p = 0.1695). The progression-free survival was longer for patients treated in the control group than for patients in the canfosfamide group (4.3 versus 2.3 months; p < 0.01). Hemato‐ logic adverse events were more frequent in the control arm, and non-hematologic adverse events were similar in both arms.

#### **6. Extending the platinum-free interval**

The cells of ovarian cancer could have intrinsic or acquired resistance to platinum compounds, which is a large clinical obstacle in the treatment of women with relapsed ovarian cancer. There are several mechanisms by which tumor cells can develop resistance to platinum, including increased efflux, enhanced DNA repair of damage caused by chemotherapy and defective cell death pathways. Some of these mechanisms may be reversible with time. It has been hypothe‐ sized that artificially extending the platinum-free interval with non-cross-resistant chemo‐ therapy may improve the likelihood of responding to platinum salts subsequently administered and prolong the overall survival [35, 50, 51].

Recently, the OVA-301 trial [35] randomized 672 women with recurrent ovarian cancer to receive trabectedin 1.1 mg/m2 plus PLD 30 mg/m2 every 21 days, or PLD 50 mg/m2 every 28 days. The primary endpoint was progression-free survival, and secondary endpoints included overall survival and safety. The PFS was higher in the combination group in the overall population of the study (7.3 versus 5.8 months; HR = 0.79, p = 0.0190) and in the platinumsensitive patients (9.2 versus 7.5 months; HR = 0.73, p = 0.0170). The most common adverse effects were hand-foot syndrome in the PLD group and neutropenia and a transient ALT increase in the PLD/trabectedin group. After a median follow-up of 47.4 months, no difference in overall survival was observed (22.2 months in the combination group versus 18.9 months in the PLD group; HR = 0.86, p = 0.0835). Despite stratification based on platinum sensitivity, the authors detected an imbalance in the mean platinum-free interval, which favored the PLD group (13.3 versus 10.6 months; p = 0.009) [36].

Furthermore, the data reported in patients with a platinum-free interval of 6 - 12 months are especially interesting. These are the patients who can obtain the most benefit from an extension in the platinum-free interval. In this population, the median PFS was 7.4 months in the PLD/ trabectedin group versus 5.5 months in the PLD group (HR = 0.65; p = 0.0152) [52]. The median OS was 22.4 months in the PLD/trabectedin group versus 16.4 months in the monotherapy arm (HR = 0.64; p = 0.0027) [36].

In the OVA-301 study, similar proportions of patients received subsequent therapy in each arm (77% and 76%), with 56% and 57% receiving platinum-based therapies in the 6 - 12 months subgroup. In this subgroup, the time from randomization to subsequent platinum-based therapy was significantly longer for patients treated with PLD/trabectedin (9.8 versus 7.9 months; p = 0.0167). Patients randomized to the combination group experienced significantly longer survival after the initiation of subsequent platinum-based therapy (13.3 versus 9.8 months; HR = 0.63, p = 0.0357) [52]. These data support the hypothesis that the enhanced survival benefits may be due to an artificial extension of the platinum-free interval. In any case, this hypothesis should be confirmed in prospective randomized trials.

When the data on patients who received platinum-based therapy as the first subsequent treatment after PLD/trabectedin or PLD in the 6 - 12 months subset were analyzed, platinum was delayed 4 months (11.5 versus 7.5 months; HR: 0.61, p = 0.0203) and the overall survival from the first platinum treatment was significantly extended by a median of 8.7 months (18.6 versus 9.9 months; HR = 0.54, p = 0.0169) [53].

The delay in platinum re-treatment could promote the recovery from toxicities, such as polyneuropathy or alopecia.

#### **7. Discussion**

**5.5. Canfosfamide versus pegylated liposomal doxorubicin or topotecan**

every 21 days.

patients [49].

days or topotecan 1.5 mg/m2

196 Ovarian Cancer - A Clinical and Translational Update

events were similar in both arms.

receive trabectedin 1.1 mg/m2

**6. Extending the platinum-free interval**

administered and prolong the overall survival [35, 50, 51].

group (13.3 versus 10.6 months; p = 0.009) [36].

canfosfamide 1000 mg/m2

A phase III clinical trial (ASSIST-1) was designed to attempt to demonstrate superiority in the overall survival (primary endpoint) and progression-free survival (secondary end‐ point) with canfosfamide versus PLD or topotecan in patients who progressed despite second-line treatment with either topotecan or PLD in platinum-refractory or -resistant

The median overall survival was 8.5 months with canfosfamide and 13.5 months in the control arm (p < 0.01). The median OS was similar between PLD and topotecan (14.2 versus 10.8 months; p = 0.1695). The progression-free survival was longer for patients treated in the control group than for patients in the canfosfamide group (4.3 versus 2.3 months; p < 0.01). Hemato‐ logic adverse events were more frequent in the control arm, and non-hematologic adverse

The cells of ovarian cancer could have intrinsic or acquired resistance to platinum compounds, which is a large clinical obstacle in the treatment of women with relapsed ovarian cancer. There are several mechanisms by which tumor cells can develop resistance to platinum, including increased efflux, enhanced DNA repair of damage caused by chemotherapy and defective cell death pathways. Some of these mechanisms may be reversible with time. It has been hypothe‐ sized that artificially extending the platinum-free interval with non-cross-resistant chemo‐ therapy may improve the likelihood of responding to platinum salts subsequently

Recently, the OVA-301 trial [35] randomized 672 women with recurrent ovarian cancer to

days. The primary endpoint was progression-free survival, and secondary endpoints included overall survival and safety. The PFS was higher in the combination group in the overall population of the study (7.3 versus 5.8 months; HR = 0.79, p = 0.0190) and in the platinumsensitive patients (9.2 versus 7.5 months; HR = 0.73, p = 0.0170). The most common adverse effects were hand-foot syndrome in the PLD group and neutropenia and a transient ALT increase in the PLD/trabectedin group. After a median follow-up of 47.4 months, no difference in overall survival was observed (22.2 months in the combination group versus 18.9 months in the PLD group; HR = 0.86, p = 0.0835). Despite stratification based on platinum sensitivity, the authors detected an imbalance in the mean platinum-free interval, which favored the PLD

Furthermore, the data reported in patients with a platinum-free interval of 6 - 12 months are especially interesting. These are the patients who can obtain the most benefit from an extension

plus PLD 30 mg/m2

on days 1 – 5, every 21 days, based on the prior therapy) or

every 21 days, or PLD 50 mg/m2

every 28

every 28

The study included 461 patients randomized to an active control arm (PLD 50 mg/m2

As previously shown, a longer platinum-free interval is the most important factor associated with a higher likelihood of response and prolongation of progression-free survival. Therefore, patients who relapse after six months of completion of chemotherapy and are responders are candidates for re-treatment with platinum salts.

The considerations in the choice of a second and subsequent line of chemotherapy in recurrent ovarian cancer may also include assessment of efficacy, cumulative toxicities and the optimal sequencing of available agents.

Currently, in patients with platinum-sensitive disease, it is preferred to administer a combi‐ nation regimen including a platinum compound and a second active drug (Table 3). The platinum compound most commonly used is carboplatin, due to its better toxicity profile. These treatments provide a high response rate and significant improvements in the quality of life and progression-free survival compared to platinum monotherapy. However, the ideal platinum combination is unknown, and several regimens are available. Recently, schemes without platinum, such as PLD/trabectedin, have been developed and can be useful. Never‐ theless, there are no data comparing these regimens to platinum-based schemes, so we must be prudent.


Recurrent platinum-resistant ovarian cancer has limited treatment options and is generally treated sequentially with multiple single-agent regimens consisting of non-platinum and non-

The most common options are PLD, topotecan and gemcitabine. These options have been compared in several phase III clinical trials (Table 4), but none of the options have proven superior. PLD and gemcitabine could be used in patients who do not desire alopecia. Addi‐ tionally, PLD is dosed less frequently than topotecan and gemcitabine, which results in

improved convenience for the patient and a reduction in the use of resources.

**Author, year Drugs Patients RR PFS OS**

n = 226 13.1% vs.

6.7%\*\*

17.8%\*

6.5%\*\*

11.7%\*\*

Gemcitabine vs. PLD n = 153 29% vs. 16%\* 20 vs. 16 w\* 51 vs.

Abbreviations: RR: response rate, PFS: progression-free survival, OS: overall survival, PLD: pegylated liposomal doxoru‐

Recently, the use of non-platinum agents in relapsed ovarian cancer to extend the platinumfree interval has gained interest. The answer to the question of whether the prolongation of the platinum-free interval increases overall survival after the reintroduction of platinum should be revealed by two phase III trials currently in progress. The MITO-8 (ClinicalTrials.gov Identifier: NCT00657878) trial compares carboplatin/paclitaxel followed by PLD versus the reverse sequence (PLD followed by carboplatin/paclitaxel), and the INOVATYON trial (ClinicalTrial.gov identifier: NCT01379989) compares the administration of carboplatin/PLD followed by treatment at the discretion of the investigator versus PLD/trabectedin followed

**Table 4.** Main phase III clinical trials including platinum-resistant relapsed ovarian cancer

23.1 vs. 14 w\* 28.4 vs.

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199

22.4 vs. 21.7 w\* 56.1 vs.

9.1 vs. 13.6 w\*\* 35.6 vs.

3.6 vs. 3.1 m\*\* 12.7 vs.

39.7 w\*\*

45.7 w\*

41.3 w\*\*

13.5 m\*\*

56 w\*

Subsequent lines of treatment will be made with available drugs.

taxane chemotherapy.

ten Bokkel Huinink W, 2004

Ferrandina G, 2008

\*Data from the whole group

bicin, w: weeks, m: months.

by a platinum-based.

\*\*Data from platinum-resistant patients

Topotecan vs. Paclitaxel

O'Byrne KJ, 2002 Paclitaxel vs. PLD n = 214 22.4% vs.

Gordon AN, 2004 PLD vs. Topotecan n = 474 12.3% vs.

Mutch DG, 2007 Gemcitabine vs. PLD n = 195 9.2% vs.

Abbreviations: RR: response rate. PFS: progression-free survival. OS: overall survival. C: carboplatin. P: paclitaxel. Gem: gemcitabine. PLD: pegylated liposomal doxorubicin, m: months.

**Table 3.** Main phase III clinical trials in platinum-sensitive relapsed ovarian cancer

The ICON4/AGO-OVAR 2.2 trial was the first clinical trial that showed the superiority of polychemotherapy to monotherapy in patients with platinum-sensitive relapsed ovarian cancer. The combination of carboplatin and paclitaxel may be used in patients who have no residual neurotoxicity, especially if the platinum-free interval is greater than one year.

A valid alternative is the administration of carboplatin plus PLD (CALYPSO), which has demonstrated similar efficacy to carboplatin/paclitaxel and a more favorable toxicity profile, with less alopecia, neurotoxicity and allergic/hypersensitivity reactions. Perhaps this is the most commonly used scheme by oncologists worldwide, now conditioned by a globally limited availability of PLD.

Although no survival benefit was achieved in the AGO-OVAR 2.5 trial with carboplatin and gemcitabine, the results of this clinical trial allow us to recommend this chemotherapy scheme as an alternative to carboplatin/paclitaxel, due to its different, and perhaps more favorable, toxicity profile. This scheme is especially useful for patients with risk factors for neurotoxicity development. The addition of an anti-angiogenic drug, such as bevacizumab (OCEANS), can improve outcomes without a significant increase in toxicity.

The incorporation of new active drugs into the treatment of patients with platinum-sensitive ovarian cancer is also important. Thus, we must be aware of the results of the phase III clinical trial, HECTOR (ClinicalTrials.gov Identifier: NCT00437307), which compares the combination of carboplatin plus topotecan with the current standard of care (carboplatin/paclitaxel, carboplatin/gemcitabine or carboplatin/PLD). The trial may be completed in 2013.

Because the response rate to platinum salts is too low in patients with platinum-resistant disease, monotherapy with a non-platinum drug is usually the choice in this setting. Compar‐ isons of the efficacy of different active drugs in phase III clinical trials show no superiority of any of them, and there is no clear recommendation for the standard treatment in these patients. Therefore, the selection of treatment for platinum-resistant patients will be based on other criteria, such as toxicity, patient preferences and physician experience. Whenever possible, patients with platinum-resistant disease should be considered for treatment in clinical trials.

Recurrent platinum-resistant ovarian cancer has limited treatment options and is generally treated sequentially with multiple single-agent regimens consisting of non-platinum and nontaxane chemotherapy.

The most common options are PLD, topotecan and gemcitabine. These options have been compared in several phase III clinical trials (Table 4), but none of the options have proven superior. PLD and gemcitabine could be used in patients who do not desire alopecia. Addi‐ tionally, PLD is dosed less frequently than topotecan and gemcitabine, which results in improved convenience for the patient and a reduction in the use of resources.


Subsequent lines of treatment will be made with available drugs.

\*Data from the whole group

**Clinical Trial Scheme Patients RR PFS OS**

AGO-OVAR 2.5 [24] C vs. C/Gem n = 356 30.9 vs. 47.2% 5.8 vs. 8.6 m 18 vs. 17.3 m CALYPSO [25] C/P vs. C/PLD n = 976 Not achieved 9.4 vs. 11.3 m 33 vs. 30.7 m

Abbreviations: RR: response rate. PFS: progression-free survival. OS: overall survival. C: carboplatin. P: paclitaxel. Gem:

The ICON4/AGO-OVAR 2.2 trial was the first clinical trial that showed the superiority of polychemotherapy to monotherapy in patients with platinum-sensitive relapsed ovarian cancer. The combination of carboplatin and paclitaxel may be used in patients who have no residual neurotoxicity, especially if the platinum-free interval is greater than one year.

A valid alternative is the administration of carboplatin plus PLD (CALYPSO), which has demonstrated similar efficacy to carboplatin/paclitaxel and a more favorable toxicity profile, with less alopecia, neurotoxicity and allergic/hypersensitivity reactions. Perhaps this is the most commonly used scheme by oncologists worldwide, now conditioned by a globally

Although no survival benefit was achieved in the AGO-OVAR 2.5 trial with carboplatin and gemcitabine, the results of this clinical trial allow us to recommend this chemotherapy scheme as an alternative to carboplatin/paclitaxel, due to its different, and perhaps more favorable, toxicity profile. This scheme is especially useful for patients with risk factors for neurotoxicity development. The addition of an anti-angiogenic drug, such as bevacizumab (OCEANS), can

The incorporation of new active drugs into the treatment of patients with platinum-sensitive ovarian cancer is also important. Thus, we must be aware of the results of the phase III clinical trial, HECTOR (ClinicalTrials.gov Identifier: NCT00437307), which compares the combination of carboplatin plus topotecan with the current standard of care (carboplatin/paclitaxel,

Because the response rate to platinum salts is too low in patients with platinum-resistant disease, monotherapy with a non-platinum drug is usually the choice in this setting. Compar‐ isons of the efficacy of different active drugs in phase III clinical trials show no superiority of any of them, and there is no clear recommendation for the standard treatment in these patients. Therefore, the selection of treatment for platinum-resistant patients will be based on other criteria, such as toxicity, patient preferences and physician experience. Whenever possible, patients with platinum-resistant disease should be considered for treatment in clinical trials.

carboplatin/gemcitabine or carboplatin/PLD). The trial may be completed in 2013.

n = 484

C vs. C/P n = 802 54% vs. 66% 9 vs. 12 m 24 vs. 29 m

8.4 vs. 12.4 m 35.2 vs. 33.3 m

57.4% vs. 78.5%

ICON4/AGO-OVAR 2.2 [20]

OCEANS [28]

limited availability of PLD.

C/Gem/Pl vs. C/Gem/Bev

198 Ovarian Cancer - A Clinical and Translational Update

gemcitabine. PLD: pegylated liposomal doxorubicin, m: months.

**Table 3.** Main phase III clinical trials in platinum-sensitive relapsed ovarian cancer

improve outcomes without a significant increase in toxicity.

\*\*Data from platinum-resistant patients

Abbreviations: RR: response rate, PFS: progression-free survival, OS: overall survival, PLD: pegylated liposomal doxoru‐ bicin, w: weeks, m: months.

**Table 4.** Main phase III clinical trials including platinum-resistant relapsed ovarian cancer

Recently, the use of non-platinum agents in relapsed ovarian cancer to extend the platinumfree interval has gained interest. The answer to the question of whether the prolongation of the platinum-free interval increases overall survival after the reintroduction of platinum should be revealed by two phase III trials currently in progress. The MITO-8 (ClinicalTrials.gov Identifier: NCT00657878) trial compares carboplatin/paclitaxel followed by PLD versus the reverse sequence (PLD followed by carboplatin/paclitaxel), and the INOVATYON trial (ClinicalTrial.gov identifier: NCT01379989) compares the administration of carboplatin/PLD followed by treatment at the discretion of the investigator versus PLD/trabectedin followed by a platinum-based.

#### **Author details**

Miguel Angel Alonso Bermejo1 , Ana Fernandez Montes1\*, Eva Perez Lopez1 , Miguel Angel Nuñez Viejo2 , Jesus Garcia Gomez1 and Jesus Garcia Mata1

\*Address all correspondence to: afm1003@hotmail.com

1 Medical Oncology Service at the University Hospital Ourense, Ourense, Spain

2 Palliative Care Department at the University Hospital Ourense, Ourense, Spain

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**Author details**

**References**

Miguel Angel Alonso Bermejo1

200 Ovarian Cancer - A Clinical and Translational Update

Miguel Angel Nuñez Viejo2

, Ana Fernandez Montes1\*, Eva Perez Lopez1

and Jesus Garcia Mata1

, Jesus Garcia Gomez1

1 Medical Oncology Service at the University Hospital Ourense, Ourense, Spain

2 Palliative Care Department at the University Hospital Ourense, Ourense, Spain

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[38] Williams, C, Simera, I, & Bryant, A. Tamoxifen for relapse of ovarian cancer. Co‐

[39] Smyth, J. F, Gourley, C, Walker, G, et al. Antiestrogen therapy is active in selected ovarian cancer cases: the use of letrozole in estrogen receptor-positive patients. Clin

[40] Kristensen, G, Kaern, J, Baekelandt, M, et al. Chemotherapy versus hormonal treat‐ ment in patients with platinum and taxane resistant ovarian cancer: a NSGO study. J

[41] ten Bokkel Huinink WGore M, Carmichael J, et al. Topotecan versus Paclitaxel for he treatment of recurrent epithelial ovarian cancer. J Clin Oncol (1997). , 15(6), 2183-93.

[42] ten Bokkel Huinink WLane SR, Ross GA, et al. Long-term survival in a phase III, randomised study of topotecan versus paclitaxel in advanced epithelial ovarian car‐

[43] Gore, M. ten Bokkel Huinnink W, Carmichael J, et al. Clinical evidence for topotecanpaclitaxel non--cross-resistance in ovarian cancer. J Clin Oncol (2001). , 19(7),

[44] Byrne, O, Bliss, K. J, & Graham, P. JD, et al. A phase III study of Doxil/Caelyx versus paclitaxel in platinum-treated, taxane-naive relapsed ovarian cancer. Proc Am Soc

[45] Gordon, A. N, Fleagle, J. T, Guthrie, D, et al. Recurrent epithelial ovarian carcinoma: a randomized phase III study of pegylated liposomal doxorrubicin versus topotecan.

[46] Gordon, A. N, Tonda, M, Sun, S, et al. Long-term survival advantage for women treated with pegylated liposomal doxorrubicin compared with topotecan in a phase 3

liposomal doxorubicin. Gynecol Oncol (2010). , 26-31.

(2010). , 20(5), 772-80.

Eur J Cancer (2012). , 2361-68.

chrane Database Syst Rev (2010). CD001034.

Cancer Res (2007). , 13(12), 3617-22.

Clin Oncol (2008). suppl)[abstract 5508].

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Clin Oncol 21:(2002). abstr 808).

J Clin Oncol (2001). , 19(14), 3312-22.

1893-900.


[33] Mirza, M. R, Lund, B, Lindegaard, J. C, et al. A phase II study of combination chemo‐ therapy in early relapsed epithelial ovarian cancer using gemcitabine and pegylated liposomal doxorubicin. Gynecol Oncol (2010). , 26-31.

with platinum-sensitive ovarian cancer in late relapse. J Clin Oncol (2010). , 28(20),

[23] Wagner, U, Marth, C, Largillier, R, et al. Final overall survival results of phase III GCIG CALYPSO trial of pegylated liposomal doxorubicin and carboplatin vs pacli‐ taxel and carboplatin in platinum-sensitive ovarian cancer patients. Br J Cancer

[24] Brundage, M, Gropp, M, Mefti, F, et al. Health-related quality of life in recurrent platinum-sensitive ovarian cancer-- results from the CALYPSO trial. Ann Oncol

[25] Aghajanian, C, Blank, S. V, Goff, B. A, et al. OCEANS: A randomized, double-blind, placebo-controlled phase III trial of chemotherapy with or without bevacizumab in patients with platinum-sensitive recurrent epithelial ovarian, primary peritoneal, or

[26] Fong, P. C, Boss, D. S, Yap, T. A, et al. Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. N Engl J Med (2009). , 361(2), 123-34.

[27] Kaye, S. B, Lubinski, J, Matulonis, U, et al. Phase II, Open-Label, Randomized, Multi‐ center Study Comparing the Efficacy and Safety of Olaparib, a Poly (ADP-Ribose) Polymerase Inhibitor, and Pegylated Liposomal Doxorubicin in Patients With BRCA1 or BRCA2 Mutations and Recurrent Ovarian Cancer. J Clin Oncol (2012). ,

[28] Ledermann, J, Harter, P, Gourley, C, et al. Olaparib maintenance therapy in plati‐ num-sensitive relapsed ovarian cancer. N Engl J Med (2012). , 366(15), 1382-92. [29] Tan, D. S, Rothermundt, C, Thomas, K, et al. BRCAness syndrome in ovarian cancer: a case-control study describing the clinical features and outcome of patients with epi‐ thelial ovarian cancer associated with BRCA1 and BRCA2 mutations. J Clin Oncol

[30] Garcia, A. A, Meara, O, & Bahador, A. A, et al. Phase II study of gemcitabine and weekly paclitaxel in recurrent platinum-resistant ovarian cancer. Gynecol Oncol

[31] Sehouli, J, Stengel, D, Oskay-oezcelik, G, et al. Nonplatinum topotecan combinations versus topotecan alone for recurrent ovarian cancer: results of a phase III study of the North-Eastern German Society of Gynecological Oncology Ovarian Cancer Study

[32] Joly, F, Petit, T, Pautier, P, et al. Weekly combination of topotecan and gemcitabine in early recurrent ovarian cancer patients: a French multicenter phase II study. Gynecol

fallopian tube cancer. J Clin Oncol (2012). , 30(17), 2039-45.

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(2012). , 23(8), 2020-7.

30(4), 372-9.

(2008). , 26(34), 5530-36.

(2004). , 93(2), 493-8.

Oncol (2009). , 115(3), 382-8.

Group. J Clin Oncol (2008). , 26(19), 3176-82.


randomized study of recurrent and refractory epithelial ovarian cancer. Gynecol On‐ col (2004). , 95(1), 1-8.

**Chapter 10**

**Molecular Mechanisms of**

Gonzalo Tapia and Ivan Diaz-Padilla

http://dx.doi.org/10.5772/55562

**1. Introduction**

Additional information is available at the end of the chapter

**Platinum Resistance in Ovarian Cancer**

In 2012, approximately 22,280 women will be diagnosed with ovarian carcinoma in the United States and roughly 15,500 will die from this disease, ranked the most common cause of death among gynecologic malignancies in developed countries [1]. Most women with epithelial ovarian cancer (EOC) present with advanced disease (stage III or IV) at the time of diagnosis. This phenomenon is mainly due to the lack of specific symptoms until disease has spread beyond the ovaries, at which time the chance of cure is dramatically reduced [2]. Current standard treatment of ovarian cancer, in both early and advanced stages, consists of complete cytoreductive surgery followed by chemotherapy, usually based on a platinum and a taxane doublet [3,4,5]. Initial response rate (RR) is high (70%-80%); but the majority of patients with advanced disease relapse within two years. Recurrent ovarian cancer is not curable, due to the development of chemoresistance [6,7]. The Gynecologic Oncology Group (GOG) adopted the definition of sensitivity to chemotherapy (or sensitivity to platinum) in EOC based on clinical criteria from retrospective case series [8]. When patients were re-challenged with a platinum compound the longer the interval from the last dose of platinum patients had received the better the response (and outcome) was. This clinical observation set the base for the current classification of platinum resistance in relapsed EOC (Figure 1), and allowed the commonly used stratification criteria in clinical trials of recurrent EOC. Platinum-resistant disease is also characterized by resistance to other cytotoxic agents, and not necessarily only resistant to platinum. However, current treatment for platinum-resistant EOC consists of chemotherapy agents whose mechanism of action is somewhat different from that of platinum compounds [9].

Since platinum compounds are the backbone in the systemic treatment of EOC, there is great interest in elucidate the molecular mechanisms contributing to platinum resistance in this disease. The present chapter will provide a comprehensive basic and translational update with

and reproduction in any medium, provided the original work is properly cited.

© 2013 Tapia and Diaz-Padilla; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,


#### **Chapter 10**

### **Molecular Mechanisms of Platinum Resistance in Ovarian Cancer**

Gonzalo Tapia and Ivan Diaz-Padilla

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55562

#### **1. Introduction**

randomized study of recurrent and refractory epithelial ovarian cancer. Gynecol On‐

[47] Mutch, D. G, Orlando, M, Goss, T, et al. Randomized phase III trial of gemcitabine compared with pegylated liposomal doxorrubicin in patients with platinum-resistant

[48] Ferrandina, G, Ludovisi, M, Lorusso, D, et al. Phase III Trial of Gemcitabine com‐ pared with Pegylated Liposomal Doxorubicin in progressive or recurrent ovarian

[49] Vergote, I, & Finkler, N. del Campo J, et al. Phase 3 randomised study of canfosfa‐ mide (Telcyta®, TLK286) versus pegylated liposomal doxorubicin or topotecan as third-line therapy in patients with platinum-refractory or-resistant ovarian cancer.

[50] Colombo, N, & Gore, M. Treatment of recurrent ovarian cancer relapsing 6-12 months post platinum-based chemotherapy. Crit Rev Oncol Hematol (2007). , 64(2),

[51] Bookman, M. A. Extending the platinum-free interval in recurrent ovarian cancer: the

[52] Poveda, A, Vergote, I, Tjulandin, S, et al. Trabectedin plus pegylated liposomal dox‐ orubicin in relapsed ovarian cancer: outcomes in the partially platinum-sensitive (platinum-free interval 6-12 months) subpopulation of OVA-301 phase III random‐

[53] Kaye, S. B, Colombo, N, Monk, B. J, et al. Trabectedin plus pegylated liposomal dox‐ orubicin in relapsed ovarian cancer delays third-line chemotherapy and prolongs the

role of Topotecan in second-line chemotherapy. Oncologist (1999).

ovarian cancer. J Clin Oncol (2007). , 25(19), 2811-8.

cancer. J Clin Oncol (2008). , 26(6), 890-6.

Eur J Cancer (2009). , 45(13), 2324-32.

ized trial. Ann Oncol (2011). , 22(1), 39-48.

platinum-free interval. Ann Oncol (2011). , 22(1), 49-58.

col (2004). , 95(1), 1-8.

204 Ovarian Cancer - A Clinical and Translational Update

129-38.

In 2012, approximately 22,280 women will be diagnosed with ovarian carcinoma in the United States and roughly 15,500 will die from this disease, ranked the most common cause of death among gynecologic malignancies in developed countries [1]. Most women with epithelial ovarian cancer (EOC) present with advanced disease (stage III or IV) at the time of diagnosis. This phenomenon is mainly due to the lack of specific symptoms until disease has spread beyond the ovaries, at which time the chance of cure is dramatically reduced [2]. Current standard treatment of ovarian cancer, in both early and advanced stages, consists of complete cytoreductive surgery followed by chemotherapy, usually based on a platinum and a taxane doublet [3,4,5]. Initial response rate (RR) is high (70%-80%); but the majority of patients with advanced disease relapse within two years. Recurrent ovarian cancer is not curable, due to the development of chemoresistance [6,7]. The Gynecologic Oncology Group (GOG) adopted the definition of sensitivity to chemotherapy (or sensitivity to platinum) in EOC based on clinical criteria from retrospective case series [8]. When patients were re-challenged with a platinum compound the longer the interval from the last dose of platinum patients had received the better the response (and outcome) was. This clinical observation set the base for the current classification of platinum resistance in relapsed EOC (Figure 1), and allowed the commonly used stratification criteria in clinical trials of recurrent EOC. Platinum-resistant disease is also characterized by resistance to other cytotoxic agents, and not necessarily only resistant to platinum. However, current treatment for platinum-resistant EOC consists of chemotherapy agents whose mechanism of action is somewhat different from that of platinum compounds [9].

Since platinum compounds are the backbone in the systemic treatment of EOC, there is great interest in elucidate the molecular mechanisms contributing to platinum resistance in this disease. The present chapter will provide a comprehensive basic and translational update with

© 2013 Tapia and Diaz-Padilla; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Other proposed mechanisms of cisplatin cytotoxicity include mitochondrial damage, de‐ creased ATPase activity, and altered cellular transport mechanisms. Mitochondria seem to play a role in the cell death. This is believed to be mediated by their interaction with nuclear DNA [13]. Additionally, mitochondria are thought to be a major target of cisplatin and mitochondrial DNA is heavily damaged by cisplatin leading to mitochondrial loss of energy production and decreasing the ATPase activity [14,15]. Another proposed mechanism of action is the transporter-mediated uptake. Entering the cells is the first step for cisplatin to exert its toxic effects. In recent years there has been increasing evidence that the cellular uptake of cisplatin is mediated, at least in part, by transport proteins. Several transporters, which are expressed on the cell membranes, have been associated with cisplatin transport across the plasma membrane and across the cell: the copper transporter 1 (Ctr1), the copper transporter

Molecular Mechanisms of Platinum Resistance in Ovarian Cancer

http://dx.doi.org/10.5772/55562

207

Despite the clear advantage in OS and PFS obtained with cisplatin–paclitaxel, it was immedi‐ ately noted that this regimen carries a significant toxicity, namely peripheral neurotoxicity and nephrotoxicity [18]. Another important limitation of cisplatin–paclitaxel chemotherapy is the difficulty in administering it as an outpatient regimen. Prior to the introduction of paclitaxel, several studies had established that cisplatin and carboplatin are therapeutically equivalent in women with advanced epithelial ovarian cancer. Furthermore, carboplatin is associated with significantly lower neurotoxicity and renal toxicity and that the combination of carboplatin and 3-h infusion paclitaxel can be given as an outpatient schedule. This was also demonstrated

Three trials have investigated the equivalence of carboplatin and cisplatin in combination with paclitaxel in the first-line setting [20,21,22]. Given the evidence of a more favorable toxicity profile and ease of delivery, the carboplatin–paclitaxel combination has become an almost universal choice in the management of ovarian cancer, and is the standard comparator in all

Even though initial responsiveness to platinum-based therapy is high in ovarian cancer, the majority of patients relapse. Several mechanisms of cellular resistance to platinum compounds have been described. These mechanisms can be classified in two groups: 1) those that the limit the formation of cytotoxic platinum-DNA adducts, and 2) those that prevent cell death occurring after platinum-DNA adduct formation [11,23]. A better understanding of the molecular basis of cisplatin resistance may lead to new antitumor strategies that will sensitize

2 (Ctr2), the P-type copper-transporting ATPases ATP7A and ATP7B [16,17].

**3. Platinum analogues and ovarian cancer therapeutics**

**4. Mechanisms of cellular resistance to platinum agents**

unresponsive ovarian cancers to cisplatin-based chemotherapy.

in a Cochrane meta-analysis [19].

the recent trials performed in this disease.

**Figure 1.** Platinum-resistance definition by the Gynecologic Oncology Group (GOG). Platinum sensitivity is classified as resistant, partially sensitive, or sensitive, according to the time relapsed since finishing first-line treatment. Probability of re-treatment response is shown for each group of patients.

regards to biological pathways implicated in the development of platinum resistance, focusing on ovarian cancer therapeutics.

#### **2. Cisplatin: Mechanism of action**

Once introduced actively into the cell mediated by a copper transporter (CTR1), the molecule is activated through a series of aquation reactions, in which one of the chloride ligands is slowly displaced by water. Aquated cisplatin avidly binds DNA, with a predilection for nucleophilic N7-sites on purine bases [10]. The first step of the reaction involves the formation of monoad‐ ducts. These monoadducts may then react further to form intra-strand and inter-strand crosslinks. The cytotoxic activity of platinum compounds has been related to binding with DNA and the production of intra-strand and inter-strand crosslinks, as well as the formation of adducts that cause conformational DNA changes, impeding the separation of both DNA strands, which subsequently impairs replication and inhibits DNA synthesis [11]. Intra-strand cross-links are the most abundant products of the interaction with DNA (around 70% of all platinum-DNA linking products). These lesions cause significant distortions in the DNA that can be recognized by one or more DNA binding proteins. These proteins can either initiate DNA damage repair or signal for apoptosis. Platinum-mediated programmed cell death is caused by cell cycle arrest in the G2-phase, although the pathways from platinum–DNA binding to apoptosis are not completely understood [12].

Other proposed mechanisms of cisplatin cytotoxicity include mitochondrial damage, de‐ creased ATPase activity, and altered cellular transport mechanisms. Mitochondria seem to play a role in the cell death. This is believed to be mediated by their interaction with nuclear DNA [13]. Additionally, mitochondria are thought to be a major target of cisplatin and mitochondrial DNA is heavily damaged by cisplatin leading to mitochondrial loss of energy production and decreasing the ATPase activity [14,15]. Another proposed mechanism of action is the transporter-mediated uptake. Entering the cells is the first step for cisplatin to exert its toxic effects. In recent years there has been increasing evidence that the cellular uptake of cisplatin is mediated, at least in part, by transport proteins. Several transporters, which are expressed on the cell membranes, have been associated with cisplatin transport across the plasma membrane and across the cell: the copper transporter 1 (Ctr1), the copper transporter 2 (Ctr2), the P-type copper-transporting ATPases ATP7A and ATP7B [16,17].

#### **3. Platinum analogues and ovarian cancer therapeutics**

regards to biological pathways implicated in the development of platinum resistance, focusing

**Figure 1.** Platinum-resistance definition by the Gynecologic Oncology Group (GOG). Platinum sensitivity is classified as resistant, partially sensitive, or sensitive, according to the time relapsed since finishing first-line treatment. Probability

Once introduced actively into the cell mediated by a copper transporter (CTR1), the molecule is activated through a series of aquation reactions, in which one of the chloride ligands is slowly displaced by water. Aquated cisplatin avidly binds DNA, with a predilection for nucleophilic N7-sites on purine bases [10]. The first step of the reaction involves the formation of monoad‐ ducts. These monoadducts may then react further to form intra-strand and inter-strand crosslinks. The cytotoxic activity of platinum compounds has been related to binding with DNA and the production of intra-strand and inter-strand crosslinks, as well as the formation of adducts that cause conformational DNA changes, impeding the separation of both DNA strands, which subsequently impairs replication and inhibits DNA synthesis [11]. Intra-strand cross-links are the most abundant products of the interaction with DNA (around 70% of all platinum-DNA linking products). These lesions cause significant distortions in the DNA that can be recognized by one or more DNA binding proteins. These proteins can either initiate DNA damage repair or signal for apoptosis. Platinum-mediated programmed cell death is caused by cell cycle arrest in the G2-phase, although the pathways from platinum–DNA

on ovarian cancer therapeutics.

206 Ovarian Cancer - A Clinical and Translational Update

**2. Cisplatin: Mechanism of action**

of re-treatment response is shown for each group of patients.

binding to apoptosis are not completely understood [12].

Despite the clear advantage in OS and PFS obtained with cisplatin–paclitaxel, it was immedi‐ ately noted that this regimen carries a significant toxicity, namely peripheral neurotoxicity and nephrotoxicity [18]. Another important limitation of cisplatin–paclitaxel chemotherapy is the difficulty in administering it as an outpatient regimen. Prior to the introduction of paclitaxel, several studies had established that cisplatin and carboplatin are therapeutically equivalent in women with advanced epithelial ovarian cancer. Furthermore, carboplatin is associated with significantly lower neurotoxicity and renal toxicity and that the combination of carboplatin and 3-h infusion paclitaxel can be given as an outpatient schedule. This was also demonstrated in a Cochrane meta-analysis [19].

Three trials have investigated the equivalence of carboplatin and cisplatin in combination with paclitaxel in the first-line setting [20,21,22]. Given the evidence of a more favorable toxicity profile and ease of delivery, the carboplatin–paclitaxel combination has become an almost universal choice in the management of ovarian cancer, and is the standard comparator in all the recent trials performed in this disease.

#### **4. Mechanisms of cellular resistance to platinum agents**

Even though initial responsiveness to platinum-based therapy is high in ovarian cancer, the majority of patients relapse. Several mechanisms of cellular resistance to platinum compounds have been described. These mechanisms can be classified in two groups: 1) those that the limit the formation of cytotoxic platinum-DNA adducts, and 2) those that prevent cell death occurring after platinum-DNA adduct formation [11,23]. A better understanding of the molecular basis of cisplatin resistance may lead to new antitumor strategies that will sensitize unresponsive ovarian cancers to cisplatin-based chemotherapy.

#### **4.1. Reduced intracellular drug accumulation**

Decreased cellular uptake of cisplatin by resistant cells is one of the major mechanisms of resistance described in vitro. The mechanism responsible for reduced cisplatin accumulation in resistant cells may be ascribed to either an inhibition in drug uptake, an increase in drug efflux, or both. Cisplatin and its analogues may accumulate within cells by passive diffusion or facilitated transport. The copper transporter-1 (CTR1) regulates the influx of cisplatin and its analogues into the cell. This is supported by the evidence in cell lines of deletion of the yeast *CTR1* gene, which encodes a high-affinity copper transporter, results in increased cisplatin resistance and reduced intracellular accumulation of cisplatin in various cell lines including ovarian cancer [24,25]. In human ovarian cancer cell lines it has been demonstrated that copper and cisplatin are competitive inhibitors for the transport of each other into the cell and cause a rapid down-regulation of *CTR1* expression mediated by internalization of this transporter from the plasma membrane and subsequent [26]. Two copper exporters, ATP7A and ATP7B, have also been proposed to be involved in cellular resistance to cisplatin [27]. ATP7A is thought to sequester platinum agents in intracellular compartments, preventing their reaction with nuclear DNA. ATP7A is over-expressed in some cisplatin-resistant ovarian carcinoma cell lines. Additionally, ovarian cancer patients with ATP7A expression have a lower survival rate than patients with undetectable levels of expression, as determined by ATP7A immunostain‐ ing [28]. Over-expression of ATP7B in primary ovarian carcinomas and ovarian carcinoma cell lines resulted in resistance to cisplatin, with only 60% of the cisplatin accumulation present in ATP7B-expressing cells compared to vector control [29].

concomitantly administered chemotherapy, thereby elevating plasma concentrations beyond acceptable toxicity [34]. It is thus clear that the Pgp overexpression can be a cause of failure of anticancer chemotherapy and be associated with worse prognosis in patients with ovarian

Molecular Mechanisms of Platinum Resistance in Ovarian Cancer

http://dx.doi.org/10.5772/55562

209

Glutathione (gamma-glutamylcysteinylglycine: GSH), the most abundant intracellular thiol, contributes (along with methionine, metallothionein and other cysteine-rich proteins) to detoxify many cellular toxins, including cisplatin and its analogues. Part of the cytoplasmic cisplatin reacts with DNA, which ultimately lead to the activation of the apoptosis cascade in response to DNA damage. However, a major fraction of intracellular cisplatin can be converted into cisplatin-thiol conjugates by GSH-S-transferase π, and these conjugates are ultimately inactivated. Both GSTπ and γ-glutamylcysteine synthetase (γ- GCS), the latter being the enzyme involved in GSH synthesis, have been associated with cisplatin resistance in ovarian,

Thus, reducing intracellular glutathione levels would seem a rational strategy to overcome platinum resistance. To that end, a novel glutathione analog prodrug, canfosfamide, initiated clinical development in ovarian cancer. Canfosfamide (TLK286) works by targeting tumors that over-express glutathione S-transferase (GST) P1-1, increasing the sensitivity of those tumors to the cytotoxic effects of canfosfamide. Following activation, the apoptotic activity of canfosfamide is mediated through the stress response pathway, resulting in the induction of cellular apoptosis. Human cancer cells exposed to canfosfamide demonstrate activation of mitogen- activated protein (MAP) kinase MKK4, p38 kinase, jun-N-terminal kinase (JNK) and caspase 3. The cytotoxic activity of canfosfamide has been demonstrated *in vitro* and *in vivo* against a variety of human cancer cell lines, including ovarian cancer cells (OVCAR3).

A phase II trial involving 34 patients with platinum-refractory or platinum-resistant ovarian cancer reported that 15% of patients had an objective response and 50% of patients had disease stabilization [40]. Three phase III trials in platinum-resistant ovarian cancer were undertaken in an attempt to define the potential role of canfosfamide in ovarian cancer therapeutics: TLK286 versus liposomal doxorubicin or topotecan (ASSIST-1,41); TLK286 plus carboplatin versus liposomal doxorubicin (ASSIST-3,42) and TLK286 plus liposomal doxorubicin versus liposomal doxorubicin alone (ASSIST-5,43). Unfortunately, none of these studies showed

The cytotoxicity of cisplatin is attributed to the formation of cisplatin-DNA adducts, and to the induction of DNA damage. The balance between DNA damage to DNA repair dictates tumor cell death or survival after cisplatin therapy. Depending on the type of damage inflicted on the DNA structure, different DNA repair mechanisms have the ability to restore these lesions and remove the platinum-DNA adducts from the tumor DNA [44]. The major pathway in the repair of DNA damage is the nucleotide excision repair (NER) system. NER is one of

superior efficacy of canfosfamide compared to standard treatment.

**4.3. Increased DNA repair**

and breast cancers, sarcomas and other malignancies [29,35,36].

**4.2. Intracellular cisplatin inactivation**

cervical and lung cancer cell lines [37,38,39].

MRP-related transport proteins are involved in the active efflux of platinum drugs. MRP is a member of the ABC (adenosine triphosphate-binding cassette) family of transport pro‐ teins that participates in the efflux of anticancer drugs from cells. Thus, it has been speculat‐ ed that deregulation of some of the MRP components may influence platinum resistance [30]. The MRP gene family is composed of at least seven members (MRP1–7) but recent reports reinforced the notion that MRP2 expression levels can be important in predicting the sensitivity of tumors to platinum-based therapies [31,32]. MDR1 encodes an integral membrane protein named P-glycoprotein (Pgp) or an ATP-binding cassette subfamily B, member 1, which acts as a drug efflux pump [33]. This protein is a transmembrane transport‐ er that resides in the plasma membrane of many cells, including cancer cells that are multidrug resistant. Pgp recognizes a wide range of anticancer drugs and was shown to reduce intracellular concentrations of a variety of cytotoxic drugs, including platinum agents. Pgp activity results in blunted chemotherapy-induced cytotoxicity in vitro and in vivo. Moreover, anticancer drugs were found to induce MDR1 gene. Since Pgp alone can medi‐ ate resistance to a whole array of drugs through drug efflux, it is an attractive target for the improvement of anticancer therapy. In theory, co-administration of transporter inhibitors with Pgp-substrate anticancer drugs could reverse MDR and improve treatment outcome. Clinical trials aimed at specifically inhibiting the function of Pgp have given mixed results, but in at least some cases this inhibition has resulted in improved tumor shrinkage and increased patient survival. Unfortunately, Pgp inhibitors such as PSC-833 (Valspodar) induced pharmacokinetic interactions that limited drug clearance and metabolism of the concomitantly administered chemotherapy, thereby elevating plasma concentrations beyond acceptable toxicity [34]. It is thus clear that the Pgp overexpression can be a cause of failure of anticancer chemotherapy and be associated with worse prognosis in patients with ovarian and breast cancers, sarcomas and other malignancies [29,35,36].

#### **4.2. Intracellular cisplatin inactivation**

**4.1. Reduced intracellular drug accumulation**

208 Ovarian Cancer - A Clinical and Translational Update

ATP7B-expressing cells compared to vector control [29].

Decreased cellular uptake of cisplatin by resistant cells is one of the major mechanisms of resistance described in vitro. The mechanism responsible for reduced cisplatin accumulation in resistant cells may be ascribed to either an inhibition in drug uptake, an increase in drug efflux, or both. Cisplatin and its analogues may accumulate within cells by passive diffusion or facilitated transport. The copper transporter-1 (CTR1) regulates the influx of cisplatin and its analogues into the cell. This is supported by the evidence in cell lines of deletion of the yeast *CTR1* gene, which encodes a high-affinity copper transporter, results in increased cisplatin resistance and reduced intracellular accumulation of cisplatin in various cell lines including ovarian cancer [24,25]. In human ovarian cancer cell lines it has been demonstrated that copper and cisplatin are competitive inhibitors for the transport of each other into the cell and cause a rapid down-regulation of *CTR1* expression mediated by internalization of this transporter from the plasma membrane and subsequent [26]. Two copper exporters, ATP7A and ATP7B, have also been proposed to be involved in cellular resistance to cisplatin [27]. ATP7A is thought to sequester platinum agents in intracellular compartments, preventing their reaction with nuclear DNA. ATP7A is over-expressed in some cisplatin-resistant ovarian carcinoma cell lines. Additionally, ovarian cancer patients with ATP7A expression have a lower survival rate than patients with undetectable levels of expression, as determined by ATP7A immunostain‐ ing [28]. Over-expression of ATP7B in primary ovarian carcinomas and ovarian carcinoma cell lines resulted in resistance to cisplatin, with only 60% of the cisplatin accumulation present in

MRP-related transport proteins are involved in the active efflux of platinum drugs. MRP is a member of the ABC (adenosine triphosphate-binding cassette) family of transport pro‐ teins that participates in the efflux of anticancer drugs from cells. Thus, it has been speculat‐ ed that deregulation of some of the MRP components may influence platinum resistance [30]. The MRP gene family is composed of at least seven members (MRP1–7) but recent reports reinforced the notion that MRP2 expression levels can be important in predicting the sensitivity of tumors to platinum-based therapies [31,32]. MDR1 encodes an integral membrane protein named P-glycoprotein (Pgp) or an ATP-binding cassette subfamily B, member 1, which acts as a drug efflux pump [33]. This protein is a transmembrane transport‐ er that resides in the plasma membrane of many cells, including cancer cells that are multidrug resistant. Pgp recognizes a wide range of anticancer drugs and was shown to reduce intracellular concentrations of a variety of cytotoxic drugs, including platinum agents. Pgp activity results in blunted chemotherapy-induced cytotoxicity in vitro and in vivo. Moreover, anticancer drugs were found to induce MDR1 gene. Since Pgp alone can medi‐ ate resistance to a whole array of drugs through drug efflux, it is an attractive target for the improvement of anticancer therapy. In theory, co-administration of transporter inhibitors with Pgp-substrate anticancer drugs could reverse MDR and improve treatment outcome. Clinical trials aimed at specifically inhibiting the function of Pgp have given mixed results, but in at least some cases this inhibition has resulted in improved tumor shrinkage and increased patient survival. Unfortunately, Pgp inhibitors such as PSC-833 (Valspodar) induced pharmacokinetic interactions that limited drug clearance and metabolism of the Glutathione (gamma-glutamylcysteinylglycine: GSH), the most abundant intracellular thiol, contributes (along with methionine, metallothionein and other cysteine-rich proteins) to detoxify many cellular toxins, including cisplatin and its analogues. Part of the cytoplasmic cisplatin reacts with DNA, which ultimately lead to the activation of the apoptosis cascade in response to DNA damage. However, a major fraction of intracellular cisplatin can be converted into cisplatin-thiol conjugates by GSH-S-transferase π, and these conjugates are ultimately inactivated. Both GSTπ and γ-glutamylcysteine synthetase (γ- GCS), the latter being the enzyme involved in GSH synthesis, have been associated with cisplatin resistance in ovarian, cervical and lung cancer cell lines [37,38,39].

Thus, reducing intracellular glutathione levels would seem a rational strategy to overcome platinum resistance. To that end, a novel glutathione analog prodrug, canfosfamide, initiated clinical development in ovarian cancer. Canfosfamide (TLK286) works by targeting tumors that over-express glutathione S-transferase (GST) P1-1, increasing the sensitivity of those tumors to the cytotoxic effects of canfosfamide. Following activation, the apoptotic activity of canfosfamide is mediated through the stress response pathway, resulting in the induction of cellular apoptosis. Human cancer cells exposed to canfosfamide demonstrate activation of mitogen- activated protein (MAP) kinase MKK4, p38 kinase, jun-N-terminal kinase (JNK) and caspase 3. The cytotoxic activity of canfosfamide has been demonstrated *in vitro* and *in vivo* against a variety of human cancer cell lines, including ovarian cancer cells (OVCAR3).

A phase II trial involving 34 patients with platinum-refractory or platinum-resistant ovarian cancer reported that 15% of patients had an objective response and 50% of patients had disease stabilization [40]. Three phase III trials in platinum-resistant ovarian cancer were undertaken in an attempt to define the potential role of canfosfamide in ovarian cancer therapeutics: TLK286 versus liposomal doxorubicin or topotecan (ASSIST-1,41); TLK286 plus carboplatin versus liposomal doxorubicin (ASSIST-3,42) and TLK286 plus liposomal doxorubicin versus liposomal doxorubicin alone (ASSIST-5,43). Unfortunately, none of these studies showed superior efficacy of canfosfamide compared to standard treatment.

#### **4.3. Increased DNA repair**

The cytotoxicity of cisplatin is attributed to the formation of cisplatin-DNA adducts, and to the induction of DNA damage. The balance between DNA damage to DNA repair dictates tumor cell death or survival after cisplatin therapy. Depending on the type of damage inflicted on the DNA structure, different DNA repair mechanisms have the ability to restore these lesions and remove the platinum-DNA adducts from the tumor DNA [44]. The major pathway in the repair of DNA damage is the nucleotide excision repair (NER) system. NER is one of five separate DNA repair mechanisms that also include mismatch repair (MMR), homologous recombination repair (HR), base excision repair (BER) and translesion synthesis. The prepon‐ derance of one repair mechanism over another may also change in different tumor types.

#### *4.3.1. Nucleotide Excision Repair (NER)*

The nucleotide excision repair (NER) pathway is predominantly responsible for repairing platinum-DNA adducts in cellular DNA. Several proteins interact in a coordinated fashion to recognize damage and further repair of the DNA (Figure 2). One of these proteins is excision repair cross-complementation group 1 (ERCC1). This 33-kD protein, mainly coupled with XPF (Xeroderma Pigmentosum-F protein) acts in the rate-limiting incision step that cleaves the DNA strand before DNA polymerases and ligases act to reconstitute double-strand integrity. Different studies with ovarian cancer cell lines have demonstrat‐ ed that high ERCC1 mRNA expression is correlated with increased capacity of cells to repair cisplatin-induced DNA damage, thus conferring resistance to the drug. Further, transfec‐ tion experiments using ERCC1 antisense vectors in both cell lines and mice have shown increased sensitivity to platinum [45,46,47].

There is growing interest in evaluating the potential role of ERCC1 as a biomarker of platinum resistance in ovarian cancer. However, despite multiple studies evaluating the association between ERCC1 protein expression or even single nucleotide polymorphisms and clinical outcome, no definitive conclusion has yet been reached regarding the predictive and/or prognostic role of ERRCC1 in the management of EOC [48,49].

#### *4.3.2. DNA mismatch repair*

The mismatch repair (MMR) system is a strand-specific DNA repair mechanism involved in the post-replicative repair of the errors made by DNA polymerases and in charge of eliminat‐ ing single-base mismatches and insertion-deletion loops that have escaped the proofreading back-up mechanisms.

Loss of function of the cellular mismatch repair system (MMR) can partially contribute to develop DNA damage tolerance. Unaltered, MMR scans newly synthesized DNA and removes mismatches that result from nucleotide incorporation errors made by the DNA polymerases. The repair process consists of 3 steps—initiation, excision, and re-synthesis that involve several proteins: MLH1, MSH2, MSH3, MSH6, and PMS2. Inactivation of MMR leads to the occurrence of unrepaired deletions in mononucleotide and dinucleotide repeats, resulting in variable length repeats. This phenomenon is called microsatellite instability (MSI), which can be caused by genetic or epigenetic inactivation and has been postulated as a potential marker for MMR deficiency. DNA methylation changes in plasma have been suggested as another rationale of chemotherapy resistance in OEC after treatment (ac‐ quired MLH1 methylation) [50].

MMR deficient human cancer cell lines tolerate cytotoxic drugs, suggesting that loss of MMR could cause platinum resistance [51]. Most MMR-deficient cancers have mutations in MLH1 or MSH2. Samimi et al [52] investigated MLH1 and MSH2 expression in paired ovarian tumor **Figure 2.** Schematic Representation of the Mismatch Repair Pathway (Adapted with permission from Diaz-Padilla I, Po‐ *veda A. Clin Ovarian Cancer Other Gynecol Malig 2010, 3(1):29-35*.) Base-base mismatches are the most frequent errors associated with microsatellites (repetitive sequences of mononucleotide, dinucleotide, or higher-order nucleotide re‐ peats distributed throughout the human genome). The mismatch repair system is responsible for the surveillance and correction of errors introduced in microsatellites. Mismatch repair proteins: MLH1, MSH2, MSH3, MSH6, PMS2. Exo 1,

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exonuclease; DNA Pol, DNA polymerase δ; PCNA, proliferating cell nuclear antigen.

five separate DNA repair mechanisms that also include mismatch repair (MMR), homologous recombination repair (HR), base excision repair (BER) and translesion synthesis. The prepon‐ derance of one repair mechanism over another may also change in different tumor types.

The nucleotide excision repair (NER) pathway is predominantly responsible for repairing platinum-DNA adducts in cellular DNA. Several proteins interact in a coordinated fashion to recognize damage and further repair of the DNA (Figure 2). One of these proteins is excision repair cross-complementation group 1 (ERCC1). This 33-kD protein, mainly coupled with XPF (Xeroderma Pigmentosum-F protein) acts in the rate-limiting incision step that cleaves the DNA strand before DNA polymerases and ligases act to reconstitute double-strand integrity. Different studies with ovarian cancer cell lines have demonstrat‐ ed that high ERCC1 mRNA expression is correlated with increased capacity of cells to repair cisplatin-induced DNA damage, thus conferring resistance to the drug. Further, transfec‐ tion experiments using ERCC1 antisense vectors in both cell lines and mice have shown

There is growing interest in evaluating the potential role of ERCC1 as a biomarker of platinum resistance in ovarian cancer. However, despite multiple studies evaluating the association between ERCC1 protein expression or even single nucleotide polymorphisms and clinical outcome, no definitive conclusion has yet been reached regarding the predictive and/or

The mismatch repair (MMR) system is a strand-specific DNA repair mechanism involved in the post-replicative repair of the errors made by DNA polymerases and in charge of eliminat‐ ing single-base mismatches and insertion-deletion loops that have escaped the proofreading

Loss of function of the cellular mismatch repair system (MMR) can partially contribute to develop DNA damage tolerance. Unaltered, MMR scans newly synthesized DNA and removes mismatches that result from nucleotide incorporation errors made by the DNA polymerases. The repair process consists of 3 steps—initiation, excision, and re-synthesis that involve several proteins: MLH1, MSH2, MSH3, MSH6, and PMS2. Inactivation of MMR leads to the occurrence of unrepaired deletions in mononucleotide and dinucleotide repeats, resulting in variable length repeats. This phenomenon is called microsatellite instability (MSI), which can be caused by genetic or epigenetic inactivation and has been postulated as a potential marker for MMR deficiency. DNA methylation changes in plasma have been suggested as another rationale of chemotherapy resistance in OEC after treatment (ac‐

MMR deficient human cancer cell lines tolerate cytotoxic drugs, suggesting that loss of MMR could cause platinum resistance [51]. Most MMR-deficient cancers have mutations in MLH1 or MSH2. Samimi et al [52] investigated MLH1 and MSH2 expression in paired ovarian tumor

*4.3.1. Nucleotide Excision Repair (NER)*

210 Ovarian Cancer - A Clinical and Translational Update

increased sensitivity to platinum [45,46,47].

*4.3.2. DNA mismatch repair*

back-up mechanisms.

quired MLH1 methylation) [50].

prognostic role of ERRCC1 in the management of EOC [48,49].

**Figure 2.** Schematic Representation of the Mismatch Repair Pathway (Adapted with permission from Diaz-Padilla I, Po‐ *veda A. Clin Ovarian Cancer Other Gynecol Malig 2010, 3(1):29-35*.) Base-base mismatches are the most frequent errors associated with microsatellites (repetitive sequences of mononucleotide, dinucleotide, or higher-order nucleotide re‐ peats distributed throughout the human genome). The mismatch repair system is responsible for the surveillance and correction of errors introduced in microsatellites. Mismatch repair proteins: MLH1, MSH2, MSH3, MSH6, PMS2. Exo 1, exonuclease; DNA Pol, DNA polymerase δ; PCNA, proliferating cell nuclear antigen.

sections from 54 ovarian cancer patients before and after platinum-based therapy by using immunohistochemical staining techniques. These authors demonstrated associations between MLH1 and MSH2 protein expression and clinical parameters known to be of prognostic significance as well as response to treatment and overall survival. MLH1 and MSH2 staining decreased significantly after platinum-based therapy. Hypermethylation of the MLH1 promoter has also been identified as a casual event in sporadic MMR-deficient malignant tumors. In ovarian cancer, it is estimated that about 10% of cases are related to this molecular pathway [53], although the methodology and definitions when assessing MSI in ovarian cancer are heterogeneous and not prospectively validated. The frequency of MMR dysfunction seems to vary depending on the histological subtype, being higher in endometrioid (19%) and mucinous (17%) subtypes. It is an assumption that MMR deficiency might be a tumor-initiating phenomenon in ovarian cancer, similar to colorectal and endometrial tumors. However, MMR deficient ovarian cancers have been only poorly characterized to date with respect to their epidemiological, molecular and clinical features.

that loss of this specific BRCA1 function in DSB repair is source of the genomic instability and

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The BRCA2 gene is located on chromosome 13q. The BRCA2 protein has its primary function in HR and its based upon its ability to bind to the strand invasion recombinase, RAD51. In fact, recruitment of RAD51 to sites of DNA damage requires BRCA2, and BRCA2-deficient cells

Despite only 5-10% of epithelial ovarian cancer has an inherited background, more than 90% of hereditary ovarian tumors bear BRCA1/2 mutations. It has been described that these tumors are generally of serous histology, and high-grade. They usually present at younger ages, and more recently, it has been described that BRCA-mutated tumors have better prognosis. [60,61,62]. It is relevant to note that up to 55% of sporadic epithelial ovarian tumors have some sort of BRCA dysfunction. This has been named as BRCAness, and it may have important clinical consequences. One of the reasons behind this better outcome relies on a higher sensitivity to platinum compounds [63,64]. However, BRCA1/2-mutated also develop plati‐ num resistance. One possible explanation is in relation with the production of secondary intragenic mutations in BRCA1/2 that restore these genetic expressions and HR function correcting the open reading frames of mutated BRCA1/2 [65,66,67]. However BRCA1/2 restoration does not explain all cases of cisplatin resistance so investigations in other mecha‐

Recently, PARP inhibitors have been developed as an important novel strategy for the treatment of BRCA mutation-associated ovarian and breast cancer. The rationale for this approach is that by inhibiting BER, these agents can prevent repair that occurs after cytotoxic chemotherapy that causes single-strand DNA breaks, and also they can work by creating "synthetic lethality" in cells which have lost one mechanism of DNA repair. In the absence of HR, inhibition of PARP results in poor repair of these lesions and apoptosis, increasing around 1000-fold the sensitivity in cells that are BRCA1 or BRCA2 deficient [68,69,70]. Although olaparib and veliparib are the most widely studied in ovarian cancer [71,72,73,74,75], other PARP inhibitors are in development such as BSI-201, AG014699, CEP9722, MK4827, E7016,

Chemotherapy resistance is the ultimate reason for tumor recurrence. Relapsed ovarian cancer is an incurable disease where chemotherapy plays a major therapeutic role. Platinum agents, likely in conjunction with taxanes, are the most active cytotoxic drugs in ovarian cancer. Traditionally, ovarian cancer recurrence has been classified according to the time elapsed from the last dose of platinum. Thus, relapses occurring more than six months from the last dose of platinum are generally re-treated with a platinum combination. Responses to a platinum rechallenge tend to be similar to the initial response, and the longer the platinum-free interval is the better the responses are. The so-called "platinum-sensitive" patients have better

nisms of chemo-resistance in BRCA-deficient ovarian cancer cells are needed.

tumorigenesis observed in this subset of BRCA1 mutation carriers.

exhibit genomic instability.

LT673, to name just a few.

**5. Conclusion and future directions**

Only a few studies have found a consistent relationship between MMR inactivation and platinum-based chemotherapy resistance (primarily down-regulation or mutations in MMR genes MLH1, MSH2 or MSH1) [50, 54,55,56].

This resistance to cisplatin can be circumvented using a DNA demethylating agent such as 2′ deoxy-5-azacytidine (decitabine; Dacoge, MGI Pharma) in combination with cisplatin or carboplatin to reverse this resistance mechanism [57]. Two phase II clinical trials have tested the combination of carboplatin and decitabine in recurrent Platinum-resistant OC patients with different conclusions [58,59].

#### *4.3.3. Homologous recombination repair pathway*

Platinum-based chemotherapy causes inter-strand DNA cross-linking which cause DNA double-strand breaks (DSBs) during DNA replication. DSBs are one of the most toxic lesions to DNA. This is because it affects both strands of the duplex, thus no intact complimentary strand is available as a template for repair. When such lesions are not repaired the cell undergoes apoptosis. If the reparation is not done appropriately, secondary lesions can occur, such as mutations and/or deletions. Cells have evolved two major pathways for the repair of DSBs: non-homologous end-joining (NHEJ) and homologous recombination (HR). The HR system is the preferred system by cells when it comes to repair DSBs. It is a highly conserved system, generally regarded as error-free, that requires an intact sister chromatid to act as template for correct repair of the break without loss of sequence information. As such, HR takes place in G2 and S phases of the cell cycle.

The *BRCA1* gene is located on chromosome 17q21. The BRCA1 protein is a component of a number of supercomplexes, each of which plays a role in DNA damage response activation, cell cycle checkpoint activation and/or DSB repair. Some of the key components of this repair process are proteins such as BRCA2, RAD51 and PALB2. Actually, the interaction between specific domains of BRCA1 and PALB2 is key in the reparation of DBSs. Thus, mutations in domains of BRCA1 can potentially abolish its PALB2-binding activity, resulting in compro‐ mised HR function. These mutations have been found in BRCA1-mutated tumors, implying that loss of this specific BRCA1 function in DSB repair is source of the genomic instability and tumorigenesis observed in this subset of BRCA1 mutation carriers.

The BRCA2 gene is located on chromosome 13q. The BRCA2 protein has its primary function in HR and its based upon its ability to bind to the strand invasion recombinase, RAD51. In fact, recruitment of RAD51 to sites of DNA damage requires BRCA2, and BRCA2-deficient cells exhibit genomic instability.

Despite only 5-10% of epithelial ovarian cancer has an inherited background, more than 90% of hereditary ovarian tumors bear BRCA1/2 mutations. It has been described that these tumors are generally of serous histology, and high-grade. They usually present at younger ages, and more recently, it has been described that BRCA-mutated tumors have better prognosis. [60,61,62]. It is relevant to note that up to 55% of sporadic epithelial ovarian tumors have some sort of BRCA dysfunction. This has been named as BRCAness, and it may have important clinical consequences. One of the reasons behind this better outcome relies on a higher sensitivity to platinum compounds [63,64]. However, BRCA1/2-mutated also develop plati‐ num resistance. One possible explanation is in relation with the production of secondary intragenic mutations in BRCA1/2 that restore these genetic expressions and HR function correcting the open reading frames of mutated BRCA1/2 [65,66,67]. However BRCA1/2 restoration does not explain all cases of cisplatin resistance so investigations in other mecha‐ nisms of chemo-resistance in BRCA-deficient ovarian cancer cells are needed.

Recently, PARP inhibitors have been developed as an important novel strategy for the treatment of BRCA mutation-associated ovarian and breast cancer. The rationale for this approach is that by inhibiting BER, these agents can prevent repair that occurs after cytotoxic chemotherapy that causes single-strand DNA breaks, and also they can work by creating "synthetic lethality" in cells which have lost one mechanism of DNA repair. In the absence of HR, inhibition of PARP results in poor repair of these lesions and apoptosis, increasing around 1000-fold the sensitivity in cells that are BRCA1 or BRCA2 deficient [68,69,70]. Although olaparib and veliparib are the most widely studied in ovarian cancer [71,72,73,74,75], other PARP inhibitors are in development such as BSI-201, AG014699, CEP9722, MK4827, E7016, LT673, to name just a few.

#### **5. Conclusion and future directions**

sections from 54 ovarian cancer patients before and after platinum-based therapy by using immunohistochemical staining techniques. These authors demonstrated associations between MLH1 and MSH2 protein expression and clinical parameters known to be of prognostic significance as well as response to treatment and overall survival. MLH1 and MSH2 staining decreased significantly after platinum-based therapy. Hypermethylation of the MLH1 promoter has also been identified as a casual event in sporadic MMR-deficient malignant tumors. In ovarian cancer, it is estimated that about 10% of cases are related to this molecular pathway [53], although the methodology and definitions when assessing MSI in ovarian cancer are heterogeneous and not prospectively validated. The frequency of MMR dysfunction seems to vary depending on the histological subtype, being higher in endometrioid (19%) and mucinous (17%) subtypes. It is an assumption that MMR deficiency might be a tumor-initiating phenomenon in ovarian cancer, similar to colorectal and endometrial tumors. However, MMR deficient ovarian cancers have been only poorly characterized to date with respect to their

Only a few studies have found a consistent relationship between MMR inactivation and platinum-based chemotherapy resistance (primarily down-regulation or mutations in MMR

This resistance to cisplatin can be circumvented using a DNA demethylating agent such as 2′ deoxy-5-azacytidine (decitabine; Dacoge, MGI Pharma) in combination with cisplatin or carboplatin to reverse this resistance mechanism [57]. Two phase II clinical trials have tested the combination of carboplatin and decitabine in recurrent Platinum-resistant OC patients with

Platinum-based chemotherapy causes inter-strand DNA cross-linking which cause DNA double-strand breaks (DSBs) during DNA replication. DSBs are one of the most toxic lesions to DNA. This is because it affects both strands of the duplex, thus no intact complimentary strand is available as a template for repair. When such lesions are not repaired the cell undergoes apoptosis. If the reparation is not done appropriately, secondary lesions can occur, such as mutations and/or deletions. Cells have evolved two major pathways for the repair of DSBs: non-homologous end-joining (NHEJ) and homologous recombination (HR). The HR system is the preferred system by cells when it comes to repair DSBs. It is a highly conserved system, generally regarded as error-free, that requires an intact sister chromatid to act as template for correct repair of the break without loss of sequence information. As such, HR

The *BRCA1* gene is located on chromosome 17q21. The BRCA1 protein is a component of a number of supercomplexes, each of which plays a role in DNA damage response activation, cell cycle checkpoint activation and/or DSB repair. Some of the key components of this repair process are proteins such as BRCA2, RAD51 and PALB2. Actually, the interaction between specific domains of BRCA1 and PALB2 is key in the reparation of DBSs. Thus, mutations in domains of BRCA1 can potentially abolish its PALB2-binding activity, resulting in compro‐ mised HR function. These mutations have been found in BRCA1-mutated tumors, implying

epidemiological, molecular and clinical features.

212 Ovarian Cancer - A Clinical and Translational Update

genes MLH1, MSH2 or MSH1) [50, 54,55,56].

*4.3.3. Homologous recombination repair pathway*

takes place in G2 and S phases of the cell cycle.

different conclusions [58,59].

Chemotherapy resistance is the ultimate reason for tumor recurrence. Relapsed ovarian cancer is an incurable disease where chemotherapy plays a major therapeutic role. Platinum agents, likely in conjunction with taxanes, are the most active cytotoxic drugs in ovarian cancer. Traditionally, ovarian cancer recurrence has been classified according to the time elapsed from the last dose of platinum. Thus, relapses occurring more than six months from the last dose of platinum are generally re-treated with a platinum combination. Responses to a platinum rechallenge tend to be similar to the initial response, and the longer the platinum-free interval is the better the responses are. The so-called "platinum-sensitive" patients have better prognosis than women whose relapse is shorter than six months. For this group of patients therapeutic options are limited and usually consist in non-platinum agents.

**References**

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The development of platinum resistance is a multifactorial and complex molecular process. Understanding the molecular basis of this mechanism would help potentially in selecting patients who are likely to have platinum-resistance tumors for alternate non-platinum containing regimens. This would spare women from unnecessary toxic effects and ineffective treatments. One potential scenario where the application of a molecular selection of patients by platinum sensitivity would be at initial presentation. A substantial number of advanced ovarian cancer patients undergo neoadjuvant chemotherapy prior to debulk‐ ing surgery. Such chemotherapy consists of platinum and a taxane doublet. Despite most patients do have a major response to primary chemotherapy, about 20-30% fail to re‐ spond or progress.

The development of platinum resistance seems a dynamic process. Patients who initially respond to platinum-based chemotherapy end up becoming resistant. This suggests that we may need to investigate the mechanisms at several time points of the disease course. It is likely that primary platinum resistance is a molecular phenomenon different from secondary (and subsequent) platinum resistance. At this point, it is key that reliable biomarkers can be identified to better define platinum resistance. The quest for a bonafide biomarker of platinum resistance in ovarian cancer has been so far disappointing. It may well be the case that several markers need to be jointly studied, since platinum resistance is not a one-step molecular event. Further validation in large (ideally prospec‐ tive) cohorts and in randomized phase III trials will be still needed. It will be difficult though to extrapolate results of platinum sensitivity when other agents are given concom‐ itantly. It is not possible to rule out the potential influence of cytotoxics with similar mechanisms of action or biologics with the potential of modifying the tumor microenviron‐ ment. Unraveling the mechanisms of resistance to platinum (and other chemotherapy agents) in ovarian cancer is a very difficult task. However, its potential clinical benefits are worth such tremendous joint basic and clinical research effort.

#### **Author details**

Gonzalo Tapia1 and Ivan Diaz-Padilla2\*

\*Address all correspondence to: ivandpadilla@hospitaldemadrid.com

1 Division of Medical Oncology, Gregorio Marañón University Hospital, Madrid, Spain

2 Division of Medical Oncology, Centro Integral Oncológico Clara Campal, Madrid, Spain

Disclosures: The authors have no conflict of interest

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The development of platinum resistance is a multifactorial and complex molecular process. Understanding the molecular basis of this mechanism would help potentially in selecting patients who are likely to have platinum-resistance tumors for alternate non-platinum containing regimens. This would spare women from unnecessary toxic effects and ineffective treatments. One potential scenario where the application of a molecular selection of patients by platinum sensitivity would be at initial presentation. A substantial number of advanced ovarian cancer patients undergo neoadjuvant chemotherapy prior to debulk‐ ing surgery. Such chemotherapy consists of platinum and a taxane doublet. Despite most patients do have a major response to primary chemotherapy, about 20-30% fail to re‐

The development of platinum resistance seems a dynamic process. Patients who initially respond to platinum-based chemotherapy end up becoming resistant. This suggests that we may need to investigate the mechanisms at several time points of the disease course. It is likely that primary platinum resistance is a molecular phenomenon different from secondary (and subsequent) platinum resistance. At this point, it is key that reliable biomarkers can be identified to better define platinum resistance. The quest for a bonafide biomarker of platinum resistance in ovarian cancer has been so far disappointing. It may well be the case that several markers need to be jointly studied, since platinum resistance is not a one-step molecular event. Further validation in large (ideally prospec‐ tive) cohorts and in randomized phase III trials will be still needed. It will be difficult though to extrapolate results of platinum sensitivity when other agents are given concom‐ itantly. It is not possible to rule out the potential influence of cytotoxics with similar mechanisms of action or biologics with the potential of modifying the tumor microenviron‐ ment. Unraveling the mechanisms of resistance to platinum (and other chemotherapy agents) in ovarian cancer is a very difficult task. However, its potential clinical benefits are

therapeutic options are limited and usually consist in non-platinum agents.

worth such tremendous joint basic and clinical research effort.

\*Address all correspondence to: ivandpadilla@hospitaldemadrid.com

1 Division of Medical Oncology, Gregorio Marañón University Hospital, Madrid, Spain

2 Division of Medical Oncology, Centro Integral Oncológico Clara Campal, Madrid, Spain

and Ivan Diaz-Padilla2\*

Disclosures: The authors have no conflict of interest

spond or progress.

214 Ovarian Cancer - A Clinical and Translational Update

**Author details**

Gonzalo Tapia1


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**Section 4**

**Molecular Pathogenesis and Targeted Therapies**

**Molecular Pathogenesis and Targeted Therapies**

**Chapter 11**

**Biological Significance of Apoptosis in Ovarian Cancer:**

An apoptotic program is present in almost all cell types. Functional characterization of the apoptotic cascade has revealed how the apoptotic program is activated in response to di‐ verse stresses such as DNA damage, signaling imbalance provoked by oncogene activation, survival factors insufficiency or hypoxia. One of the hallmarks of tumor cells is their ability to resist apoptosis. The concept that apoptosis serves as a barrier to cancer development has been well established (Evan and Littlewood, 1998; Hengartner, 2000; Lowe et al., 2004; Adams and Cory, 2007). This is especially relevant for ovarian cancer (OC) where most pa‐ tients presenting with advanced OC (most commonly high grade serous OC) will respond to the initial chemotherapy treatment suggesting that most tumor cells present are sensitive to chemotherapy. However, only 10-15% of these patients maintain a complete response to the initial therapy implying that a fraction of the tumor cells escaped apoptosis induced by che‐ motherapeutic drugs. Thus, one of the main obstacles to an effective treatment in OC is the failure of the initial chemotherapy to eradicate a sufficient number of tumor cells to prevent disease recurrence. Attenuation of apoptosis in those tumor cells contributes to the resist‐

ance to subsequent therapy and likely plays an important role in OC progression.

This chapter focuses on the molecular pathways that lead to apoptotic resistance and the need to move towards new targeted treatment in OC. Particular attention will be given to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling cascade. TRAIL is a cytokine that triggers apoptosis in a wide variety of tumor cells with apparent little effect on normal cells. We will discuss the various mechanisms that OC cells may de‐ velop to suppress TRAIL cytotoxicity. Furthermore, we will review the emerging TRAIL-tar‐

and reproduction in any medium, provided the original work is properly cited.

© 2013 Goncharenko-Khaider et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**TRAIL Therapeutic Targeting**

Nadzeya Goncharenko-Khaider, Denis Lane,

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53380

**1. Introduction**

Isabelle Matte, Claudine Rancourt and Alain Piché

### **Biological Significance of Apoptosis in Ovarian Cancer: TRAIL Therapeutic Targeting**

Nadzeya Goncharenko-Khaider, Denis Lane, Isabelle Matte, Claudine Rancourt and Alain Piché

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53380

#### **1. Introduction**

An apoptotic program is present in almost all cell types. Functional characterization of the apoptotic cascade has revealed how the apoptotic program is activated in response to di‐ verse stresses such as DNA damage, signaling imbalance provoked by oncogene activation, survival factors insufficiency or hypoxia. One of the hallmarks of tumor cells is their ability to resist apoptosis. The concept that apoptosis serves as a barrier to cancer development has been well established (Evan and Littlewood, 1998; Hengartner, 2000; Lowe et al., 2004; Adams and Cory, 2007). This is especially relevant for ovarian cancer (OC) where most pa‐ tients presenting with advanced OC (most commonly high grade serous OC) will respond to the initial chemotherapy treatment suggesting that most tumor cells present are sensitive to chemotherapy. However, only 10-15% of these patients maintain a complete response to the initial therapy implying that a fraction of the tumor cells escaped apoptosis induced by che‐ motherapeutic drugs. Thus, one of the main obstacles to an effective treatment in OC is the failure of the initial chemotherapy to eradicate a sufficient number of tumor cells to prevent disease recurrence. Attenuation of apoptosis in those tumor cells contributes to the resist‐ ance to subsequent therapy and likely plays an important role in OC progression.

This chapter focuses on the molecular pathways that lead to apoptotic resistance and the need to move towards new targeted treatment in OC. Particular attention will be given to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling cascade. TRAIL is a cytokine that triggers apoptosis in a wide variety of tumor cells with apparent little effect on normal cells. We will discuss the various mechanisms that OC cells may de‐ velop to suppress TRAIL cytotoxicity. Furthermore, we will review the emerging TRAIL-tar‐

© 2013 Goncharenko-Khaider et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

geting strategies for treating OC and provide information about the latest clinical studies of TRAIL agonists that are being conducted for the treatment of OC.

activity. In specific conditions, its structure allows c-FLIP to be recruited to the DISC where it inhibits the processing and activation of pro-caspase-8. Although many isoforms of c-FLIP have been identified, only three are expressed in human cells (Djerbi et al., 2001). They con‐ sist of two short variants, c-FLIPS and c-FLIPR, and a long splice variant, c-FLIPL. Both c-FLIPL and c-FLIPS contain two DEDs and compete with pro-caspase-8 for association with FADD (Bagnoli et al., 2010). Depending on the level of c-FLIPL expression, its function at the DISC will vary. When present in high amounts, c-FLIPL will exert an anti-apoptotic effect at the DISC (Krueger et al., 2001). When present in low amounts, it may heterodimerize with caspase-8 at the DISC and promotes apoptosis (Chang et al., 2002). c-FLIP is thus seen as a major inhibitor of the extrinsic pathway of apoptosis. In so-called type II cells, less caspase-8 is activated at the DISC and efficient apoptosis requires further signal amplification via the intrinsic or mitochondrial pathway. This is achieved by caspase-8-mediated Bid cleavage to generate a truncated form of Bid (tBid) which subsequently engages Bax/Bak to activate the

Biological Significance of Apoptosis in Ovarian Cancer: TRAIL Therapeutic Targeting

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229

The intrinsic pathway is usually triggered in response to DNA damage, hypoxia or onco‐ gene overexpression. As a sensor of cellular stress, p53 is a critical initiator of the intrinsic pathway. In response to cellular damage, p53 translocates from the cytoplasm to the nucleus where it promotes the transcription of pro-apoptotic members of the Bcl-2 family. Pro-apop‐ totic Bcl-2 family members Bax and Bak form pores in the outer mitochondrial membrane causing the release of cytochrome c and other apoptogenic factors such as apoptosis induc‐ ing factor (AIF) and SMAC/DIABLO into the cytoplasm. The released of cytochrome c, along with apoptosis protease activating factor-1 (APAF-1) and pro-caspase-9 form the apoptosome. Within the apoptosome, clustered pro-caspase-9 gets activated and cleaves downstream effector caspases, leading to the hallmark of apoptosis (Youle and Strasser, 2008; Brunelle and Letai, 2009). The release of SMAC/DIABLO from the mitochondria pro‐ motes apoptosis by binding to and neutralizing members of the family of inhibitor of apop‐ tosis proteins (IAPs), which can block caspase-3 activity through its baculovirus IAP repeat domains. Although the extrinsic and intrinsic pathways are activated by different mecha‐ nisms, these two pathways are interconnected (Figure 1). In type II cells, activated caspase-8 cleaves pro-apoptotic Bcl-2 family member Bid to form truncated Bid (tBid), which can then interact with Bax/Bak. This interaction increases the release of cytochrome c from the mito‐ chondria. Thus, Bid provides a connection between extrinsic and intrinsic pathways (so called mitochondrial amplification loop). The reasons that determine whether tumor cells re‐ ly on type I or II signaling are not well understood but resistance has been attributed to dys‐ function of different steps in the TRAIL-induced apoptosis pathway and/or elevation of survival signals (Zhang and Fang, 2005). In particular, it has been proposed that the levels of c-FLIP and XIAP relative to caspase-8 and SMAC/DIABLO might be important determi‐

Bcl-2 family proteins are involved in the regulation of apoptosis by controlling mitochondri‐ al membrane permeability. Several studies have demonstrated that these proteins can inter‐ act with each other and these interactions can neutralize their pro- or anti-apoptotic functions. The balance between anti- and pro-apoptotic members dictates the fate of cell sur‐

mitochondria.

nants (Kim et al., 2000).

#### **2. Treatment for ovarian cancer**

Because of the limited efficacy of current treatments for advanced OC, novel and more effec‐ tive therapies are being investigated. An emerging option for the treatment of OC is the tar‐ geting of the TRAIL signaling cascade. Because of its unique ability to trigger apoptosis in cancer cells and spare normal cells, in contrast to other cytokines such as FasL and TNFα, TRAIL is an attractive and promising treatment for cancer therapy. Preclinical studies in mice provided the first evidence that the soluble recombinant TRAIL suppresses the growth of human tumor xenografts with no apparent systemic toxicity (Walczak et al., 1999; Ashke‐ nazi et al., 1999). More recently, recombinant TRAIL has entered clinical trials for the treat‐ ment of various malignancies (Ashkenazi, 2008; Ashkenazi et al., 2008; Abdulghani and El-Deiry, 2010; Hellwig and Rehm, 2012). In addition to soluble TRAIL, several agonistic antibodies targeting TRAIL R1 or TRAIL R2 death receptors have been developed and en‐ tered into clinical trials that included OC patients (Ashkenazi et al., 2008; Hellwig and Rehm, 2012). As for standard chemotherapy, tumor cells have developed various mecha‐ nisms to escape the apoptosis induced by TRAIL. This underscores the need to understand the mechanisms of TRAIL resistance, and based on this knowledge, identify and validate novel combinations that could be used with TRAIL to potentiate its therapeutic efficacy. For example, TRAIL resistance has been often associated with overexpression of anti-apoptotic proteins. Therefore, the identification of combination treatments that abrogate anti-apoptotic protein function is promising.

#### **3. Apoptosis overview**

Deregulation of the apoptotic cascade not only plays a key role in the pathogenesis and pro‐ gression of cancer, but also leads to resistance to chemotherapy. There are two major cellular death pathways that transduce the effects of various death inducers: the extrinsic and the intrinsic pathway (Figure 1). The extrinsic pathway is triggered when TRAIL binds to TRAIL R1 or TRAIL R2. Receptor trimerization, along with the subsequent oligomerization and clustering of the receptors, leads to the recruitment of the adaptor protein Fas-associat‐ ed protein with death domain (FADD). FADD allows the recruitment of the inactive procaspase-8 or –caspase-10 via a shared death effector domain (DED) leading to the formation of the death-inducing signaling complex (DISC). Depending on the cell type, apoptosis acti‐ vation through the extrinsic pathways may or may not depend on the intrinsic pathway. For example, in type I cells, upon DISC activation, sufficient caspase-8 is activated and, in turn, directly activates the effector caspases (caspase-3, -6, -7) leading to the execution of apopto‐ sis (Abdulghani and El-Deiry, 2010). FLICE-inhibitory protein (c-FLIP) shares structural ho‐ mology with pro-caspase-8 and possesses a death effector domain that lacks protease activity. In specific conditions, its structure allows c-FLIP to be recruited to the DISC where it inhibits the processing and activation of pro-caspase-8. Although many isoforms of c-FLIP have been identified, only three are expressed in human cells (Djerbi et al., 2001). They con‐ sist of two short variants, c-FLIPS and c-FLIPR, and a long splice variant, c-FLIPL. Both c-FLIPL and c-FLIPS contain two DEDs and compete with pro-caspase-8 for association with FADD (Bagnoli et al., 2010). Depending on the level of c-FLIPL expression, its function at the DISC will vary. When present in high amounts, c-FLIPL will exert an anti-apoptotic effect at the DISC (Krueger et al., 2001). When present in low amounts, it may heterodimerize with caspase-8 at the DISC and promotes apoptosis (Chang et al., 2002). c-FLIP is thus seen as a major inhibitor of the extrinsic pathway of apoptosis. In so-called type II cells, less caspase-8 is activated at the DISC and efficient apoptosis requires further signal amplification via the intrinsic or mitochondrial pathway. This is achieved by caspase-8-mediated Bid cleavage to generate a truncated form of Bid (tBid) which subsequently engages Bax/Bak to activate the mitochondria.

geting strategies for treating OC and provide information about the latest clinical studies of

Because of the limited efficacy of current treatments for advanced OC, novel and more effec‐ tive therapies are being investigated. An emerging option for the treatment of OC is the tar‐ geting of the TRAIL signaling cascade. Because of its unique ability to trigger apoptosis in cancer cells and spare normal cells, in contrast to other cytokines such as FasL and TNFα, TRAIL is an attractive and promising treatment for cancer therapy. Preclinical studies in mice provided the first evidence that the soluble recombinant TRAIL suppresses the growth of human tumor xenografts with no apparent systemic toxicity (Walczak et al., 1999; Ashke‐ nazi et al., 1999). More recently, recombinant TRAIL has entered clinical trials for the treat‐ ment of various malignancies (Ashkenazi, 2008; Ashkenazi et al., 2008; Abdulghani and El-Deiry, 2010; Hellwig and Rehm, 2012). In addition to soluble TRAIL, several agonistic antibodies targeting TRAIL R1 or TRAIL R2 death receptors have been developed and en‐ tered into clinical trials that included OC patients (Ashkenazi et al., 2008; Hellwig and Rehm, 2012). As for standard chemotherapy, tumor cells have developed various mecha‐ nisms to escape the apoptosis induced by TRAIL. This underscores the need to understand the mechanisms of TRAIL resistance, and based on this knowledge, identify and validate novel combinations that could be used with TRAIL to potentiate its therapeutic efficacy. For example, TRAIL resistance has been often associated with overexpression of anti-apoptotic proteins. Therefore, the identification of combination treatments that abrogate anti-apoptotic

Deregulation of the apoptotic cascade not only plays a key role in the pathogenesis and pro‐ gression of cancer, but also leads to resistance to chemotherapy. There are two major cellular death pathways that transduce the effects of various death inducers: the extrinsic and the intrinsic pathway (Figure 1). The extrinsic pathway is triggered when TRAIL binds to TRAIL R1 or TRAIL R2. Receptor trimerization, along with the subsequent oligomerization and clustering of the receptors, leads to the recruitment of the adaptor protein Fas-associat‐ ed protein with death domain (FADD). FADD allows the recruitment of the inactive procaspase-8 or –caspase-10 via a shared death effector domain (DED) leading to the formation of the death-inducing signaling complex (DISC). Depending on the cell type, apoptosis acti‐ vation through the extrinsic pathways may or may not depend on the intrinsic pathway. For example, in type I cells, upon DISC activation, sufficient caspase-8 is activated and, in turn, directly activates the effector caspases (caspase-3, -6, -7) leading to the execution of apopto‐ sis (Abdulghani and El-Deiry, 2010). FLICE-inhibitory protein (c-FLIP) shares structural ho‐ mology with pro-caspase-8 and possesses a death effector domain that lacks protease

TRAIL agonists that are being conducted for the treatment of OC.

**2. Treatment for ovarian cancer**

228 Ovarian Cancer - A Clinical and Translational Update

protein function is promising.

**3. Apoptosis overview**

The intrinsic pathway is usually triggered in response to DNA damage, hypoxia or onco‐ gene overexpression. As a sensor of cellular stress, p53 is a critical initiator of the intrinsic pathway. In response to cellular damage, p53 translocates from the cytoplasm to the nucleus where it promotes the transcription of pro-apoptotic members of the Bcl-2 family. Pro-apop‐ totic Bcl-2 family members Bax and Bak form pores in the outer mitochondrial membrane causing the release of cytochrome c and other apoptogenic factors such as apoptosis induc‐ ing factor (AIF) and SMAC/DIABLO into the cytoplasm. The released of cytochrome c, along with apoptosis protease activating factor-1 (APAF-1) and pro-caspase-9 form the apoptosome. Within the apoptosome, clustered pro-caspase-9 gets activated and cleaves downstream effector caspases, leading to the hallmark of apoptosis (Youle and Strasser, 2008; Brunelle and Letai, 2009). The release of SMAC/DIABLO from the mitochondria pro‐ motes apoptosis by binding to and neutralizing members of the family of inhibitor of apop‐ tosis proteins (IAPs), which can block caspase-3 activity through its baculovirus IAP repeat domains. Although the extrinsic and intrinsic pathways are activated by different mecha‐ nisms, these two pathways are interconnected (Figure 1). In type II cells, activated caspase-8 cleaves pro-apoptotic Bcl-2 family member Bid to form truncated Bid (tBid), which can then interact with Bax/Bak. This interaction increases the release of cytochrome c from the mito‐ chondria. Thus, Bid provides a connection between extrinsic and intrinsic pathways (so called mitochondrial amplification loop). The reasons that determine whether tumor cells re‐ ly on type I or II signaling are not well understood but resistance has been attributed to dys‐ function of different steps in the TRAIL-induced apoptosis pathway and/or elevation of survival signals (Zhang and Fang, 2005). In particular, it has been proposed that the levels of c-FLIP and XIAP relative to caspase-8 and SMAC/DIABLO might be important determi‐ nants (Kim et al., 2000).

Bcl-2 family proteins are involved in the regulation of apoptosis by controlling mitochondri‐ al membrane permeability. Several studies have demonstrated that these proteins can inter‐ act with each other and these interactions can neutralize their pro- or anti-apoptotic functions. The balance between anti- and pro-apoptotic members dictates the fate of cell sur‐ vival or death. Pro-apoptotic Bcl-2 members can be divided into 2 groups according to their function and the number of BH domains that they possess. Proteins containing BH domains 1-3 are known as multidomain pro-apoptotic proteins such as Bax, Bak and Bok (Youle and Strasser, 2008). BH-3-only pro-apoptotic proteins such as Bik, Bid, Bad, Bim, Bmf, Noxa, Pu‐ ma and others can form heterodimers with the multidomain proteins Bax and Bak leading to the activation of the mitochondria. Anti-apoptotic proteins such as Bcl-2, Bcl-XL and Mcl-1 can also form hetero-dimeric interactions with Bax and Bak, thereby neutralizing their proapoptotic activity. Anti-apoptotic proteins can form hetero-dimers with BH-3-only proteins and this interaction neutralizes the pro-survival function of anti-apoptotic proteins.

pression has been correlated with poor prognostic for patients with OC (Shigemasa et al., 2002). Elevated expression of c-FLIPL has been reported in a substantial percentage of OC tissues from patients with advanced diseases (Mezzanzanica et al., 2004; Horak et al., 2005a) and has been associated with adverse outcome in some studies (Ouellet et al, 2007; Bagnoli

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231

In patients with OC, high TRAIL expression in either tumor or stromal cells is a predictor of overall survival (Lancaster et al., 2003; Horak et al., 2005a). Interestingly in Horak's study, almost 50% of the tumor analyzed expressed elevated level of c-FLIPL and about 80% of tu‐ mors displayed low expression of TRAIL R1 and/or TRAIL R2, which could contribute to protect OC cells from TRAIL-induced apoptosis. Loss of TRAIL expression has been associ‐ ated with worse outcome (Duiker et al., 2010). Furthermore, this group reported that epige‐ netic silencing of TRAIL R1 occurred in 8% to 27% OC tumor samples (Horak et al., 2005b). Higher expression of TRAIL receptors in OC cells has been associated with a worse outcome (Ouellet et al., 2007; Dong et al., 2008) but other studies have found no correlation between

The mechanisms of resistance to TRAIL can be divided into three categories based on their mode of acquisition: intrinsic resistance, acquired resistance and environment-mediated re‐ sistance (Goncharenko-Khaider et al., 2012). Each of them will be discussed separately.

Intrinsic resistance is observed when tumor cells are resistant to a specific drug without pre‐ vious exposure to this drug. The incidence of intrinsic resistance to TRAIL among patients presenting with OC is not known but intrinsic TRAIL resistance among OC cell lines and primary OC cells is roughly 50% (Cuello et al., 2001a; Vignati et al., 2002; Siervo-Sassi et al., 2003; Lane et al., 2004). Multiple mechanisms have been described for intrinsic TRAIL resist‐ ance in OC cells because the susceptibility to TRAIL-induced apoptosis can be regulated at multiple levels in the apoptotic signaling cascade. The loss of TRAIL R1 expression by epi‐ genetic silencing correlated with resistance to TRAIL-induced apoptosis in OC cells (Horak et al., 2005b). Aberrant methylation of TRAIL receptors has been reported in up to 40% of OC tumors (Shivapurkar et al., 2004). Despite these observations in OC tissues, the levels of TRAIL receptors or decoy receptors do not usually correlate with sensitivity or resistance to TRAIL in OC cell lines (Cuello et al., 2001a; Vignati et al., 2002; Lane et al., 2004). However, the modulation of TRAIL receptors expression may sensitize tumor cells to TRAIL. For ex‐ ample, celestrol-induced upregulation of TRAIL R1 and TRAIL R2 enhances TRAIL-induced

As mentioned earlier, c-FLIP is an important modulator of TRAIL sensitivity. Therefore, it is not surprising that c-FLIP overexpression has been associated with intrinsic TRAIL resist‐ ance in OC cells. A number of studies have demonstrated that the down-regulation of c-

et al., 2009) whereas others have found no such association (Duiker et al., 2010).

TRAIL R1 or TRAIL R2 expression and survival (Duiker et al., 2010).

**6. Resistance in OC cells**

**6.1. Intrinsic resistance**

apoptosis (Zhu et al., 2010).

#### **4. TRAIL and its receptors**

TRAIL is a member of the TNF ligand superfamily of cytokines and is a type II transmem‐ brane protein, which is anchored to the plasma membrane and presented to the cell surface. The extracellular domain of TRAIL can be shed from the cell surface by cysteine proteases to produce soluble TRAIL. Both the soluble and the membrane-bounded TRAIL can trigger apoptosis by interacting with its cognate death receptors expressed by target cells. Of the five human TRAIL receptors that have been identified, both TRAIL R1 (DR4) and TRAIL R2 (DR5) contain a functional death domain in their intracellular portion, unlike decoy recep‐ tors TRAIL R3 (DcR1) and TRAIL R4 (DcR2), which lack a functional death domain and are thus incapable of transmitting an apoptotic signal (Pan et al., 1997a; Pan et al., 1997b; Sheri‐ dan et al., 1997; Marsters et al., 1997). Soluble TRAIL also binds with low affinity to soluble osteoprotegerin (OPG), which is a decoy receptor for RANKL that blocks the RANK-RANKL interaction (Hofbauer et al., 2000). OPG negatively regulates osteoclastogenesis and soluble OPG can act as a scavenger for soluble TRAIL and therefore inhibits TRAIL-induced apoptosis (Vitovski et al., 2007).

#### **5. Expression of apoptosis-related proteins in ovarian cancer**

Because the susceptibility of tumor cells to apoptosis appears to be determined, at least in part, by the ratio between pro- and anti-apoptotic proteins, the expression pattern of antiapoptotic proteins, Bcl-2, Bcl-XL and Mcl-1 has been assessed in OC tissues. For example, higher Bcl-2 expression has been generally associated with a favorable outcome in OC (Hen‐ riksen et al., 1995; Herod et al., 1996; Marx et al., 1997; Marone et al., 1998). This apparent paradox may be explained by the observation that high Bcl-2 expression delays cell cycle progression by promoting accumulation of cells in S phases without affecting the rate of apoptosis in OC cells (Bélanger et al., 2005). Bcl-XL expression is generally higher in OC tis‐ sues when compared to normal tissues (Marone et al., 1998) but has not been consistently associated with worse outcome (Shigemasa et al., 2002; Williams et al., 2005). This could be related to the observation that the ability of Bcl-XL to attenuate apoptosis appears to be cell context-dependent in OC (Dodier and Piché, 2006). In at least one study, increased Mcl-1 ex‐ pression has been correlated with poor prognostic for patients with OC (Shigemasa et al., 2002). Elevated expression of c-FLIPL has been reported in a substantial percentage of OC tissues from patients with advanced diseases (Mezzanzanica et al., 2004; Horak et al., 2005a) and has been associated with adverse outcome in some studies (Ouellet et al, 2007; Bagnoli et al., 2009) whereas others have found no such association (Duiker et al., 2010).

In patients with OC, high TRAIL expression in either tumor or stromal cells is a predictor of overall survival (Lancaster et al., 2003; Horak et al., 2005a). Interestingly in Horak's study, almost 50% of the tumor analyzed expressed elevated level of c-FLIPL and about 80% of tu‐ mors displayed low expression of TRAIL R1 and/or TRAIL R2, which could contribute to protect OC cells from TRAIL-induced apoptosis. Loss of TRAIL expression has been associ‐ ated with worse outcome (Duiker et al., 2010). Furthermore, this group reported that epige‐ netic silencing of TRAIL R1 occurred in 8% to 27% OC tumor samples (Horak et al., 2005b). Higher expression of TRAIL receptors in OC cells has been associated with a worse outcome (Ouellet et al., 2007; Dong et al., 2008) but other studies have found no correlation between TRAIL R1 or TRAIL R2 expression and survival (Duiker et al., 2010).

#### **6. Resistance in OC cells**

vival or death. Pro-apoptotic Bcl-2 members can be divided into 2 groups according to their function and the number of BH domains that they possess. Proteins containing BH domains 1-3 are known as multidomain pro-apoptotic proteins such as Bax, Bak and Bok (Youle and Strasser, 2008). BH-3-only pro-apoptotic proteins such as Bik, Bid, Bad, Bim, Bmf, Noxa, Pu‐ ma and others can form heterodimers with the multidomain proteins Bax and Bak leading to the activation of the mitochondria. Anti-apoptotic proteins such as Bcl-2, Bcl-XL and Mcl-1 can also form hetero-dimeric interactions with Bax and Bak, thereby neutralizing their proapoptotic activity. Anti-apoptotic proteins can form hetero-dimers with BH-3-only proteins

and this interaction neutralizes the pro-survival function of anti-apoptotic proteins.

**5. Expression of apoptosis-related proteins in ovarian cancer**

Because the susceptibility of tumor cells to apoptosis appears to be determined, at least in part, by the ratio between pro- and anti-apoptotic proteins, the expression pattern of antiapoptotic proteins, Bcl-2, Bcl-XL and Mcl-1 has been assessed in OC tissues. For example, higher Bcl-2 expression has been generally associated with a favorable outcome in OC (Hen‐ riksen et al., 1995; Herod et al., 1996; Marx et al., 1997; Marone et al., 1998). This apparent paradox may be explained by the observation that high Bcl-2 expression delays cell cycle progression by promoting accumulation of cells in S phases without affecting the rate of apoptosis in OC cells (Bélanger et al., 2005). Bcl-XL expression is generally higher in OC tis‐ sues when compared to normal tissues (Marone et al., 1998) but has not been consistently associated with worse outcome (Shigemasa et al., 2002; Williams et al., 2005). This could be related to the observation that the ability of Bcl-XL to attenuate apoptosis appears to be cell context-dependent in OC (Dodier and Piché, 2006). In at least one study, increased Mcl-1 ex‐

TRAIL is a member of the TNF ligand superfamily of cytokines and is a type II transmem‐ brane protein, which is anchored to the plasma membrane and presented to the cell surface. The extracellular domain of TRAIL can be shed from the cell surface by cysteine proteases to produce soluble TRAIL. Both the soluble and the membrane-bounded TRAIL can trigger apoptosis by interacting with its cognate death receptors expressed by target cells. Of the five human TRAIL receptors that have been identified, both TRAIL R1 (DR4) and TRAIL R2 (DR5) contain a functional death domain in their intracellular portion, unlike decoy recep‐ tors TRAIL R3 (DcR1) and TRAIL R4 (DcR2), which lack a functional death domain and are thus incapable of transmitting an apoptotic signal (Pan et al., 1997a; Pan et al., 1997b; Sheri‐ dan et al., 1997; Marsters et al., 1997). Soluble TRAIL also binds with low affinity to soluble osteoprotegerin (OPG), which is a decoy receptor for RANKL that blocks the RANK-RANKL interaction (Hofbauer et al., 2000). OPG negatively regulates osteoclastogenesis and soluble OPG can act as a scavenger for soluble TRAIL and therefore inhibits TRAIL-induced

**4. TRAIL and its receptors**

230 Ovarian Cancer - A Clinical and Translational Update

apoptosis (Vitovski et al., 2007).

The mechanisms of resistance to TRAIL can be divided into three categories based on their mode of acquisition: intrinsic resistance, acquired resistance and environment-mediated re‐ sistance (Goncharenko-Khaider et al., 2012). Each of them will be discussed separately.

#### **6.1. Intrinsic resistance**

Intrinsic resistance is observed when tumor cells are resistant to a specific drug without pre‐ vious exposure to this drug. The incidence of intrinsic resistance to TRAIL among patients presenting with OC is not known but intrinsic TRAIL resistance among OC cell lines and primary OC cells is roughly 50% (Cuello et al., 2001a; Vignati et al., 2002; Siervo-Sassi et al., 2003; Lane et al., 2004). Multiple mechanisms have been described for intrinsic TRAIL resist‐ ance in OC cells because the susceptibility to TRAIL-induced apoptosis can be regulated at multiple levels in the apoptotic signaling cascade. The loss of TRAIL R1 expression by epi‐ genetic silencing correlated with resistance to TRAIL-induced apoptosis in OC cells (Horak et al., 2005b). Aberrant methylation of TRAIL receptors has been reported in up to 40% of OC tumors (Shivapurkar et al., 2004). Despite these observations in OC tissues, the levels of TRAIL receptors or decoy receptors do not usually correlate with sensitivity or resistance to TRAIL in OC cell lines (Cuello et al., 2001a; Vignati et al., 2002; Lane et al., 2004). However, the modulation of TRAIL receptors expression may sensitize tumor cells to TRAIL. For ex‐ ample, celestrol-induced upregulation of TRAIL R1 and TRAIL R2 enhances TRAIL-induced apoptosis (Zhu et al., 2010).

As mentioned earlier, c-FLIP is an important modulator of TRAIL sensitivity. Therefore, it is not surprising that c-FLIP overexpression has been associated with intrinsic TRAIL resist‐ ance in OC cells. A number of studies have demonstrated that the down-regulation of cFLIPL (through different means) enhances TRAIL-induced apoptosis in resistant OC cells (Lane et al., 2004; Clarke et al., 2007; Syed et al. 2007; Park et al., 2009). In addition, the knockdown of c-FLIPL inhibited human OC cell lines migratory phenotype in a TRAIL-de‐ pendent manner *in vitro* and inhibited the invasion of tumor cells into the peritoneal cavity (El-Gazzar et al., 2010a).

For example, in an OC cell line model, resistance to the anti-TRAIL-R2 antibody TRA-8 was induced by repeated exposure to non-apoptosis-inducing doses of the antibody (Li et al., 2006). Interestingly, the apoptotic responses induced by TRAIL, a TRAIL-R1 agonist anti‐ body (2E12), and other apoptotic stimuli were not impaired. Lane et al. demonstrated that TRAIL acquired resistance was due to a rapid degradation of active caspase-3 subunits by the proteasome in the TRAIL resistant variant OC cells OVCAR3 (Lane et al., 2006). Interest‐

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One reassuring finding of these studies in OC and other in different tumor types is the fact that acquired TRAIL resistance does not confer cross-resistance to chemotherapeutic drugs such as cisplatin. In fact, combining standard chemotherapy with TRAIL treatment appears to be beneficial because treatment with platinum compounds upregulates the expression of TRAIL death receptors regardless of the p53 status which leads to increase apoptosis in OC

Environment-mediated drug resistance (*de novo* resistance) is a form of resistance by which tumor cells are transiently protected from drug-induced apoptosis via the induction of sur‐ vival signaling pathways (Meads et al., 2009). Soluble factors in the tumor environment may engage cell surface receptor to activate survival pathways. Evidence is accumulating that the tumor environment affects both tumor progression and response to chemotherapy in OC. The accumulation of peritoneal fluid that develops during OC progression, which contains a large mass of the tumor cells, represents a unique form of tumor environment. The floating malignant cells are capable of surviving and proliferating despite lacking immediate prox‐ imity to blood vessels presumably due to the permissive attributes of this environment. There are several indirect evidences to suggest that ascites alter drug resistance in tumor cells. Proteomic profiling of tumor cells from ascites before and after chemotherapy showed an increase in the activation of survival pathways such as Akt pathway (Davidson et al., 2006). Moreover, OC ascites attenuate TRAIL and drug-induced apoptosis *in vitro* (Lane et al., 2007; Lane et al., 2010a; Lane et al., 2010b). OC ascites contains significant levels of bioac‐ tive lipids such as lysophosphatidic acid (LPA), which exceed levels required to activate LPA receptors (Yamada et al., 2004; Lane et al., 2010a). LPA, one of the ligands of G-protein coupled receptors, has been shown to induce cell survival signaling pathways through dif‐ ferent mechanisms including PI3K/Akt activation and regulation of DR4 and c-FLIP (Tanyi et al., 2003; Kang et al., 2004; Ishdorj et al., 2008). Furthermore, LPA inhibits cisplatin-in‐ duced apoptosis (Tanyi et al., 2003). The role of LPA, as a component of ascites, in modulat‐ ing drug resistance in OC cells remains however uncertain. For example, the blockade of LPA cascade did not altered TRAIL-induced apoptosis in OC cells (Lane et al., 2010a) and incubation of OC cells with LPA did not protect them from TRAIL-induced apoptosis (Lane

A wide variety of cytokines can be measured in OC ascites and interleukin-6 (IL-6) and interleukin-8 (IL-8) are among the most abundant (Giuntoli et al., 2009; Lane et al., 2011; Matte et al., 2012). A number of studies have reported an association between serum lev‐

ingly, TRAIL resistant OVCAR3 cells remained sensitive to chemotherapeutic drugs.

cells (El-Gazzar et al., 2010b).

et al., 2010).

**6.3. Environment-mediated resistance**

Activation of the PI3K/Akt promotes cell survival and resistance to chemotherapy in OC cells (Fraser et al., 2008; Abedini et al., 2010). The constitutive activation of Akt in OC cell lines and primary tumor cells also promotes resistance to TRAIL (Goncharenko-Khaider et al., 2010). There is a close correlation between the activation of Akt in OC cells and the de‐ gree of resistance to TRAIL (Goncharenko-Khaider et al., 2010; Lane et al., 2010). The inhibi‐ tion of Akt phosphorylation reversed cellular resistance to TRAIL whereas the transfection of Akt in tumor cells with low Akt basal activity enhanced TRAIL resistance (Goncharenko-Khaider et al., 2010). Akt confers resistance, in part, by modulating TRAIL-induced Bid cleavage (Goncharenko-Khaider et al., 2010). The role of Akt in TRAIL resistance among OC cells is also supported by the observation that the inhibition of Akt activation by trastuzu‐ mab (Cuello et al., 2001b), an ErbB2 receptor inhibitor, or by a small molecule that inhibits hPEBP4 (Qiu et al., 2010), enhanced TRAIL-induced apoptosis.

TRAIL triggers changes in mitochondrial membrane permeability which results in the re‐ lease of pro-apoptotic proteins such as cytochrome *c* and SMAC/DIABLO from the mito‐ chondria. In a cohort of 75 patients, Mao et al. demonstrated decreased expression of SMAC/ DIABLO and increased expression of XIAP in OC compared to normal ovarian tissues (Mao et al., 2007). However, they observed no difference in SMAC/DIABLO and XIAP expression between TRAIL sensitive and resistant cell lines. To assess the biological relevance of these observations, they stably transfected TRAIL resistant OC cell lines with a SMAC/DIABLO expression vector and showed enhanced TRAIL-induced apoptosis in transfected cells. Simi‐ larly, the treatment of TRAIL resistant OC cells with a small molecule SMAC/DIABLO mim‐ ic enhanced TRAIL- and TRAIL R1 or R2 agonist-induced apoptosis (Petrucci et al., 2007). Others have found a lack of correlation between XIAP protein expression and TRAIL sensi‐ tivity (Goncharenko-Khaider et al., 2010). Furthermore, down-regulation of XIAP in TRAIL resistant OC cells failed to enhance TRAIL-induced apoptosis (Goncharenko-Khaider et al., 2010) suggesting that XIAP is not a major factor contributing to TRAIL resistance in OC.

In summary, intrinsic TRAIL resistance appears to be multi-factorial and can be influenced by the activation of survival pathways such as Akt. In this context, the identification of in‐ formative and validated biomarkers of TRAIL resistance will be important for selecting pa‐ tients and predicting the clinical outcome.

#### **6.2. Acquired resistance**

Acquired resistance is a mechanism by which tumor cells that were initially sensitive to a drug adapted to survive to prolonged exposure to this drug. Acquired drug resistance con‐ stitutes a major problem in the management of OC. This type of resistance is believed to be caused by sequential genetic alterations in tumor cells often associated with sub-lethal expo‐ sure to apoptosis-inducing drugs that eventually result in a therapy-resistant phenotype. For example, in an OC cell line model, resistance to the anti-TRAIL-R2 antibody TRA-8 was induced by repeated exposure to non-apoptosis-inducing doses of the antibody (Li et al., 2006). Interestingly, the apoptotic responses induced by TRAIL, a TRAIL-R1 agonist anti‐ body (2E12), and other apoptotic stimuli were not impaired. Lane et al. demonstrated that TRAIL acquired resistance was due to a rapid degradation of active caspase-3 subunits by the proteasome in the TRAIL resistant variant OC cells OVCAR3 (Lane et al., 2006). Interest‐ ingly, TRAIL resistant OVCAR3 cells remained sensitive to chemotherapeutic drugs.

One reassuring finding of these studies in OC and other in different tumor types is the fact that acquired TRAIL resistance does not confer cross-resistance to chemotherapeutic drugs such as cisplatin. In fact, combining standard chemotherapy with TRAIL treatment appears to be beneficial because treatment with platinum compounds upregulates the expression of TRAIL death receptors regardless of the p53 status which leads to increase apoptosis in OC cells (El-Gazzar et al., 2010b).

#### **6.3. Environment-mediated resistance**

FLIPL (through different means) enhances TRAIL-induced apoptosis in resistant OC cells (Lane et al., 2004; Clarke et al., 2007; Syed et al. 2007; Park et al., 2009). In addition, the knockdown of c-FLIPL inhibited human OC cell lines migratory phenotype in a TRAIL-de‐ pendent manner *in vitro* and inhibited the invasion of tumor cells into the peritoneal cavity

Activation of the PI3K/Akt promotes cell survival and resistance to chemotherapy in OC cells (Fraser et al., 2008; Abedini et al., 2010). The constitutive activation of Akt in OC cell lines and primary tumor cells also promotes resistance to TRAIL (Goncharenko-Khaider et al., 2010). There is a close correlation between the activation of Akt in OC cells and the de‐ gree of resistance to TRAIL (Goncharenko-Khaider et al., 2010; Lane et al., 2010). The inhibi‐ tion of Akt phosphorylation reversed cellular resistance to TRAIL whereas the transfection of Akt in tumor cells with low Akt basal activity enhanced TRAIL resistance (Goncharenko-Khaider et al., 2010). Akt confers resistance, in part, by modulating TRAIL-induced Bid cleavage (Goncharenko-Khaider et al., 2010). The role of Akt in TRAIL resistance among OC cells is also supported by the observation that the inhibition of Akt activation by trastuzu‐ mab (Cuello et al., 2001b), an ErbB2 receptor inhibitor, or by a small molecule that inhibits

TRAIL triggers changes in mitochondrial membrane permeability which results in the re‐ lease of pro-apoptotic proteins such as cytochrome *c* and SMAC/DIABLO from the mito‐ chondria. In a cohort of 75 patients, Mao et al. demonstrated decreased expression of SMAC/ DIABLO and increased expression of XIAP in OC compared to normal ovarian tissues (Mao et al., 2007). However, they observed no difference in SMAC/DIABLO and XIAP expression between TRAIL sensitive and resistant cell lines. To assess the biological relevance of these observations, they stably transfected TRAIL resistant OC cell lines with a SMAC/DIABLO expression vector and showed enhanced TRAIL-induced apoptosis in transfected cells. Simi‐ larly, the treatment of TRAIL resistant OC cells with a small molecule SMAC/DIABLO mim‐ ic enhanced TRAIL- and TRAIL R1 or R2 agonist-induced apoptosis (Petrucci et al., 2007). Others have found a lack of correlation between XIAP protein expression and TRAIL sensi‐ tivity (Goncharenko-Khaider et al., 2010). Furthermore, down-regulation of XIAP in TRAIL resistant OC cells failed to enhance TRAIL-induced apoptosis (Goncharenko-Khaider et al., 2010) suggesting that XIAP is not a major factor contributing to TRAIL resistance in OC.

In summary, intrinsic TRAIL resistance appears to be multi-factorial and can be influenced by the activation of survival pathways such as Akt. In this context, the identification of in‐ formative and validated biomarkers of TRAIL resistance will be important for selecting pa‐

Acquired resistance is a mechanism by which tumor cells that were initially sensitive to a drug adapted to survive to prolonged exposure to this drug. Acquired drug resistance con‐ stitutes a major problem in the management of OC. This type of resistance is believed to be caused by sequential genetic alterations in tumor cells often associated with sub-lethal expo‐ sure to apoptosis-inducing drugs that eventually result in a therapy-resistant phenotype.

hPEBP4 (Qiu et al., 2010), enhanced TRAIL-induced apoptosis.

tients and predicting the clinical outcome.

**6.2. Acquired resistance**

(El-Gazzar et al., 2010a).

232 Ovarian Cancer - A Clinical and Translational Update

Environment-mediated drug resistance (*de novo* resistance) is a form of resistance by which tumor cells are transiently protected from drug-induced apoptosis via the induction of sur‐ vival signaling pathways (Meads et al., 2009). Soluble factors in the tumor environment may engage cell surface receptor to activate survival pathways. Evidence is accumulating that the tumor environment affects both tumor progression and response to chemotherapy in OC. The accumulation of peritoneal fluid that develops during OC progression, which contains a large mass of the tumor cells, represents a unique form of tumor environment. The floating malignant cells are capable of surviving and proliferating despite lacking immediate prox‐ imity to blood vessels presumably due to the permissive attributes of this environment. There are several indirect evidences to suggest that ascites alter drug resistance in tumor cells. Proteomic profiling of tumor cells from ascites before and after chemotherapy showed an increase in the activation of survival pathways such as Akt pathway (Davidson et al., 2006). Moreover, OC ascites attenuate TRAIL and drug-induced apoptosis *in vitro* (Lane et al., 2007; Lane et al., 2010a; Lane et al., 2010b). OC ascites contains significant levels of bioac‐ tive lipids such as lysophosphatidic acid (LPA), which exceed levels required to activate LPA receptors (Yamada et al., 2004; Lane et al., 2010a). LPA, one of the ligands of G-protein coupled receptors, has been shown to induce cell survival signaling pathways through dif‐ ferent mechanisms including PI3K/Akt activation and regulation of DR4 and c-FLIP (Tanyi et al., 2003; Kang et al., 2004; Ishdorj et al., 2008). Furthermore, LPA inhibits cisplatin-in‐ duced apoptosis (Tanyi et al., 2003). The role of LPA, as a component of ascites, in modulat‐ ing drug resistance in OC cells remains however uncertain. For example, the blockade of LPA cascade did not altered TRAIL-induced apoptosis in OC cells (Lane et al., 2010a) and incubation of OC cells with LPA did not protect them from TRAIL-induced apoptosis (Lane et al., 2010).

A wide variety of cytokines can be measured in OC ascites and interleukin-6 (IL-6) and interleukin-8 (IL-8) are among the most abundant (Giuntoli et al., 2009; Lane et al., 2011; Matte et al., 2012). A number of studies have reported an association between serum lev‐ els of IL-6 and prognosis, where elevated levels correlated with a poor relapse-free and overall survival (Plante et al., 1994; Scambia et al., 1995; Tempfer et al., 1997). Interesting‐ ly, it was recently shown that elevated ascites levels of IL-6, but not IL-8, were an inde‐ pendent predictor of shorter progression-free survival (Lane et al., 2011). Whether IL-6 is a critical soluble factor in ascites-mediated TRAIL resistance is unclear but a recent study suggests that IL-6 may indeed be an important component of the tumor environment that support tumor growth (Kulbe et al., 2012). Recently, high levels of IL-10, OPG and leptin in ascites were found to correlate with shorter PFS (Matte et al., 2012). Furthermore, in this study, Il-10 neutralizing antibodies attenuated the protective effect of ascites against TRAIL-induced apoptosis suggesting that IL-10 is one of the factors in ascites that pro‐ mote ascites-induced TRAIL resistance.

ically (Table 1). These antibodies have a significantly increased half-life and consequently

**Name Targets Compagny Clinical stage**

TRAIL R1 and TRAIL R2

**8. Therapeutic potential of TRAIL agonistic agents in ovarian cancer:**

The anti-tumor activity of dulanermin has been extensively evaluated in preclinical models (Ashkenazi et al., 1999; Hylander et al., 2005; Pollack et al., 2001). Furthermore, preclinical *in vitro* studies have demonstrated that OC cell lines displayed variable sensitivity to recombi‐ nant human TRAIL (Cuello et al., 2001; Vignati et al., 2002; Siervo-Sassi et al., 2003; Lane et al., 2004). TRAIL-resistant cell lines usually remain sensitive to chemotherapy and converse‐ ly, cisplatin-resistant cell lines may be sensitive to TRAIL. Collectively, these results suggest that both platinum-sensitive and platinum-resistant OC are candidates for TRAIL-targeting therapy (Tomek et al., 2004). To increase cancer cell-directed toxicity of TRAIL, fusion pro‐ teins of rsTRAIL with target moiety to epidermal growth factor receptor (EGFR) have been developed and were shown to have superior pro-apoptotic activity compared to soluble TRAIL in tumor cells that expressed high levels of EGFR such as the OC cell line OVCAR3

Mapatumumab (TRM-1, HGS-ETR1) TRAIL R1 Human Genome

TRAIL R2 Genetech Phase II

Biological Significance of Apoptosis in Ovarian Cancer: TRAIL Therapeutic Targeting

TRAIL R2 Amgen Phase I/II

TRAIL R2 Human Genome

Sciences

Sciences

TRAIL R2 Daiichi Sankyo Phase I/II

TRAIL R2 Novartis Phase I

Amgen/Genetech Phase I/II

**development**

235

http://dx.doi.org/10.5772/53380

Phase I

Phase II

their bioavailability is increased at the tumor site.

Apomab/Drozitumab (PRO95780) (human monoclonal antibody agonist)

(human monoclonal antibody agonist)

(humanized monoclonal antibody agonist)

(humanized monoclonal antibody agonist)

**Table 1.** TRAIL-targeting agents

**Preclinical studies**

**8.1. Monotherapy**

(Bremer et al., 2008).

Conatumumab (AMG 655)

Lexatumumab (HGS-ETR2) (monoclonal antibody agonist)

Tigatuzumab (CS-1008)

Dulanermin (rs TRAIL)

LBY-135

The role of integrins in mediating cell proliferation, migration and survival in ovarian can‐ cer is well established (Carreiras et al., 1999; Cruet-Hennequart et al., 2003; Lane et al., 2008). Integrins transmit signals directly through ligation-dependent recruitment of non-receptor tyrosine kinases from the focal adhesion kinase (FAK) leading to the activation of several cell signaling pathways including the PI3K/Akt pathway (Stupack and Cheresh, 2002). Re‐ cently, it has been shown that the PI3K/Akt cascade is activated by OC ascites (Lane et al., 2010a). The ability of different ascites to induce Akt phosphorylation in tumor cells strongly correlates with their ability to inhibit TRAIL-induced apoptosis. The PI3K/Akt pathway most likely couples signals from ascites-activated cell surface receptors which regulate the expression and/or phosphorylation of apoptosis-regulating targets. Ascites-induced activa‐ tion of αvβ5 integrins leads to focal adhesion kinase (FAK) phosphorylation and FAK indu‐ ces the activation of Akt (Lane et al., 2010a). This leads to Akt-mediated up-regulation of c-FLIPs expression in ovarian cancer cells (Lane et al., 2007).

Collectively, these data support the role of ascites to promote resistance to TRAIL-induced apoptosis, at least *in vitro*. Whether this is relevant *in vivo* remains unclear for the moment. However, the prosurvival activity of ascites against TRAIL-induced apoptosis has been as‐ sociated with shorter PFS in women with OC suggesting that ascites-mediated resistance might be clinically relevant (Lane et al., 2010b).

#### **7. TRAIL targeting agents**

Different strategies have been used to activate the TRAIL signaling pathway in cancer thera‐ py. A variety of recombinant forms of soluble TRAIL have been developed and fused with different tags (Pitti et al., 1996; Schneider et al., 2000; Ganten et al., 2006). Major limitations however of recombinant soluble TRAIL (rsTRAIL) include the short half-life *in vivo* and rela‐ tive lack of specificity as rsTRAIL can also bind decoy receptors TRAIL R3 and TRAIL R4. Despite these potential limitations, rsTRAIL (dulanermin) has entered phase I and phase II clinical trials. Alternatively, various humanized TRAIL receptor agonist antibodies have been developed which target TRAIL R1 (Mapatumumab) or TRAIL R2 (Apomab, Conatu‐ mumab, Lexatumumab, Tigatuzumab and LBY-135), and are currently being evaluated clin‐ ically (Table 1). These antibodies have a significantly increased half-life and consequently their bioavailability is increased at the tumor site.


**Table 1.** TRAIL-targeting agents

els of IL-6 and prognosis, where elevated levels correlated with a poor relapse-free and overall survival (Plante et al., 1994; Scambia et al., 1995; Tempfer et al., 1997). Interesting‐ ly, it was recently shown that elevated ascites levels of IL-6, but not IL-8, were an inde‐ pendent predictor of shorter progression-free survival (Lane et al., 2011). Whether IL-6 is a critical soluble factor in ascites-mediated TRAIL resistance is unclear but a recent study suggests that IL-6 may indeed be an important component of the tumor environment that support tumor growth (Kulbe et al., 2012). Recently, high levels of IL-10, OPG and leptin in ascites were found to correlate with shorter PFS (Matte et al., 2012). Furthermore, in this study, Il-10 neutralizing antibodies attenuated the protective effect of ascites against TRAIL-induced apoptosis suggesting that IL-10 is one of the factors in ascites that pro‐

The role of integrins in mediating cell proliferation, migration and survival in ovarian can‐ cer is well established (Carreiras et al., 1999; Cruet-Hennequart et al., 2003; Lane et al., 2008). Integrins transmit signals directly through ligation-dependent recruitment of non-receptor tyrosine kinases from the focal adhesion kinase (FAK) leading to the activation of several cell signaling pathways including the PI3K/Akt pathway (Stupack and Cheresh, 2002). Re‐ cently, it has been shown that the PI3K/Akt cascade is activated by OC ascites (Lane et al., 2010a). The ability of different ascites to induce Akt phosphorylation in tumor cells strongly correlates with their ability to inhibit TRAIL-induced apoptosis. The PI3K/Akt pathway most likely couples signals from ascites-activated cell surface receptors which regulate the expression and/or phosphorylation of apoptosis-regulating targets. Ascites-induced activa‐ tion of αvβ5 integrins leads to focal adhesion kinase (FAK) phosphorylation and FAK indu‐ ces the activation of Akt (Lane et al., 2010a). This leads to Akt-mediated up-regulation of c-

Collectively, these data support the role of ascites to promote resistance to TRAIL-induced apoptosis, at least *in vitro*. Whether this is relevant *in vivo* remains unclear for the moment. However, the prosurvival activity of ascites against TRAIL-induced apoptosis has been as‐ sociated with shorter PFS in women with OC suggesting that ascites-mediated resistance

Different strategies have been used to activate the TRAIL signaling pathway in cancer thera‐ py. A variety of recombinant forms of soluble TRAIL have been developed and fused with different tags (Pitti et al., 1996; Schneider et al., 2000; Ganten et al., 2006). Major limitations however of recombinant soluble TRAIL (rsTRAIL) include the short half-life *in vivo* and rela‐ tive lack of specificity as rsTRAIL can also bind decoy receptors TRAIL R3 and TRAIL R4. Despite these potential limitations, rsTRAIL (dulanermin) has entered phase I and phase II clinical trials. Alternatively, various humanized TRAIL receptor agonist antibodies have been developed which target TRAIL R1 (Mapatumumab) or TRAIL R2 (Apomab, Conatu‐ mumab, Lexatumumab, Tigatuzumab and LBY-135), and are currently being evaluated clin‐

mote ascites-induced TRAIL resistance.

234 Ovarian Cancer - A Clinical and Translational Update

FLIPs expression in ovarian cancer cells (Lane et al., 2007).

might be clinically relevant (Lane et al., 2010b).

**7. TRAIL targeting agents**

### **8. Therapeutic potential of TRAIL agonistic agents in ovarian cancer: Preclinical studies**

#### **8.1. Monotherapy**

The anti-tumor activity of dulanermin has been extensively evaluated in preclinical models (Ashkenazi et al., 1999; Hylander et al., 2005; Pollack et al., 2001). Furthermore, preclinical *in vitro* studies have demonstrated that OC cell lines displayed variable sensitivity to recombi‐ nant human TRAIL (Cuello et al., 2001; Vignati et al., 2002; Siervo-Sassi et al., 2003; Lane et al., 2004). TRAIL-resistant cell lines usually remain sensitive to chemotherapy and converse‐ ly, cisplatin-resistant cell lines may be sensitive to TRAIL. Collectively, these results suggest that both platinum-sensitive and platinum-resistant OC are candidates for TRAIL-targeting therapy (Tomek et al., 2004). To increase cancer cell-directed toxicity of TRAIL, fusion pro‐ teins of rsTRAIL with target moiety to epidermal growth factor receptor (EGFR) have been developed and were shown to have superior pro-apoptotic activity compared to soluble TRAIL in tumor cells that expressed high levels of EGFR such as the OC cell line OVCAR3 (Bremer et al., 2008).

#### **8.2. Combination therapy**

Several studies demonstrated that the combination of TRAIL with cisplatin was more effi‐ cient than either molecule alone in various OC cell lines *in vitro* (Cuello et al., 2001; Vignati et al., 2002; Siervo-Sassi et al., 2003; Tomek et al., 2004; Liu et al., 2006). In a mouse model of OC, treatment with rhTRAIL-DR5 or rhTRAIL in combination with cisplatin significantly re‐ duced tumor growth compared to rhTRAIL-DR5 alone (97% and 85% reduction in the com‐ bination arms versus 63% reduction in the rhTRAIL-DR5 arm alone) (Duiker et al., 2009). In this study, the beneficial effect of combined treatment was related to the observation that cisplatin strongly enhanced TRAIL R2 surface expression. Similar to cisplatin, proteasome inhibitors and nelfinavir, an HIV protease inhibitor, up-regulate TRAIL R2 and enhance the sensitivity of ovarian cancer cells and tissue explants to an apoptosis-inducing TRAIL recep‐ tor antibody (Saulle et al., 2007; Brüning et al., 2008; Brüning et al., 2009; Pasquini et al., 2010). For example, mapatumumab (TRAIL R1 agonist) and lexatumumab (TRAIL R2 ago‐ nist) were more efficient than TRAIL to induce apoptosis in primary OC cells and enhanced apoptosis induced by the proteasome inhibitor bortezomid (Pasquini et al., 2010). Using a model of acquired cisplatin resistant cell lines, Duiker et al. showed that cisplatin enhances TRAIL-induced apoptosis in cisplatin-resistant ovarian cancer cells, and induction of cas‐ pase-8 protein expression is the key factor of TRAIL sensitization (Duiker et al., 2011). Estes et al. evaluated the cytotoxicity of TRAIL R2 agonist (TRA-8) in nineteen chemotherapy-na‐ ive primary ovarian tumor samples (stage III/IV) (Estes et al., 2007). Using a similar ex vivo model, increased cytotoxicity was observed when TRA-8 was used in combination with che‐ motherapeutic drugs (Frederick et al., 2009). The potential of TRA-8 was further evaluated in a xenograft mouse model of OC (Bevis et al., 2011). When used alone, TRA-8 produced only a modest benefit in terms of tumor growth inhibition. However, animals treated with the combination of carboplatin, docetaxel and TRA-8 demonstrated a better outcome when compared to carboplatin and docetaxel only.

**9. Clinical trials with TRAIL targeting agents in OC patients**

untreated, advanced-stage NSCLC Completed Phase II NCT00480831

Bevacizumab for first-line metastatic colorectal cancer Completed Phase I/II NCT00625651

first-line treatment of unresectable soft tissue sarcoma Completed Phase I/II NCT00626704

**Name Status**

A study of PRO95780 in patients with previously

A study of PRO95780 in combination with Rituximab in patients with NHL that has progressed following previous

A study of PRO95780 in combination with Cetuximab and Irinotecan chemotherapy or the FOLFIRI regimen with Bevacizumab in patients with previously treated

A study of PRO95780 administered in combination with the FOLFOX regimen and Bevacizumab in patients with previously untreated, locally advanced, recurrent, and

Phase I/II study of Conatumumab and Gemcitabine Hydrochloride followed by Conatumumab, Capecitabine, and 3-dimensional conformal radiotherapy in patients

A phase 1b/2 study of AMG 655 in combination with Paclitaxel and Carboplatin for the first-line treatment of

Phase 1b/2 study of AMG 655 with mFOLFOX6 and

Phase 1b/2 study of AMG 655 with Doxorubicin for the

with locally advanced pancreatic cancer

with OC.

**Apomab/Drozitumab**

Rituximab therapy

metastatic colorectal cancer

metastatic colorectal Cancer

**Conatumumab**

advanced NSCLC

A large number of phase I/II clinical trials have been undertaken with TRAIL targeting agents either as monotherapy or in combination with chemotherapeutic drugs in a wide range of solid and haematological malignancies (Table 2). For the purpose of this discussion, we have only considered clinical studies with TRAIL targeting agents that included patients

> **Clinical stage**

Biological Significance of Apoptosis in Ovarian Cancer: TRAIL Therapeutic Targeting

Completed Phase II NCT00517049

Completed Phase I NCT00497497

Completed Phase I NCT00851136

Completed Phase I/II NCT00534027

Phase I NCT01017822

Approved – not yet active

**Clinical Trials Identifier**

http://dx.doi.org/10.5772/53380

237

Because TRAIL cytotoxicity in OC cells relies on the activation of both the extrinsic and the intrinsic apoptosis pathways, the combination of TRAIL with pro-apoptotic proteins is of in‐ terest. For example, SMAC/DIABLO or LBW242, a SMAC/DIABLO mimic, sensitizes OC cell lines to the antitumor effects of TRAIL and anticancer drugs commonly used in clinic (Mao et al., 2007; Petrucci et al., 2007; Petrucci et al., 2012). These observations suggest that the LBW242 could be of value for the development of experimental strategies for treatment of ovarian cancer. Radicicol, an Hsp90 inhibitor, potentiate the apoptotic effect of TRAIL on ovarian carcinoma cell lines by increasing the activation of the caspase-8- and Bid-depend‐ ent pathway and the mitochondria-mediated apoptotic pathway, leading to caspase activa‐ tion (Kim et al., 2012).

The enhanced efficacy of TRAIL in combination with other agents in preclinical models is encouraging and suggests that combination therapies with TRAIL probably represent the best clinical option at this point. Because TRAIL resistance in OC can be induced by various pathways, a combination of molecules that targets critical steps in the TRAIL signaling cas‐ cade is likely to be the most efficient approach.

#### **9. Clinical trials with TRAIL targeting agents in OC patients**

**8.2. Combination therapy**

236 Ovarian Cancer - A Clinical and Translational Update

compared to carboplatin and docetaxel only.

cade is likely to be the most efficient approach.

tion (Kim et al., 2012).

Several studies demonstrated that the combination of TRAIL with cisplatin was more effi‐ cient than either molecule alone in various OC cell lines *in vitro* (Cuello et al., 2001; Vignati et al., 2002; Siervo-Sassi et al., 2003; Tomek et al., 2004; Liu et al., 2006). In a mouse model of OC, treatment with rhTRAIL-DR5 or rhTRAIL in combination with cisplatin significantly re‐ duced tumor growth compared to rhTRAIL-DR5 alone (97% and 85% reduction in the com‐ bination arms versus 63% reduction in the rhTRAIL-DR5 arm alone) (Duiker et al., 2009). In this study, the beneficial effect of combined treatment was related to the observation that cisplatin strongly enhanced TRAIL R2 surface expression. Similar to cisplatin, proteasome inhibitors and nelfinavir, an HIV protease inhibitor, up-regulate TRAIL R2 and enhance the sensitivity of ovarian cancer cells and tissue explants to an apoptosis-inducing TRAIL recep‐ tor antibody (Saulle et al., 2007; Brüning et al., 2008; Brüning et al., 2009; Pasquini et al., 2010). For example, mapatumumab (TRAIL R1 agonist) and lexatumumab (TRAIL R2 ago‐ nist) were more efficient than TRAIL to induce apoptosis in primary OC cells and enhanced apoptosis induced by the proteasome inhibitor bortezomid (Pasquini et al., 2010). Using a model of acquired cisplatin resistant cell lines, Duiker et al. showed that cisplatin enhances TRAIL-induced apoptosis in cisplatin-resistant ovarian cancer cells, and induction of cas‐ pase-8 protein expression is the key factor of TRAIL sensitization (Duiker et al., 2011). Estes et al. evaluated the cytotoxicity of TRAIL R2 agonist (TRA-8) in nineteen chemotherapy-na‐ ive primary ovarian tumor samples (stage III/IV) (Estes et al., 2007). Using a similar ex vivo model, increased cytotoxicity was observed when TRA-8 was used in combination with che‐ motherapeutic drugs (Frederick et al., 2009). The potential of TRA-8 was further evaluated in a xenograft mouse model of OC (Bevis et al., 2011). When used alone, TRA-8 produced only a modest benefit in terms of tumor growth inhibition. However, animals treated with the combination of carboplatin, docetaxel and TRA-8 demonstrated a better outcome when

Because TRAIL cytotoxicity in OC cells relies on the activation of both the extrinsic and the intrinsic apoptosis pathways, the combination of TRAIL with pro-apoptotic proteins is of in‐ terest. For example, SMAC/DIABLO or LBW242, a SMAC/DIABLO mimic, sensitizes OC cell lines to the antitumor effects of TRAIL and anticancer drugs commonly used in clinic (Mao et al., 2007; Petrucci et al., 2007; Petrucci et al., 2012). These observations suggest that the LBW242 could be of value for the development of experimental strategies for treatment of ovarian cancer. Radicicol, an Hsp90 inhibitor, potentiate the apoptotic effect of TRAIL on ovarian carcinoma cell lines by increasing the activation of the caspase-8- and Bid-depend‐ ent pathway and the mitochondria-mediated apoptotic pathway, leading to caspase activa‐

The enhanced efficacy of TRAIL in combination with other agents in preclinical models is encouraging and suggests that combination therapies with TRAIL probably represent the best clinical option at this point. Because TRAIL resistance in OC can be induced by various pathways, a combination of molecules that targets critical steps in the TRAIL signaling cas‐ A large number of phase I/II clinical trials have been undertaken with TRAIL targeting agents either as monotherapy or in combination with chemotherapeutic drugs in a wide range of solid and haematological malignancies (Table 2). For the purpose of this discussion, we have only considered clinical studies with TRAIL targeting agents that included patients with OC.



**Name Status**

Study of Mapatumumab in combination with Bortezomib (Velcade) and Bortezomib alone in subjects with relapsed

A Study of Mapatumumab in combination with Paclitaxel

Study of Mapatumumab in combination with Sorafenib

An imaging and pharmacodynamic trial of CS-1008 in

Open-label study of CS1008 for subjects with untreated

Combination chemotherapy with CS-1008 to treat

CS-1008 with Carboplatin/Paclitaxel in chemotherapy

CS1008- in combination with Sorafenib compared to

Abraxane with or without Tigatuzumab in patients with

Study of CS-1008 in combination with FOLFIRI in patients

Abbreviations: NHL, non Hodgkin lymphoma; NSCLC, non-small cell lung cancer

**Table 2.** Active or completed clinical trials with TRAIL targeting agents

Study of CS-1008 in patients with advanced solid malignancies and lymphomas (without leukemic

patients with metastatic colorectal cancer

and Carboplatin in Subjects With NSCLC Completed Phase II NCT00583830

in subjects with advanced hepatocellular carcinoma Completed Phase II NCT01258608

and unresectable pancreatic cancer Completed Phase II NCT00521404

ovarian cancer Completed Phase II NCT00945191

naive subjects with metastatic or unresectable NSCLC Completed Phase II NCT00991796

Sorafenib alone in subjects with advanced liver cancer Completed Phase II NCT01033240

metastatic, triple negative breast cancer Completed Phase II NCT01307891

who have failed other treatments Completed Phase I NCT01124630

TRAIL-based treatment strategies that entered clinical studies have included dulanermin. In a phase I study involving 71 patients with advanced or metastatic solid tumors or non-Hodgkin lymphoma (NHL), dulanermin appeared safe and well tolerated (Herbst et al., 2010). Partial response and stable disease were observed in 3% and 53% of patients respec‐ tively in this study. Additonal clinical studies with dulanermin in combination with other drugs have been performed most often in patients with lung cancer (Soria et al., 2010; Soria

or refractory multiple myeloma

**Tigatuzumab**

component)

et al., 2011).

**Clinical stage**

Biological Significance of Apoptosis in Ovarian Cancer: TRAIL Therapeutic Targeting

Completed Phase II NCT00315757

Active Phase I NCT01220999

Completed Phase I NCT00320827

**Clinical Trials Identifier**

http://dx.doi.org/10.5772/53380

239


**Table 2.** Active or completed clinical trials with TRAIL targeting agents

**Name Status**

colorectal cancer study Completed Phase I/II NCT00630786

refractory solid tumors Completed Phase I/II NCT00819169

With Bortezomib or Vorinostat Completed Phase I NCT00791011

subject with relapsed or refractory NSCLC Completed Phase II NCT00092924

subjects with relapsed or refractory NHL Completed Phase II NCT00094848

A study of AMG 655 or AMG 479 in combination with Gemcitabine for treatment of metastatic pancreatic

238 Ovarian Cancer - A Clinical and Translational Update

AMG655/Panitumumab combination in metastatic

AMG 655 in combination with AMG 479 in advanced,

Phase 2 safety & efficacy of FOLFIRI in combination with AMG 479 or AMG 655 vs FOLFIRI in KRAS-mutant

Phase 1b Lymphoma Study of AMG 655 in Combination

A study of AMG 951 [rhApo2L/TRAIL] in subjects with previously untreated NSCLC treated with chemotherapy

A study of Dulanermin administered in combination with Camptosar®/Erbitux® chemotherapy or FOLFIRI (with or without Bevacizumab) in subjects with previously treated

A study of Dulanermin administered in combination with the FOLFOX regimen and Bevacizumab in patients with previously untreated, locally advanced, recurrent, or

recombinant interferon gamma in pediatric patients with relapsed or refractory solid tumors or lymphoma

Mapatumumab, Cisplatin and radiotherapy for advanced

Study of TRM-1 (TRAIL-R1 monoclonal antibody) in

Study of TRM-1 (TRAIL-R1 monoclonal antibody) in

Phase I study of Lexatumumab with or without

metastatic colorectal carcinoma

cancer

**Dulanermin**

+/- Bevacizumab

metastatic colorectal cancer

metastatic colorectal cancer

**Lexatumumab**

Mapatumumab

cervical cancer

**Clinical stage**

Completed Phase I/II NCT00630552

Completed Phase II NCT00813605

Completed Phase II NCT00508625

Completed Phase I NCT00671372

Completed Phase I NCT00873756

Completed Phase I NCT00428272

Active Phase I/II NCT01088347

**Clinical Trials Identifier**

TRAIL-based treatment strategies that entered clinical studies have included dulanermin. In a phase I study involving 71 patients with advanced or metastatic solid tumors or non-Hodgkin lymphoma (NHL), dulanermin appeared safe and well tolerated (Herbst et al., 2010). Partial response and stable disease were observed in 3% and 53% of patients respec‐ tively in this study. Additonal clinical studies with dulanermin in combination with other drugs have been performed most often in patients with lung cancer (Soria et al., 2010; Soria et al., 2011).

Although there have been several published early-phase trials with antibody targeting TRAIL-R1 or TRAIL-R2, only two have included patients with OC. The feasibility of intrave‐ nous mapatumumab administration, as a single-agent, has been examined in a phase I phar‐ macokinetic and biological correlative study in patients with advanced solid malignancies refractory to standard therapy (Tolcher et al., 2007). Of the 49 patients enrolled in the study, two had advanced OC. Mapatumumab dosing ranged from 0.01 to 10 mg/kg and was ad‐ ministered every 2-4 weeks. Overall, mapatumumab was well tolerated and toxicity was generally limited to grade 1-2 events. No objective response was observed for mapatumu‐ mab in this unselected phase I study. Hotte et al. evaluated the safety and tolerability of ma‐ patumumab in a phase I clinical trial involving 41 patients with malignant solid tumors refractory to conventional therapy in which 22% of the patients had OC (Hotte et al., 2008). Mapatumumab was administered intravenously every 4 weeks and patients received a me‐ dian of 2 cycles (range, 1-33) with mapatumumab doses ranging from 0.01 to 20 mg/kg. The patient that received 33 cycles of mapatumumab had a diagnosis of borderline OC. She ex‐ perienced no cumulative toxicity. Indeed, mapatumumab was generally well tolerated and common adverse events included fatigue, hypotension, nausea and fever. No objective re‐ sponse was observed. Conatumumab (AMG 655), a TRAIL R2-specific antibody is currently being evaluated in patients with advanced refractory solid tumors that includes ovarian tu‐ mors in combination with ganitumab, a fully human monoclonal antibody against insulinlike growth factor receptor 1 (National Cancer Institute (NCI) Clinical Trials Identifier Number : NCT00819169).

Of the two studies published with mapatumumab in combination with chemotherapy, one included a patient with OC (primary peritoneal carcinoma) (Leong et al., 2009). A phase II using tigatuzumab (CS-1008), a humanized TRAIL-R2 antibody, in combination with pacli‐ taxel and carboplatin is underway (NCI Clinical Trials Identifier Number: NCT00945191).

**Figure 1.** Apoptotic pathways. Binding of TRAIL to death receptors (TRAIL R1, TRAIL R2) leads to the recruitment of the adaptor molecule, FADD. Pro-caspase-8 binds to FADD leading to DISC formation and resulting in its activation. Activated caspase-8 directly activates executioner caspases (caspase-3, -6, and -7) (type I cells) or cleaves Bid (type II cells). Translocation of the truncated Bid (tBid) to the mitochondria promotes the assembly of Bax-Bak oligomers and mitochondria outer membrane permeability changes. Cytochrome c is released into cytosol resulting in apoptosome assembly. Active caspase-9 then propagates a proteolytic cascade of effector caspases activation that leads to mor‐ phological hallmarks of apoptosis. Further cleavage of pro-caspase-8 by effector caspases generates a mitochondrial amplification loop that further enhances apoptosis. When FLIP levels are elevated in cells, caspase-8 preferentially re‐ cruits FLIP to form a caspase-8-FLIP heterodimer which does not trigger apoptosis. Chemotherapeutic drugs such as cisplatin cause DNA damage which is sensed by the ataxia telangiectasia mutated homolog (ATM) leading to the acti‐ vation of p53 dependent activation of genes such as PUMA and Noxa which can bind to anti-apoptotic proteins Bcl-2/

Biological Significance of Apoptosis in Ovarian Cancer: TRAIL Therapeutic Targeting

http://dx.doi.org/10.5772/53380

241

Bcl-XL thereby opposing their effect. This leads to mitochondrial permeabilization and activation.

\*Address all correspondence to: Alain.Piche@USherbrooke.ca

Santé, Université de Sherbrooke, Sherbrooke, Canada

Nadzeya Goncharenko-Khaider, Denis Lane, Isabelle Matte, Claudine Rancourt and

Département de Microbiologie et Infectiologie, Faculté de Médecine et des Sciences de la

**Author details**

Alain Piché\*

#### **10. Conclusions and future directions**

The inherent properties of TRAIL or its agonists offer a new targeted therapy for OC. Pre‐ clinical studies using TRAIL or its agonists have demonstrated the therapeutic potential of these molecules and formed the basis of ongoing phase I/II clinical trials. Although these treatments appear to be clinically well tolerated so far, intrinsic, acquired and environmentmediated resistance may limit the effectiveness of these approaches. However, the develop‐ ment of combination treatments appears to be capable of overcoming, at least in part, some of these limitations. As the search for more effective treatment for OC continues, the mor‐ bidity and mortality will hopefully improve. TRAIL treatment strategies have been used so far in the context of salvage treatment and the optimal patient population that will mostly benefit from these treatments remains to be defined. Although significant progress has been made in our understanding of the molecular basis of TRAIL resistance in OC, efforts should continue to further improve this knowledge as this will likely lead to the development of specific biomarkers of resistance and more efficient targeted therapies.

**Figure 1.** Apoptotic pathways. Binding of TRAIL to death receptors (TRAIL R1, TRAIL R2) leads to the recruitment of the adaptor molecule, FADD. Pro-caspase-8 binds to FADD leading to DISC formation and resulting in its activation. Activated caspase-8 directly activates executioner caspases (caspase-3, -6, and -7) (type I cells) or cleaves Bid (type II cells). Translocation of the truncated Bid (tBid) to the mitochondria promotes the assembly of Bax-Bak oligomers and mitochondria outer membrane permeability changes. Cytochrome c is released into cytosol resulting in apoptosome assembly. Active caspase-9 then propagates a proteolytic cascade of effector caspases activation that leads to mor‐ phological hallmarks of apoptosis. Further cleavage of pro-caspase-8 by effector caspases generates a mitochondrial amplification loop that further enhances apoptosis. When FLIP levels are elevated in cells, caspase-8 preferentially re‐ cruits FLIP to form a caspase-8-FLIP heterodimer which does not trigger apoptosis. Chemotherapeutic drugs such as cisplatin cause DNA damage which is sensed by the ataxia telangiectasia mutated homolog (ATM) leading to the acti‐ vation of p53 dependent activation of genes such as PUMA and Noxa which can bind to anti-apoptotic proteins Bcl-2/ Bcl-XL thereby opposing their effect. This leads to mitochondrial permeabilization and activation.

#### **Author details**

Although there have been several published early-phase trials with antibody targeting TRAIL-R1 or TRAIL-R2, only two have included patients with OC. The feasibility of intrave‐ nous mapatumumab administration, as a single-agent, has been examined in a phase I phar‐ macokinetic and biological correlative study in patients with advanced solid malignancies refractory to standard therapy (Tolcher et al., 2007). Of the 49 patients enrolled in the study, two had advanced OC. Mapatumumab dosing ranged from 0.01 to 10 mg/kg and was ad‐ ministered every 2-4 weeks. Overall, mapatumumab was well tolerated and toxicity was generally limited to grade 1-2 events. No objective response was observed for mapatumu‐ mab in this unselected phase I study. Hotte et al. evaluated the safety and tolerability of ma‐ patumumab in a phase I clinical trial involving 41 patients with malignant solid tumors refractory to conventional therapy in which 22% of the patients had OC (Hotte et al., 2008). Mapatumumab was administered intravenously every 4 weeks and patients received a me‐ dian of 2 cycles (range, 1-33) with mapatumumab doses ranging from 0.01 to 20 mg/kg. The patient that received 33 cycles of mapatumumab had a diagnosis of borderline OC. She ex‐ perienced no cumulative toxicity. Indeed, mapatumumab was generally well tolerated and common adverse events included fatigue, hypotension, nausea and fever. No objective re‐ sponse was observed. Conatumumab (AMG 655), a TRAIL R2-specific antibody is currently being evaluated in patients with advanced refractory solid tumors that includes ovarian tu‐ mors in combination with ganitumab, a fully human monoclonal antibody against insulinlike growth factor receptor 1 (National Cancer Institute (NCI) Clinical Trials Identifier

Of the two studies published with mapatumumab in combination with chemotherapy, one included a patient with OC (primary peritoneal carcinoma) (Leong et al., 2009). A phase II using tigatuzumab (CS-1008), a humanized TRAIL-R2 antibody, in combination with pacli‐ taxel and carboplatin is underway (NCI Clinical Trials Identifier Number: NCT00945191).

The inherent properties of TRAIL or its agonists offer a new targeted therapy for OC. Pre‐ clinical studies using TRAIL or its agonists have demonstrated the therapeutic potential of these molecules and formed the basis of ongoing phase I/II clinical trials. Although these treatments appear to be clinically well tolerated so far, intrinsic, acquired and environmentmediated resistance may limit the effectiveness of these approaches. However, the develop‐ ment of combination treatments appears to be capable of overcoming, at least in part, some of these limitations. As the search for more effective treatment for OC continues, the mor‐ bidity and mortality will hopefully improve. TRAIL treatment strategies have been used so far in the context of salvage treatment and the optimal patient population that will mostly benefit from these treatments remains to be defined. Although significant progress has been made in our understanding of the molecular basis of TRAIL resistance in OC, efforts should continue to further improve this knowledge as this will likely lead to the development of

specific biomarkers of resistance and more efficient targeted therapies.

Number : NCT00819169).

240 Ovarian Cancer - A Clinical and Translational Update

**10. Conclusions and future directions**

Nadzeya Goncharenko-Khaider, Denis Lane, Isabelle Matte, Claudine Rancourt and Alain Piché\*

\*Address all correspondence to: Alain.Piche@USherbrooke.ca

Département de Microbiologie et Infectiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Canada

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Biological Significance of Apoptosis in Ovarian Cancer: TRAIL Therapeutic Targeting

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**Chapter 12**

**Protein Kinase G-Iα Hyperactivation and VASP**

**Migration and Platinum Resistance**

Janica C. Wong and Ronald R. Fiscus

http://dx.doi.org/10.5772/53468

**1. Introduction**

Additional information is available at the end of the chapter

**Phosphorylation in Promoting Ovarian Cancer Cell**

Platinum-based drugs such as cisplatin (*cis*-diammine-dichloro-platinum, also commonly known as CDDP) have dominated the drug therapy of ovarian cancer during the past three decades [1]. Cisplatin interacts with DNA to form intrastrand crosslink adducts, and its mo‐ lecular mechanism involves regulation of p53 and the mitogen-activated protein kinase (MAPK) signaling pathway [2]. The phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway is crucial for regulation of survival and for progression and chemoresistance in ovarian cancer, leading to the development of new chemotherapeutic inhibitors targeting the PI3K/Akt pathway and the downstream serine/threonine protein kinase mTOR. [3]. In‐ hibition of PI3K pathway signaling using PI3K or mTOR inhibitors has been shown to sensi‐ tize ovarian cancer cell lines to the apoptosis-inducing effect of platinum compounds [4, 5]. In addition, activation of the PI3K/Akt/mTOR pathway in ovarian cancer cell lines contrib‐ utes to cisplatin resistance [6]. The anti-apoptotic, pro-angiogenic effects of PI3K/Akt/mTOR may be mediated, at least in part, through a downstream signaling pathway involving en‐ dogenous endothelial-form nitric oxide synthase (eNOS, also called NOS3), and subsequent‐ ly soluble guanylyl cyclase (sGC) and protein kinase G (PKG). Studies have shown that Akt activates eNOS by phosphorylating human eNOS at Ser1177 (equivalent to bovine eNOS at Ser1179), leading to an increase in nitric oxide (NO) production in endothelial cells [7, 8]. In the cases of vascular endothelial growth factor (VEGF) [9, 10], sphingosine 1-phosphate [11, 12], and estrogen [13, 14], there are vast evidences suggesting PI3K-activation of Akt is re‐ sponsible for regulating the phosphorylation and activation of eNOS. In bovine aortic endo‐ thelial cells, eNOS co-immunoprecipitates with Akt, indicating that the two enzymes associate *in vivo*, and Akt directly activates eNOS, increasing eNOS activity by 15-20 fold

> © 2013 Wong and Fiscus; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,


## **Protein Kinase G-Iα Hyperactivation and VASP Phosphorylation in Promoting Ovarian Cancer Cell Migration and Platinum Resistance**

Janica C. Wong and Ronald R. Fiscus

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53468

#### **1. Introduction**

[98] Tanyi JL, Hasegawa Y, Lapushin R, Morris AJ, Wolf JK, Berchuck A, Lu K, Smith DI, Kalli K, Hartmann LC, McCune K, Fishman D, Broaddus R, Cheng KW, Atkinson EN, Yamal JM, Bast RC, Felix EA, Newman RA, Mills GB. Role of decreased levels of lipid phosphate phosphatase-1 in accumulation of lysophosphatidic acid in ovarian

[99] Tempfer C, Zeisler H, Sliutz G, Haeusler G, Hanzal E, Kainz C. Serum evaluation of

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[101] Vignati S, Codegoni A, Polato F, Broggini M. TRAIL activity in human ovarian can‐ cer cells: potentiation of the action of cytotoxic drugs. Eur J Cancer 2002:38:177-183.

[102] Vitovski S, Phillips JS, Sayers J, Croucher PI. Investigating the interaction between osteoprotegerin and receptor activator of NF-KappaB or tumor-necrosis factor-relat‐ ed apoptosis-inducing ligand: evidence for a pivotal role for osteoprotegerin in regu‐

[103] Walczak H, Miller RE, Ariail K, Gliniak B, Griffith TS, Kubin M, Chin W, Jones J, Woodward A, Le T, Smith C, Smolak P, Goodwin RG, Rauch CT, Schuh JC, Lynch DH. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand

[104] Williams J, Lucas PC, Griffith KA, Choi M, Fogoros S, Hu YY, Liu JR. Expression of Bcl-xL in ovarian carcinoma is associated with chemoresistance and recurrent dis‐

[105] Yamada T, Sato K, Komachi M, Malchinkhuu E, Tobo M, Kimura T, Kuwabara A, Yanagita Y, Ikeya T, Tanahashi Y, Ogawa T, Ohwada S, Morishita Y, Ohta H, Im DS, Tamoto K, Tomura H, Okajima F. Lysophosphatidic acid (LPA) in malignant ascites stimulates motility of human pancreatic cancer cells through LPA1. J Biol Chem 2004;

[106] Youle RJ and Strasser A. The BCL-2 protein family: opposing activities that mediate

[107] Zhang L, Fang B. Mechanisms of resistance to TRAIL-induced apoptosis in cancer.

[108] Zhu H, Liu XW, Ding WJ, Xu DQ, Zhao YC, Lu W, He QJ, Yang B. Up-regulation of death receptor 4 and 5 by celastrol enhances the anti-cancer activity of TRAIL/

lating two distinct pathways. J Biol Chem 2007;282:31601-31609.

interleukin 6 in ovarian cancer patients. Gynecol Oncol 1997;66:27-30.

cancer. Clin Cancer Res 2003;9:3534-3545.

250 Ovarian Cancer - A Clinical and Translational Update

receptor-1. J Clin Oncol 2007;25:1390-1395.

in vivo. Nat Med 1999;5:157-163.

ease. Gynecol Oncol 2005;96:287-295.

cell death. Nat Rev Mol Cell Biol 2008;9:47-59.

Cancer Gene Ther 2005;12:228–37.

Apo-2L. Cancer Lett 2010;297:155-164.

279:6595-6605.

Platinum-based drugs such as cisplatin (*cis*-diammine-dichloro-platinum, also commonly known as CDDP) have dominated the drug therapy of ovarian cancer during the past three decades [1]. Cisplatin interacts with DNA to form intrastrand crosslink adducts, and its mo‐ lecular mechanism involves regulation of p53 and the mitogen-activated protein kinase (MAPK) signaling pathway [2]. The phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway is crucial for regulation of survival and for progression and chemoresistance in ovarian cancer, leading to the development of new chemotherapeutic inhibitors targeting the PI3K/Akt pathway and the downstream serine/threonine protein kinase mTOR. [3]. In‐ hibition of PI3K pathway signaling using PI3K or mTOR inhibitors has been shown to sensi‐ tize ovarian cancer cell lines to the apoptosis-inducing effect of platinum compounds [4, 5]. In addition, activation of the PI3K/Akt/mTOR pathway in ovarian cancer cell lines contrib‐ utes to cisplatin resistance [6]. The anti-apoptotic, pro-angiogenic effects of PI3K/Akt/mTOR may be mediated, at least in part, through a downstream signaling pathway involving en‐ dogenous endothelial-form nitric oxide synthase (eNOS, also called NOS3), and subsequent‐ ly soluble guanylyl cyclase (sGC) and protein kinase G (PKG). Studies have shown that Akt activates eNOS by phosphorylating human eNOS at Ser1177 (equivalent to bovine eNOS at Ser1179), leading to an increase in nitric oxide (NO) production in endothelial cells [7, 8]. In the cases of vascular endothelial growth factor (VEGF) [9, 10], sphingosine 1-phosphate [11, 12], and estrogen [13, 14], there are vast evidences suggesting PI3K-activation of Akt is re‐ sponsible for regulating the phosphorylation and activation of eNOS. In bovine aortic endo‐ thelial cells, eNOS co-immunoprecipitates with Akt, indicating that the two enzymes associate *in vivo*, and Akt directly activates eNOS, increasing eNOS activity by 15-20 fold

© 2013 Wong and Fiscus; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

[15]. This signaling pathway has been shown to play an essential role in promoting angio‐ genesis or tumor vascularization [16]. In a very recent study, microgravity stimulated tube formation and migration in human umbilical vein endothelial cells (HUVEC), and the proc‐ ess was mediated through the PI3K-Akt-eNOS signal pathway [17].

Our early studies of the NO/cyclic GMP (cGMP)/PKG signaling pathway have identified PKG as a key mediator of vasodilation and anti-hypertensive effects induced by NO as well as atrial natriuretic peptide (ANP) [18-21]. Recent studies from our laboratory have shown that the PKG-Iα splice variant of PKG, at basal or moderately elevated activity, plays an important cytoprotective role in preventing spontaneous apoptosis and promot‐ ing cell proliferation in many types of mammalian cells, including neural cells [22-27], human ovarian cancer cells [28-30], primary murine vascular smooth muscle cells [31] and murine bone marrow mesenchymal (stromal) stem cells [32]. Evidence from our lab‐ oratory suggested that basal activation of PKG-Iα leads to increased attachment of cells to the extracellular matrix and increased cell migration, shown in bone marrow-derived mesenchymal (stromal) stem cells [32]. We have identified certain intracellular proteins that are directly phosphorylated and functionally regulated by PKG-Iα, including 1) the apoptosis-regulating protein BAD [26], 2) vasodilator-stimulated phosphoprotein (VASP) [28, 31, 32], 3) the oncogenic tyrosine kinase c-Src [28, 33] and 4) the transcription factor cAMP responsive element binding protein (CREB) [24, 34], which may contribute to the exaggerated proliferation, enhanced chemoresistance and increased cell migration and in‐ vasion in ovarian cancer cells (Figure 1). Our recent studies have shown that cisplatin regulates the endogenous expression of nitric oxide synthases (NOSs) in human ovarian cancer cells, upregulating inducible nitric oxide synthase (iNOS, also called NOS2) ex‐ pression but dramatically downregulating the expression of eNOS and neural-form nitric oxide synthase (nNOS, also called NOS1), which is involved in determining cisplatin re‐ sistance in ovarian cancer cells [30]. Our studies show that the chemoresistance/cytopro‐ tective effects of endogenous eNOS involve hyperactivation of PKG-Iα in the ovarian cancer cells [28].

**Figure 1.** Model of the biological role of PKG-Iα in ovarian cancer cells illustrating the effects of growth factors (e.g. EGF), which stimulates both the PI3K/Akt pathway, enhancing eNOS activity and low-level NO generation and the ac‐ tivation of c-Src. The low, physiological levels of NO activate sGC, elevating cGMP levels that enhance the activation of PKG-Iα. PKG-Iα is further activated (hyperactivated) by the combined effects of cGMP allosteric stimulation and the tyrosine phosphorylation by c-Src. PKG-Iα phosphorylates several downstream proteins, including c-Src, Bad, CREB and VASP, leading to enhanced cell proliferation and cytoprotection, contributing to chemoresistance in ovarian cancer

Protein Kinase G-Iα Hyperactivation and VASP Phosphorylation in Promoting Ovarian Cancer Cell…

http://dx.doi.org/10.5772/53468

253

**2. Phosphorylation of the vasodilator-stimulated phosphoprotein (VASP)**

Vasodilator-stimulated phosphoprotein (VASP) was first described in 1987 as a protein phosphorylated in platelets in response to vasodilators such as sodium nitroprusside, nitro‐ glycerin and various prostaglandins that elevate cAMP and cGMP [35]. VASP belongs to the Ena/VASP family which includes VASP, Mena (mammalian enabled) and EVL (Ena VASPlike). The Ena/VASP family proteins function as anti-capping proteins [36, 37], regulating the actin cytoskeleton dynamics [38-42] and are therefore important for actin-based adhe‐ sion [43, 44], migration [45-47] and cell–cell interaction [48-50]. Many studies from others have suggested the involvement of VASP in invasion, angiogenesis and tumorigenesis. In an *in vitro* model of capillary morphogenesis using human umbilical vein endothelial cells (HUVECs) in three-dimensional collagen gels, the differentiated endothelial cells showed 2 to 3-fold increase in migration with increased VASP mRNA and protein expression [51]. A study on human placenta development showed that VASP may participate in vasculogene‐ sis and endothelial sprouting during placental vasculogenesis, and VASP expression was stimulated by vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) [52]. NIH-3T3 fibroblast deficient in VASP showed loss of contact inhibition, and continued cell division past confluence, while overproduction of VASP by transfection in NIH-3T3 fibro‐

**at Ser239 as a useful indicator of endogeneous PKG kinase activity**

cells and increased cell migration and invasion.

Studies from our laboratory suggest that PKG-Iα promotes proliferation in ovarian cancer cells, which involves the enhancement of the tyrosine kinase activity of c-Src [28], an onco‐ genic protein often overexpressed and/or hyperactivated in many types of cancer cells. We showed that PKG-Iα plays a key role in activating c-Src and promoting cell proliferation, us‐ ing the short interfering RNA (siRNA) or RNA interference (RNAi) technique, to knock‐ down the expression of PKG-Iα in ovarian cancer cells [28]. We found that epidermal growth factor (EGF)-induced activation of c-Src tyrosine kinase activity causes tyrosine phosphorylation of PKG-Iα, increasing the serine/threonine kinase activity of PKG-Iα and its growth-promoting effects in ovarian cancer cells [28]. Later, we have found that PKG-Iα directly phosphorylates c-Src at Ser17, which enhances the tyrosine kinase activity of c-Src in both *in vitro* and intact-cell experiments [33]. This novel interaction between PKG-Iα and c-Src causes reciprocal phosphorylation, which means PKG-Iα and c-Src phosphorylate each other, potentially setting up an "oncogenic reinforcement" resulting in exaggerated DNA synthesis and cell proliferation (Figure 1).

[15]. This signaling pathway has been shown to play an essential role in promoting angio‐ genesis or tumor vascularization [16]. In a very recent study, microgravity stimulated tube formation and migration in human umbilical vein endothelial cells (HUVEC), and the proc‐

Our early studies of the NO/cyclic GMP (cGMP)/PKG signaling pathway have identified PKG as a key mediator of vasodilation and anti-hypertensive effects induced by NO as well as atrial natriuretic peptide (ANP) [18-21]. Recent studies from our laboratory have shown that the PKG-Iα splice variant of PKG, at basal or moderately elevated activity, plays an important cytoprotective role in preventing spontaneous apoptosis and promot‐ ing cell proliferation in many types of mammalian cells, including neural cells [22-27], human ovarian cancer cells [28-30], primary murine vascular smooth muscle cells [31] and murine bone marrow mesenchymal (stromal) stem cells [32]. Evidence from our lab‐ oratory suggested that basal activation of PKG-Iα leads to increased attachment of cells to the extracellular matrix and increased cell migration, shown in bone marrow-derived mesenchymal (stromal) stem cells [32]. We have identified certain intracellular proteins that are directly phosphorylated and functionally regulated by PKG-Iα, including 1) the apoptosis-regulating protein BAD [26], 2) vasodilator-stimulated phosphoprotein (VASP) [28, 31, 32], 3) the oncogenic tyrosine kinase c-Src [28, 33] and 4) the transcription factor cAMP responsive element binding protein (CREB) [24, 34], which may contribute to the exaggerated proliferation, enhanced chemoresistance and increased cell migration and in‐ vasion in ovarian cancer cells (Figure 1). Our recent studies have shown that cisplatin regulates the endogenous expression of nitric oxide synthases (NOSs) in human ovarian cancer cells, upregulating inducible nitric oxide synthase (iNOS, also called NOS2) ex‐ pression but dramatically downregulating the expression of eNOS and neural-form nitric oxide synthase (nNOS, also called NOS1), which is involved in determining cisplatin re‐ sistance in ovarian cancer cells [30]. Our studies show that the chemoresistance/cytopro‐ tective effects of endogenous eNOS involve hyperactivation of PKG-Iα in the ovarian

Studies from our laboratory suggest that PKG-Iα promotes proliferation in ovarian cancer cells, which involves the enhancement of the tyrosine kinase activity of c-Src [28], an onco‐ genic protein often overexpressed and/or hyperactivated in many types of cancer cells. We showed that PKG-Iα plays a key role in activating c-Src and promoting cell proliferation, us‐ ing the short interfering RNA (siRNA) or RNA interference (RNAi) technique, to knock‐ down the expression of PKG-Iα in ovarian cancer cells [28]. We found that epidermal growth factor (EGF)-induced activation of c-Src tyrosine kinase activity causes tyrosine phosphorylation of PKG-Iα, increasing the serine/threonine kinase activity of PKG-Iα and its growth-promoting effects in ovarian cancer cells [28]. Later, we have found that PKG-Iα directly phosphorylates c-Src at Ser17, which enhances the tyrosine kinase activity of c-Src in both *in vitro* and intact-cell experiments [33]. This novel interaction between PKG-Iα and c-Src causes reciprocal phosphorylation, which means PKG-Iα and c-Src phosphorylate each other, potentially setting up an "oncogenic reinforcement" resulting in exaggerated DNA

ess was mediated through the PI3K-Akt-eNOS signal pathway [17].

252 Ovarian Cancer - A Clinical and Translational Update

cancer cells [28].

synthesis and cell proliferation (Figure 1).

**Figure 1.** Model of the biological role of PKG-Iα in ovarian cancer cells illustrating the effects of growth factors (e.g. EGF), which stimulates both the PI3K/Akt pathway, enhancing eNOS activity and low-level NO generation and the ac‐ tivation of c-Src. The low, physiological levels of NO activate sGC, elevating cGMP levels that enhance the activation of PKG-Iα. PKG-Iα is further activated (hyperactivated) by the combined effects of cGMP allosteric stimulation and the tyrosine phosphorylation by c-Src. PKG-Iα phosphorylates several downstream proteins, including c-Src, Bad, CREB and VASP, leading to enhanced cell proliferation and cytoprotection, contributing to chemoresistance in ovarian cancer cells and increased cell migration and invasion.

#### **2. Phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) at Ser239 as a useful indicator of endogeneous PKG kinase activity**

Vasodilator-stimulated phosphoprotein (VASP) was first described in 1987 as a protein phosphorylated in platelets in response to vasodilators such as sodium nitroprusside, nitro‐ glycerin and various prostaglandins that elevate cAMP and cGMP [35]. VASP belongs to the Ena/VASP family which includes VASP, Mena (mammalian enabled) and EVL (Ena VASPlike). The Ena/VASP family proteins function as anti-capping proteins [36, 37], regulating the actin cytoskeleton dynamics [38-42] and are therefore important for actin-based adhe‐ sion [43, 44], migration [45-47] and cell–cell interaction [48-50]. Many studies from others have suggested the involvement of VASP in invasion, angiogenesis and tumorigenesis. In an *in vitro* model of capillary morphogenesis using human umbilical vein endothelial cells (HUVECs) in three-dimensional collagen gels, the differentiated endothelial cells showed 2 to 3-fold increase in migration with increased VASP mRNA and protein expression [51]. A study on human placenta development showed that VASP may participate in vasculogene‐ sis and endothelial sprouting during placental vasculogenesis, and VASP expression was stimulated by vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) [52]. NIH-3T3 fibroblast deficient in VASP showed loss of contact inhibition, and continued cell division past confluence, while overproduction of VASP by transfection in NIH-3T3 fibro‐ blasts resulted in neoplastic transformation, suggesting a role of VASP in tumorigenesis and/or cancer progression [53]. In human osteocarcinoma specimans, higher VASP expres‐ sion was associated with metastasis and increased migration, and VASP expression was regulated by Rac1 [54]. In lung adenocarcinomas tissues, VASP expression was increased compared to normal lung tissues, and was significantly increased with more advanced tu‐ mor stage [55]. Elevated VASP expression was also reported in human breast cancer tissues [56] and was implicated on invasion and migration in breast cancer cells involving the Rac1 pathway [57]. Moreover, it was shown that in mice lacking VASP, melanoma growth was greatly impaired [58]. In gastric cancer cells, VASP was upregulated by epidermal growth factor (EGF) and promoted migration and invasion. Using microRNA (miRNA) expression profiling of the paired normal/tumor gastric tissues, the same group identified miR-610 as a novel miRNA regulated by EGF that targets VASP in gastric cancer cells [59].

tivity in ovarian cancer cells. The EGF-stimulated increase in PKG-Iα kinase activity (indicated by VASP Ser239 phosphorylation) was blocked by both SKI-1 and SU6656 (SFK inhibitors). Us‐ ing the specific PKG-Iα kinase inhibitor DT-2 and small interfering RNA (siRNA) PKG-Iα gene knockdown, we showed that the inhibition of endogenous PKG-Iα kinase activity reduced VASP Ser239 phosphorylation and DNA synthesis rate in ovarian cancer cells [28]. New data from our laboratory show that the knockdown of PKG-Iα expression inhibits the EGF-stimu‐ lated increases in VASP Ser239 phosphorylation and Src/SFK autophosphorylation at the equivalent of Tyr416 (the phosphorylation site for activating the tyrosine kinase activity) in

Protein Kinase G-Iα Hyperactivation and VASP Phosphorylation in Promoting Ovarian Cancer Cell…

http://dx.doi.org/10.5772/53468

255

**Figure 2.** A, EGF (10 ng/mL) elevated VASP Ser239 phosphorylation and Src Tyr416 phosphorylation in A2780cp cells, assessed by Western blot analysis. Gene knockdown of PKG-Iα by PKG-Iα-siRNA partially inhibited the basal VASP phosphorylation and Src/SFK autophosphorylation and completely inhibited the EGF-stimulated increases in VASP phosphorylation and Src/SFK autophosphorylation. The Western blot shown is representative of four experiments. B, Quantification of the relative levels of VASP and Src phosphorylation from Western blot. Bar graphs show mean ± SEM from four independent experiments. \*, P< 0.05, compared with no EGF control; #, P<0.05; ##, P<0.01; ###, P < 0.001,

**3. Role of PKG in invasion/migration in A2780cp ovarian cancer cells**

The role of NO/cGMP/PKG pathway in invasion/migration in cancer cells is largely unknown. However, a significant number of reports have shown that the NO/cGMP/PKG pathway plays a key role in endothelial cell migration and angiogenesis, involving the downstream activation of the mitogen-activated protein kinase (MAPK) family. It has been shown that NO promotes endo‐ thelial cell migration and neovascularization by activating the PI3K/Akt signaling pathway in a PKG-dependent manner [70]. Activation of the NO/cGMP/PKG pathway also promoted endo‐ thelial cell angiogenesis and increased extracellular signal regulated kinase 1/2 (ERK1/2) and p38 phosphorylation [71, 72], which were blocked by soluble guanylyl cyclase (sGC) inhibitor, 1H-

compared with negative control.

A2780cp (cisplatin-resistant, mutated p53) ovarian cancer cells (see Figure 2 below).

VASP has been reported to be phosphorylated by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) [35, 60]. VASP was found to be primarily present as a 46 kDa membrane-associated protein in its dephosphorylated form in platelets, and VASP is converted to an apparent 50 kDa phosphoprotein upon phosphorylation, as observed on West‐ ern blot [61, 62]. VASP contains three phosphorylation sites, Ser157, Ser239 and Thr274, all of which can be phosphorylated by either PKA or PKG [63]. Ser157 is the preferred site of phos‐ phorylation for PKA, Ser239 is the preferred site for PKG, and Ser157 was the site responsible for the phosphorylation-induced mobility shift of VASP on Western blots [63]. Because it was well-characterized that VASP at Ser239 is the preferred phosphorylation site for PKG *in vitro* and in mammalian cells, VASP phosphorylation at Ser239 has been proposed to be a useful in‐ dicator of endogenous PKG kinase activity [61, 63, 64]. In fact, VASP at Ser239 was shown to be a functional biomarker of endothelial nitric oxide/cyclic GMP signaling [65], and could be used to indicate defective nitric oxide/cGMP signaling and endothelial dysfunction [66]. In colon cancer cells, VASP Ser239 phosphorylation was used as a biomarker for the action of the anticancer drug Exisulind, an inhibitor of type-5 phosphodiesterase (PDE-5) that elevates cGMP and stimulates PKG activation, and that constitutively activated mutants of PKG resulted in di‐ rect *in vivo* phosphorylation of VASP Ser239 [67].

We had shown that the endogenous NO/cGMP signaling pathway in ovarian cancer cells caus‐ es a constitutive downregulation of p53 protein expression, which likely contributes to the che‐ moresistance and exaggerated cell proliferation in these cells [29]. Furthermore, we have previously identified that PKG-Iα is the predominant isoform of PKG in both OV2008 (cispla‐ tin-sensitive, wild-type p53) and A2780cp (cisplatin-resistant, mutated p53) ovarian cancer cells as determined by Western blot analysis as well as using the new, ultrasensitive Nano‐ Pro100 capillary electrophoresis-based nano-fluidic protein analysis system [28, 68, 69]. Our more recent data now show that the chemoresistance and exaggerated cell proliferation are likely mediated by the constitutive hyperactivation of PKG-Iα (reflected in the high levels of VASP phosphorylation at Ser239) in ovarian cancer cells, and that the PKG-Iα is already acti‐ vated to approximately 90% of maximal activity, described in our previous book chapter [68]. In our recent study, epidermal growth factor (EGF)-induced activation of Src family kinase (SFK) was found to tyrosine-phosphorylate PKG-Iα increasing its serine/threonine kinase ac‐ tivity in ovarian cancer cells. The EGF-stimulated increase in PKG-Iα kinase activity (indicated by VASP Ser239 phosphorylation) was blocked by both SKI-1 and SU6656 (SFK inhibitors). Us‐ ing the specific PKG-Iα kinase inhibitor DT-2 and small interfering RNA (siRNA) PKG-Iα gene knockdown, we showed that the inhibition of endogenous PKG-Iα kinase activity reduced VASP Ser239 phosphorylation and DNA synthesis rate in ovarian cancer cells [28]. New data from our laboratory show that the knockdown of PKG-Iα expression inhibits the EGF-stimu‐ lated increases in VASP Ser239 phosphorylation and Src/SFK autophosphorylation at the equivalent of Tyr416 (the phosphorylation site for activating the tyrosine kinase activity) in A2780cp (cisplatin-resistant, mutated p53) ovarian cancer cells (see Figure 2 below).

blasts resulted in neoplastic transformation, suggesting a role of VASP in tumorigenesis and/or cancer progression [53]. In human osteocarcinoma specimans, higher VASP expres‐ sion was associated with metastasis and increased migration, and VASP expression was regulated by Rac1 [54]. In lung adenocarcinomas tissues, VASP expression was increased compared to normal lung tissues, and was significantly increased with more advanced tu‐ mor stage [55]. Elevated VASP expression was also reported in human breast cancer tissues [56] and was implicated on invasion and migration in breast cancer cells involving the Rac1 pathway [57]. Moreover, it was shown that in mice lacking VASP, melanoma growth was greatly impaired [58]. In gastric cancer cells, VASP was upregulated by epidermal growth factor (EGF) and promoted migration and invasion. Using microRNA (miRNA) expression profiling of the paired normal/tumor gastric tissues, the same group identified miR-610 as a

VASP has been reported to be phosphorylated by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) [35, 60]. VASP was found to be primarily present as a 46 kDa membrane-associated protein in its dephosphorylated form in platelets, and VASP is converted to an apparent 50 kDa phosphoprotein upon phosphorylation, as observed on West‐ ern blot [61, 62]. VASP contains three phosphorylation sites, Ser157, Ser239 and Thr274, all of which can be phosphorylated by either PKA or PKG [63]. Ser157 is the preferred site of phos‐ phorylation for PKA, Ser239 is the preferred site for PKG, and Ser157 was the site responsible for the phosphorylation-induced mobility shift of VASP on Western blots [63]. Because it was well-characterized that VASP at Ser239 is the preferred phosphorylation site for PKG *in vitro* and in mammalian cells, VASP phosphorylation at Ser239 has been proposed to be a useful in‐ dicator of endogenous PKG kinase activity [61, 63, 64]. In fact, VASP at Ser239 was shown to be a functional biomarker of endothelial nitric oxide/cyclic GMP signaling [65], and could be used to indicate defective nitric oxide/cGMP signaling and endothelial dysfunction [66]. In colon cancer cells, VASP Ser239 phosphorylation was used as a biomarker for the action of the anticancer drug Exisulind, an inhibitor of type-5 phosphodiesterase (PDE-5) that elevates cGMP and stimulates PKG activation, and that constitutively activated mutants of PKG resulted in di‐

We had shown that the endogenous NO/cGMP signaling pathway in ovarian cancer cells caus‐ es a constitutive downregulation of p53 protein expression, which likely contributes to the che‐ moresistance and exaggerated cell proliferation in these cells [29]. Furthermore, we have previously identified that PKG-Iα is the predominant isoform of PKG in both OV2008 (cispla‐ tin-sensitive, wild-type p53) and A2780cp (cisplatin-resistant, mutated p53) ovarian cancer cells as determined by Western blot analysis as well as using the new, ultrasensitive Nano‐ Pro100 capillary electrophoresis-based nano-fluidic protein analysis system [28, 68, 69]. Our more recent data now show that the chemoresistance and exaggerated cell proliferation are likely mediated by the constitutive hyperactivation of PKG-Iα (reflected in the high levels of VASP phosphorylation at Ser239) in ovarian cancer cells, and that the PKG-Iα is already acti‐ vated to approximately 90% of maximal activity, described in our previous book chapter [68]. In our recent study, epidermal growth factor (EGF)-induced activation of Src family kinase (SFK) was found to tyrosine-phosphorylate PKG-Iα increasing its serine/threonine kinase ac‐

novel miRNA regulated by EGF that targets VASP in gastric cancer cells [59].

rect *in vivo* phosphorylation of VASP Ser239 [67].

254 Ovarian Cancer - A Clinical and Translational Update

**Figure 2.** A, EGF (10 ng/mL) elevated VASP Ser239 phosphorylation and Src Tyr416 phosphorylation in A2780cp cells, assessed by Western blot analysis. Gene knockdown of PKG-Iα by PKG-Iα-siRNA partially inhibited the basal VASP phosphorylation and Src/SFK autophosphorylation and completely inhibited the EGF-stimulated increases in VASP phosphorylation and Src/SFK autophosphorylation. The Western blot shown is representative of four experiments. B, Quantification of the relative levels of VASP and Src phosphorylation from Western blot. Bar graphs show mean ± SEM from four independent experiments. \*, P< 0.05, compared with no EGF control; #, P<0.05; ##, P<0.01; ###, P < 0.001, compared with negative control.

#### **3. Role of PKG in invasion/migration in A2780cp ovarian cancer cells**

The role of NO/cGMP/PKG pathway in invasion/migration in cancer cells is largely unknown. However, a significant number of reports have shown that the NO/cGMP/PKG pathway plays a key role in endothelial cell migration and angiogenesis, involving the downstream activation of the mitogen-activated protein kinase (MAPK) family. It has been shown that NO promotes endo‐ thelial cell migration and neovascularization by activating the PI3K/Akt signaling pathway in a PKG-dependent manner [70]. Activation of the NO/cGMP/PKG pathway also promoted endo‐ thelial cell angiogenesis and increased extracellular signal regulated kinase 1/2 (ERK1/2) and p38 phosphorylation [71, 72], which were blocked by soluble guanylyl cyclase (sGC) inhibitor, 1H- [1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), or PKG inhibitor DT-3 [73-75]. Moreover, the mitogenic effect of vascular endothelial growth factor (VEGF) on endothelial cells appears to be mediated by endogenous NO (from eNOS) and cGMP, which results in PKG activation and PKGmediated downstream stimulation of MEK and ERK [76, 77]. Although it has not yet been report‐ ed which isoform of PKG is involved in the multiple pro-angiogenesis responses of endothelial cells, our recent studies suggest that endothelial cells express predominantly the PKG-Iα isoform (unpublished observations by J.C. Wong and R.R. Fiscus), which likely mediates the stimulation of downstream growth-promoting and pro-angiogenesis pathways in endothelial cells.

As stated above, we have previously determined that PKG-Iα is the predominant isoform in A2780cp ovarian cancer cells [30, 68, 69]. To study whether PKG-Iα plays a role in cell mi‐ gration/invasion in ovarian cancer cells, we performed experiments using small interfering RNA (siRNA) gene knockdown against PKG-Iα in transwell migration studies. Figure 3 shows that siRNA gene knockdown of PKG-Iα dramatically decreases no EGF as well as EGF-stimulated cell migration (as reflected by the quantity of migrated cells at the bottom of the transwell, stained with crystal violet) in A2780cp cisplatin-resistant ovarian cancer cells. These data confirm the role of endogenous PKG-Iα activity, potentially via VASP Ser239

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257

**Figure 3.** PKG-Iα siRNA gene knockdown in A2780cp cells decreased both basal and EGF-stimulated cell migration as‐ sessed by *in vitro* cell migration (invasion) assay. Migration of cells was assessed using transwells (Corning) with 8 µM pore polycarbonated inserts, coated with growth factor-reduced matrigel (BD Bioscience). The upper chamber contained 4 x

 cells and the lower chamber contained 0.6 ml of complete medium with or without EGF. Migration through the mem‐ brane was determined after 24 h of incubation at 37°C. Cells remaining on the topside of the transwell membrane were re‐

moved using a cotton swab, and cells migrated to bottom were stained with 0.5% crystal violet.

104

phosphorylation, in promoting cell migration/invasion in ovarian cancer.

Interestingly, in colon cancer cells, recent studies showed that activation of PKG inhibited cell mi‐ gration [78], and cGMP-dependent VASP phosphorylation suppressed the number and length of locomotory (filopodia) and invasive (invadopodia) actin-based organelles [79], suggesting a role of VASP Ser239 in invasion and migration. Our studies suggest that the opposite roles of PKG in regulating apoptosis, proliferation and migration reported by others are likely dependent on cell type, growth conditions (presence of different growth factors), as well as the differential ex‐ pression of PKG-Iα and PKG-Iβ isoforms. The two splice variants of PKG-I, PKG-Iα and PKG-Iβ, are activated by different concentration ranges of NO and are localized to different subcellular lo‐ cations within cells. Therefore, the two PKG-I isoforms can phosphorylate different sets of down‐ stream target proteins and can mediate completely different biological responses. The very different biological roles of the two PKG-I isoforms are reviewed in further detail elsewhere in an‐ other recent book chapter from our laboratory [68]. For example, PKG-Iα (Kact = 0.1 µM by cGMP allosteric activation) is activated at low, physiological levels of NO, whereas PKG-Iβ is activated at higher, pathological levels of NO and requires at least 10-times higher levels of cGMP for acti‐ vation (Kact = 1 µM) [68, 72, 80, 81].

In our hypothesis, the PKG-Iα and PKG-Iβ isoforms mediate opposite biological effects on cell proliferation and apoptosis, based on observations in two types of cells that express one isoform of PKG-I or the other. Our studies have shown that human ovarian cancer cells express predomi‐ nantly the PKG-Iα isoform, and that the activation of this kinase by endogenous low-level NO (0.01 – 1 nM), generated by endogenous eNOS and nNOS, would selectively activate the PKG-Iα isoform within ovarian cancer cells from our laboratory, promoting DNA synthesis/cell prolifer‐ ation and suppressing apoptosis, thus contributing to chemoresistance [28, 30, 68]. Studies in our laboratory, using both normal and malignant cells, including vascular smooth muscle cells, bone marrow-derived mesenchymal (stromal) stem cells and neuroblastoma cells, have suggested that a major role of the low-level-NO/cGMP/PKG-Iα signaling pathway is to protect these cells against the toxic/pro-apoptotic effects of high-level NO, as might occur during inflammation and exposure of cells to pro-inflammatory cytokines [22, 24, 25, 31, 32]. In contrast, based in part on published data from the laboratories of Weinstein and Thompson, it appears that when PKG-Iβ is activated by the higher levels of NO, the growth-inhibitory and pro-apoptotic effects of PKG-Iβ predominate over the growth-stimulatory and anti-apoptotic effects mediated by PKG-Iα. Their laboratories have shown that in colon cancer cells, PKG-Iβ is the predominant PKG-I isoform ex‐ pressed. Upon activation, PKG-Iβ phosphorylates two downstream target proteins, β-catenin and MEKK1, resulting in inhibition of cell proliferation and induction of apoptosis [78, 82, 83].

As stated above, we have previously determined that PKG-Iα is the predominant isoform in A2780cp ovarian cancer cells [30, 68, 69]. To study whether PKG-Iα plays a role in cell mi‐ gration/invasion in ovarian cancer cells, we performed experiments using small interfering RNA (siRNA) gene knockdown against PKG-Iα in transwell migration studies. Figure 3 shows that siRNA gene knockdown of PKG-Iα dramatically decreases no EGF as well as EGF-stimulated cell migration (as reflected by the quantity of migrated cells at the bottom of the transwell, stained with crystal violet) in A2780cp cisplatin-resistant ovarian cancer cells. These data confirm the role of endogenous PKG-Iα activity, potentially via VASP Ser239 phosphorylation, in promoting cell migration/invasion in ovarian cancer.

[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), or PKG inhibitor DT-3 [73-75]. Moreover, the mitogenic effect of vascular endothelial growth factor (VEGF) on endothelial cells appears to be mediated by endogenous NO (from eNOS) and cGMP, which results in PKG activation and PKGmediated downstream stimulation of MEK and ERK [76, 77]. Although it has not yet been report‐ ed which isoform of PKG is involved in the multiple pro-angiogenesis responses of endothelial cells, our recent studies suggest that endothelial cells express predominantly the PKG-Iα isoform (unpublished observations by J.C. Wong and R.R. Fiscus), which likely mediates the stimulation

of downstream growth-promoting and pro-angiogenesis pathways in endothelial cells.

vation (Kact = 1 µM) [68, 72, 80, 81].

256 Ovarian Cancer - A Clinical and Translational Update

Interestingly, in colon cancer cells, recent studies showed that activation of PKG inhibited cell mi‐ gration [78], and cGMP-dependent VASP phosphorylation suppressed the number and length of locomotory (filopodia) and invasive (invadopodia) actin-based organelles [79], suggesting a role of VASP Ser239 in invasion and migration. Our studies suggest that the opposite roles of PKG in regulating apoptosis, proliferation and migration reported by others are likely dependent on cell type, growth conditions (presence of different growth factors), as well as the differential ex‐ pression of PKG-Iα and PKG-Iβ isoforms. The two splice variants of PKG-I, PKG-Iα and PKG-Iβ, are activated by different concentration ranges of NO and are localized to different subcellular lo‐ cations within cells. Therefore, the two PKG-I isoforms can phosphorylate different sets of down‐ stream target proteins and can mediate completely different biological responses. The very different biological roles of the two PKG-I isoforms are reviewed in further detail elsewhere in an‐ other recent book chapter from our laboratory [68]. For example, PKG-Iα (Kact = 0.1 µM by cGMP allosteric activation) is activated at low, physiological levels of NO, whereas PKG-Iβ is activated at higher, pathological levels of NO and requires at least 10-times higher levels of cGMP for acti‐

In our hypothesis, the PKG-Iα and PKG-Iβ isoforms mediate opposite biological effects on cell proliferation and apoptosis, based on observations in two types of cells that express one isoform of PKG-I or the other. Our studies have shown that human ovarian cancer cells express predomi‐ nantly the PKG-Iα isoform, and that the activation of this kinase by endogenous low-level NO (0.01 – 1 nM), generated by endogenous eNOS and nNOS, would selectively activate the PKG-Iα isoform within ovarian cancer cells from our laboratory, promoting DNA synthesis/cell prolifer‐ ation and suppressing apoptosis, thus contributing to chemoresistance [28, 30, 68]. Studies in our laboratory, using both normal and malignant cells, including vascular smooth muscle cells, bone marrow-derived mesenchymal (stromal) stem cells and neuroblastoma cells, have suggested that a major role of the low-level-NO/cGMP/PKG-Iα signaling pathway is to protect these cells against the toxic/pro-apoptotic effects of high-level NO, as might occur during inflammation and exposure of cells to pro-inflammatory cytokines [22, 24, 25, 31, 32]. In contrast, based in part on published data from the laboratories of Weinstein and Thompson, it appears that when PKG-Iβ is activated by the higher levels of NO, the growth-inhibitory and pro-apoptotic effects of PKG-Iβ predominate over the growth-stimulatory and anti-apoptotic effects mediated by PKG-Iα. Their laboratories have shown that in colon cancer cells, PKG-Iβ is the predominant PKG-I isoform ex‐ pressed. Upon activation, PKG-Iβ phosphorylates two downstream target proteins, β-catenin and MEKK1, resulting in inhibition of cell proliferation and induction of apoptosis [78, 82, 83].

**Figure 3.** PKG-Iα siRNA gene knockdown in A2780cp cells decreased both basal and EGF-stimulated cell migration as‐ sessed by *in vitro* cell migration (invasion) assay. Migration of cells was assessed using transwells (Corning) with 8 µM pore polycarbonated inserts, coated with growth factor-reduced matrigel (BD Bioscience). The upper chamber contained 4 x 104 cells and the lower chamber contained 0.6 ml of complete medium with or without EGF. Migration through the mem‐ brane was determined after 24 h of incubation at 37°C. Cells remaining on the topside of the transwell membrane were re‐ moved using a cotton swab, and cells migrated to bottom were stained with 0.5% crystal violet.

### **4. Inhibition of the PKG-Iα signaling pathway enhances sensitivity of ovarian cancer cells to cisplatin-induced apoptosis – Potential involvement of cAMP-response-element-binding protein (CREB) and inhibitor of apoptosis proteins (IAPs)**

cGMP caused significant protection against cisplatin-induced apoptosis, even at higher concentrations of cisplatin. Interestingly, when the same treatments were used on PKG-Iα knockdown cells, the cytoprotective effects of 8-Br-cGMP against cisplatin-induced apopto‐ sis was completely abolished, confirming that the cytoprotection (chemoresistance) was

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259

To investigate whether such synergism occurs in ovarian cancer cells, we tested the com‐ bined treatment of the specific PKG-Iα kinase inhibitor, DT-2, and cisplatin in the A2780cp cisplatin-resistant ovarian cancer cell line. Our new preliminary data presented in this book chapter (illustrated in Figure 4) verified the synergistic effects of DT-2 and cisplatin. Figure 4 shows the level of apoptosis in A2780cp cells after a 24-hr co-treatment of DT-2 (5 or 10 µM) and cisplatin (2 µM). The Cell Death Detection ELISAPLUS assay (Roche Applied Science), based on quantitative sandwich-enzyme-immunoassay-principle with monoclonal antibod‐ ies directed against DNA and histones, were used to quantify apoptotic fragments. DT-2 (5 µM) or cisplatin (2 µM) alone did not cause significant increase in apoptosis. However, com‐ bined treatment of DT-2 (5 or 10 µM) and cisplatin (2 µM) significantly (###P<0.001) in‐

**Figure 4.** Synergistic effect of DT-2 with cisplatin in A2780cp human ovarian cancer cells. Combined treatment of DT-2 (5 or 10 µM) and cisplatin (2 µM) significantly (###P<0.001) increased apoptosis, compared to cisplatin (2 µM) alone. \*\*P<0.01, \*\*\*P<0.001, compared to no DT-2 control. Statistical analysis was performed by one-way ANOVA, fol‐ lowed by Newman-Keuls Multiple Comparison Test using GraphPad (PRISM software). Results were expressed as the

mediated by PKG-Iα [34].

creased apoptosis, showing a synergistic effect.

mean ± SEM of at least six different samples.

Platinum-based drugs such as cisplatin have dominated the drug therapy of ovarian cancer during the past three decades [1]. Cisplatin interacts with DNA to form intrastrand crosslink adducts, and its molecular mechanism involves regulation of p53 and the mitogen-activated protein kinase (MAPK) signaling pathway [2]. It has been shown that inhibition of ERK1/2 activation with the mitogen-activated protein kinase/ERK kinase 1 (MEK1) inhibitor PD98059 resulted in decreased p53 protein half-life and diminished accumulation of p53 protein during exposure to cisplatin [84]. Our data have shown that human ovarian cancer cells express all of the key components of the NO/cGMP/PKG signaling pathway, including all three isoforms of NOSs, thus providing an endogenous source of NO [30]. Furthermore, ovarian cancer cells continuously produce NO at low physiological levels, activating the heme-dependent soluble guanylyl cyclase (sGC) [29], elevating cGMP levels sufficiently enough to cause continuous high-level activation of PKG [28]. Our data suggested that such basal sGC/cGMP activity regulates p53 expression, and promotes cell survival in part through regulation of caspase-3 [29] (now thought to be mediated by downstream hyperac‐ tivation of PKG-Iα).

Cisplatin is also widely employed in chemotherapy on treating solid tumors such as lung cancer. Recently, we showed that, in NCI-H460 and A549 non-small cell lung cancer (NSCLC) cells, PKG-Iα phosphorylates cAMP-response-element-binding protein (CREB) at Ser133 [34]. CREB was first shown to be phosphorylated by PKG *in vitro* by Colbran et al., which showed that PKG effectively phosphorylates CREB at Ser133, although at a slower rate compared to PKA [85]. Interestingly, NO was shown to regulate the c-fos promoter in‐ volving soluble guanylyl cyclase (sGC) and PKG [86] in a CREB-dependent manner [87]. They also showed that transfection of PKG in baby hamster kidney (BHK) cells activated the c-fos promoter [88], which required nuclear translocation of PKG and phosphorylation of CREB at Ser133 by PKG [87, 89, 90]. In our recent study, inhibition of the sGC/PKG-Iα sig‐ naling pathway by ODQ (sGC inhibitor), DT-2 (PKG-Iα kinase inhibitor) and PKG-Iα-siR‐ NA gene knockdown showed that PKG-Iα kinase activity is necessary for maintaining higher levels of CREB phosphorylation at Ser133 and the protein expression of certain inhib‐ itor of apoptosis proteins (IAPs), specifically c-IAP1, livin and survivin, as well as the antiapoptotic Bcl-2 family member Mcl-1, preventing spontaneous apoptosis and promoting colony formation [34]. In the same study, we discovered that DT-2 and cisplatin have a syn‐ ergistic effect on the induction of apoptosis, with DT-2 dramatically enhancing the proapoptotic effects of cisplatin in A549 cells (a NSCLC cell line that requires higher levels of cisplatin to induce apoptosis). We also showed that prior activation of PKG-Iα by 8-bromocGMP (8-Br-GMP), a cell-permeable cGMP analog that directly activates PKG [22, 24], has cytoprotective effects against cisplatin. PKG-Iα activity stimulated by 8-Br-cGMP was re‐ flected by increased VASP phosphorylation at Ser239. Pretreatment of A549 cells with 8-BrcGMP caused significant protection against cisplatin-induced apoptosis, even at higher concentrations of cisplatin. Interestingly, when the same treatments were used on PKG-Iα knockdown cells, the cytoprotective effects of 8-Br-cGMP against cisplatin-induced apopto‐ sis was completely abolished, confirming that the cytoprotection (chemoresistance) was mediated by PKG-Iα [34].

**4. Inhibition of the PKG-Iα signaling pathway enhances sensitivity of**

**involvement of cAMP-response-element-binding protein (CREB) and**

Platinum-based drugs such as cisplatin have dominated the drug therapy of ovarian cancer during the past three decades [1]. Cisplatin interacts with DNA to form intrastrand crosslink adducts, and its molecular mechanism involves regulation of p53 and the mitogen-activated protein kinase (MAPK) signaling pathway [2]. It has been shown that inhibition of ERK1/2 activation with the mitogen-activated protein kinase/ERK kinase 1 (MEK1) inhibitor PD98059 resulted in decreased p53 protein half-life and diminished accumulation of p53 protein during exposure to cisplatin [84]. Our data have shown that human ovarian cancer cells express all of the key components of the NO/cGMP/PKG signaling pathway, including all three isoforms of NOSs, thus providing an endogenous source of NO [30]. Furthermore, ovarian cancer cells continuously produce NO at low physiological levels, activating the heme-dependent soluble guanylyl cyclase (sGC) [29], elevating cGMP levels sufficiently enough to cause continuous high-level activation of PKG [28]. Our data suggested that such basal sGC/cGMP activity regulates p53 expression, and promotes cell survival in part through regulation of caspase-3 [29] (now thought to be mediated by downstream hyperac‐

Cisplatin is also widely employed in chemotherapy on treating solid tumors such as lung cancer. Recently, we showed that, in NCI-H460 and A549 non-small cell lung cancer (NSCLC) cells, PKG-Iα phosphorylates cAMP-response-element-binding protein (CREB) at Ser133 [34]. CREB was first shown to be phosphorylated by PKG *in vitro* by Colbran et al., which showed that PKG effectively phosphorylates CREB at Ser133, although at a slower rate compared to PKA [85]. Interestingly, NO was shown to regulate the c-fos promoter in‐ volving soluble guanylyl cyclase (sGC) and PKG [86] in a CREB-dependent manner [87]. They also showed that transfection of PKG in baby hamster kidney (BHK) cells activated the c-fos promoter [88], which required nuclear translocation of PKG and phosphorylation of CREB at Ser133 by PKG [87, 89, 90]. In our recent study, inhibition of the sGC/PKG-Iα sig‐ naling pathway by ODQ (sGC inhibitor), DT-2 (PKG-Iα kinase inhibitor) and PKG-Iα-siR‐ NA gene knockdown showed that PKG-Iα kinase activity is necessary for maintaining higher levels of CREB phosphorylation at Ser133 and the protein expression of certain inhib‐ itor of apoptosis proteins (IAPs), specifically c-IAP1, livin and survivin, as well as the antiapoptotic Bcl-2 family member Mcl-1, preventing spontaneous apoptosis and promoting colony formation [34]. In the same study, we discovered that DT-2 and cisplatin have a syn‐ ergistic effect on the induction of apoptosis, with DT-2 dramatically enhancing the proapoptotic effects of cisplatin in A549 cells (a NSCLC cell line that requires higher levels of cisplatin to induce apoptosis). We also showed that prior activation of PKG-Iα by 8-bromocGMP (8-Br-GMP), a cell-permeable cGMP analog that directly activates PKG [22, 24], has cytoprotective effects against cisplatin. PKG-Iα activity stimulated by 8-Br-cGMP was re‐ flected by increased VASP phosphorylation at Ser239. Pretreatment of A549 cells with 8-Br-

**ovarian cancer cells to cisplatin-induced apoptosis – Potential**

**inhibitor of apoptosis proteins (IAPs)**

258 Ovarian Cancer - A Clinical and Translational Update

tivation of PKG-Iα).

To investigate whether such synergism occurs in ovarian cancer cells, we tested the com‐ bined treatment of the specific PKG-Iα kinase inhibitor, DT-2, and cisplatin in the A2780cp cisplatin-resistant ovarian cancer cell line. Our new preliminary data presented in this book chapter (illustrated in Figure 4) verified the synergistic effects of DT-2 and cisplatin. Figure 4 shows the level of apoptosis in A2780cp cells after a 24-hr co-treatment of DT-2 (5 or 10 µM) and cisplatin (2 µM). The Cell Death Detection ELISAPLUS assay (Roche Applied Science), based on quantitative sandwich-enzyme-immunoassay-principle with monoclonal antibod‐ ies directed against DNA and histones, were used to quantify apoptotic fragments. DT-2 (5 µM) or cisplatin (2 µM) alone did not cause significant increase in apoptosis. However, com‐ bined treatment of DT-2 (5 or 10 µM) and cisplatin (2 µM) significantly (###P<0.001) in‐ creased apoptosis, showing a synergistic effect.

**Figure 4.** Synergistic effect of DT-2 with cisplatin in A2780cp human ovarian cancer cells. Combined treatment of DT-2 (5 or 10 µM) and cisplatin (2 µM) significantly (###P<0.001) increased apoptosis, compared to cisplatin (2 µM) alone. \*\*P<0.01, \*\*\*P<0.001, compared to no DT-2 control. Statistical analysis was performed by one-way ANOVA, fol‐ lowed by Newman-Keuls Multiple Comparison Test using GraphPad (PRISM software). Results were expressed as the mean ± SEM of at least six different samples.

Based on our study of the roles of sGC/PKG-Iα/CREB/IAPs in cisplatin resistant non-small lung cancer cells, we have proposed the anti-apoptotic role of PKG-Iα observed in A2780cp cells is likely mediated through PKG-Iα downstream phosphorylation of CREB at Ser133 and activation of certain IAPs. IAPs have been shown to regulate apoptosis and tumorigene‐ sis [91]. Although how CREB regulates apoptosis through IAPs is largely unknown, it was shown that CREB phosphorylation is a key event in the induction of certain IAPs, c-IAP2 and livin, via multiple protein kinases, PKA, ERK1/2 and p38 MAPK, in colon cancer cells [92, 93]. In ovarian cancer cells, X-linked inhibitor of apoptosis protein (XIAP) has been shown to control ovarian tumor growth and regulate Akt activity and caspase-3 in cisplatininduced apoptosis [94-96], and the ability of cisplatin to down-regulate XIAP may be an im‐ portant determinant of chemosensitivity [97]. Down-regulation of XIAP sensitized cells to cisplatin in the presence of wild-type p53, and both XIAP and Akt modulated cisplatin sen‐ sitivity individually but that XIAP required Akt for its full function [98]. Inhibition of PI3K/Akt/mTOR signaling has been shown to activate apoptosis and inhibit migration and invasion in ovarian cancer cells [3, 4, 99-104]. Furthermore, inhibition of PI3K pathway sig‐ naling using PI3K or mTOR inhibitors has been shown to sensitize ovarian cancer cell lines to induction of apoptosis by platinum compounds [4, 5]. Several recent evidences have sug‐ gested that such effects involve the matrix-metalloproteinases (MMPs) [105-107], which are zinc-dependent endopeptidases capable of degradation of extracellular matrix proteins.

dependent on endogenous PKG-Iα kinase activity, contributing to decreased caspase-3 ac‐

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261

**Figure 5.** Cellular model of the involvement of the NO/cGMP/PKG-Iα signaling pathway in promoting chemoresist‐ ance, tumor growth and angiogenesis in ovarian cancer. A special role of VASP phosphorylation at Ser239 may con‐

As illustrated in the model of Figure 5, we have also identified an important role of c-Src/PKG-Iα interaction in promoting DNA synthesis and cell proliferation in human ovarian cancer cells. Previous studies have shown that PKG-I binds Raf-1 and promotes downstream activa‐ tion of MEK and ERK1/2 in endothelial cells [76]. In ovarian cancer cells, we proposed that PKG-Iα binds to Raf-1 at the internal surface of the plasma membrane, bringing PKG-Iα in close proximity to one of its downstream target proteins c-Src. This leads to downstream acti‐ vation of the Raf-1/MEK/ERK signaling pathway, promoting cell proliferation. We found that PKG-Iα directly phosphorylates c-Src at Ser17, which enhances the tyrosine kinase activity of c-Src in both *in vitro* and intact-cell experiments [33]. Our recent studies have shown a clear role of the PKG-Iα-mediated phosphorylation of c-Src at Ser17 in preventing apoptosis and pro‐ moting proliferation, attachment and migration in the mesothelioma and NSCLC cells. It is very likely that a similar PKG-Iα catalyzed phosphorylation of c-Src at Ser17 occurs in human ovarian cancer cells, which can explain the dependence of the c-Src activation by EGF on the presence of PKG-Iα [28]. Epidermal growth factor (EGF)-induced activation of c-Src tyrosine

tivity and inhibition of apoptosis [26].

tribute to enhanced ovarian cancer cell migration and invasion.

#### **5. Overall model of NO/cGMP/PKG-Iα signaling pathway in ovarian cancer**

Figure 5 illustrates our overall model showing the involvement of the NO/cGMP/PKG-Iα pathway in promoting cell proliferation and suppressing apoptosis in human ovarian cancer cells, which would contribute to enhanced tumor growth and chemoresistance. Our early studies of the NO/cGMP/PKG pathway have identified PKG as a key mediator of vasodila‐ tion and anti-hypertensive effects induced by NO as well as atrial natriuretic peptide (ANP) [18-21]. Recent studies from our laboratory have shown that basal or moderately elevated PKG-Iα activity plays a cytoprotective role in preventing spontaneous apoptosis and pro‐ moting cell proliferation in many types of mammalian cells, including neural cells [22-27], human ovarian cancer cells [28-30], primary murine vascular smooth muscle cells [31] and murine bone marrow stromal cells [32]. We found that murine bone marrow-derived mesen‐ chymal (stromal) stem cells endogenously produced ANP and that basal NO/cGMP/PKG-Iα activity and autocrine ANP/cGMP/PKG-Iα activity are necessary for preserving cell survival and promoting cell proliferation and migration in the OP9 bone marrow stromal cell line [32]. Recently, we have identified certain intracellular proteins phosphorylated by PKG-Iα, including BAD [26], vasodilator-stimulated phosphoprotein (VASP) [28, 31, 32], c-Src [28] and cAMP responsive element binding protein (CREB) [34]. We have recently shown that PKG-Iα directly phosphorylates BAD at Ser155, using *in vitro* experiments, and have further shown that a large part of the Ser155 phosphorylation of BAD within neuroblastoma cells is dependent on endogenous PKG-Iα kinase activity, contributing to decreased caspase-3 ac‐ tivity and inhibition of apoptosis [26].

Based on our study of the roles of sGC/PKG-Iα/CREB/IAPs in cisplatin resistant non-small lung cancer cells, we have proposed the anti-apoptotic role of PKG-Iα observed in A2780cp cells is likely mediated through PKG-Iα downstream phosphorylation of CREB at Ser133 and activation of certain IAPs. IAPs have been shown to regulate apoptosis and tumorigene‐ sis [91]. Although how CREB regulates apoptosis through IAPs is largely unknown, it was shown that CREB phosphorylation is a key event in the induction of certain IAPs, c-IAP2 and livin, via multiple protein kinases, PKA, ERK1/2 and p38 MAPK, in colon cancer cells [92, 93]. In ovarian cancer cells, X-linked inhibitor of apoptosis protein (XIAP) has been shown to control ovarian tumor growth and regulate Akt activity and caspase-3 in cisplatininduced apoptosis [94-96], and the ability of cisplatin to down-regulate XIAP may be an im‐ portant determinant of chemosensitivity [97]. Down-regulation of XIAP sensitized cells to cisplatin in the presence of wild-type p53, and both XIAP and Akt modulated cisplatin sen‐ sitivity individually but that XIAP required Akt for its full function [98]. Inhibition of PI3K/Akt/mTOR signaling has been shown to activate apoptosis and inhibit migration and invasion in ovarian cancer cells [3, 4, 99-104]. Furthermore, inhibition of PI3K pathway sig‐ naling using PI3K or mTOR inhibitors has been shown to sensitize ovarian cancer cell lines to induction of apoptosis by platinum compounds [4, 5]. Several recent evidences have sug‐ gested that such effects involve the matrix-metalloproteinases (MMPs) [105-107], which are zinc-dependent endopeptidases capable of degradation of extracellular matrix proteins.

**5. Overall model of NO/cGMP/PKG-Iα signaling pathway in ovarian**

Figure 5 illustrates our overall model showing the involvement of the NO/cGMP/PKG-Iα pathway in promoting cell proliferation and suppressing apoptosis in human ovarian cancer cells, which would contribute to enhanced tumor growth and chemoresistance. Our early studies of the NO/cGMP/PKG pathway have identified PKG as a key mediator of vasodila‐ tion and anti-hypertensive effects induced by NO as well as atrial natriuretic peptide (ANP) [18-21]. Recent studies from our laboratory have shown that basal or moderately elevated PKG-Iα activity plays a cytoprotective role in preventing spontaneous apoptosis and pro‐ moting cell proliferation in many types of mammalian cells, including neural cells [22-27], human ovarian cancer cells [28-30], primary murine vascular smooth muscle cells [31] and murine bone marrow stromal cells [32]. We found that murine bone marrow-derived mesen‐ chymal (stromal) stem cells endogenously produced ANP and that basal NO/cGMP/PKG-Iα activity and autocrine ANP/cGMP/PKG-Iα activity are necessary for preserving cell survival and promoting cell proliferation and migration in the OP9 bone marrow stromal cell line [32]. Recently, we have identified certain intracellular proteins phosphorylated by PKG-Iα, including BAD [26], vasodilator-stimulated phosphoprotein (VASP) [28, 31, 32], c-Src [28] and cAMP responsive element binding protein (CREB) [34]. We have recently shown that PKG-Iα directly phosphorylates BAD at Ser155, using *in vitro* experiments, and have further shown that a large part of the Ser155 phosphorylation of BAD within neuroblastoma cells is

**cancer**

260 Ovarian Cancer - A Clinical and Translational Update

**Figure 5.** Cellular model of the involvement of the NO/cGMP/PKG-Iα signaling pathway in promoting chemoresist‐ ance, tumor growth and angiogenesis in ovarian cancer. A special role of VASP phosphorylation at Ser239 may con‐ tribute to enhanced ovarian cancer cell migration and invasion.

As illustrated in the model of Figure 5, we have also identified an important role of c-Src/PKG-Iα interaction in promoting DNA synthesis and cell proliferation in human ovarian cancer cells. Previous studies have shown that PKG-I binds Raf-1 and promotes downstream activa‐ tion of MEK and ERK1/2 in endothelial cells [76]. In ovarian cancer cells, we proposed that PKG-Iα binds to Raf-1 at the internal surface of the plasma membrane, bringing PKG-Iα in close proximity to one of its downstream target proteins c-Src. This leads to downstream acti‐ vation of the Raf-1/MEK/ERK signaling pathway, promoting cell proliferation. We found that PKG-Iα directly phosphorylates c-Src at Ser17, which enhances the tyrosine kinase activity of c-Src in both *in vitro* and intact-cell experiments [33]. Our recent studies have shown a clear role of the PKG-Iα-mediated phosphorylation of c-Src at Ser17 in preventing apoptosis and pro‐ moting proliferation, attachment and migration in the mesothelioma and NSCLC cells. It is very likely that a similar PKG-Iα catalyzed phosphorylation of c-Src at Ser17 occurs in human ovarian cancer cells, which can explain the dependence of the c-Src activation by EGF on the presence of PKG-Iα [28]. Epidermal growth factor (EGF)-induced activation of c-Src tyrosine kinase activity was found to cause tyrosine phosphorylation of PKG-Iα, increasing the serine/ threonine kinase activity of PKG-Iα (indicated by phosphorylation of the PKG substrate VASP at Ser239) and its growth-promoting effects in ovarian cancer cells [28]. In human ovarian can‐ cer cells, the c-Src-mediated tyrosine-phosphorylation of the EGF receptor was found to be highly dependent on PKG-Iα kinase activity [28].

**7. Conclusions**

**Acknowledgements**

**Author details**

**References**

10;25(20):2938-43.

cogene. 2003 Oct 20;22(47):7265-79.

Nevada, USA, awarded to Dr. Fiscus.

Janica C. Wong1,2,3 and Ronald R. Fiscus1,3

Health Sciences, Henderson, Nevada, and South Jordan, USA

2 Department of Chemistry, University of Nevada, Las Vegas, Nevada, USA

The NO/cGMP/PKG-Iα pathway and the downstream phosphorylation of the actin-fila‐ ment/focal-adhesion-regulating protein VASP at Ser239 appear to promote migration/inva‐ sion and the downstream phosphorylation of BAD at ser155, CREB at ser133 and c-Src at ser17 appear to promote DNA synthesis, cell proliferation and platinum resistance in ovari‐ an cancer cells. The unique features of this signaling pathway in ovarian cancer cells may provide a novel therapeutic target for disrupting tumor growth and the metastasis and sec‐

Protein Kinase G-Iα Hyperactivation and VASP Phosphorylation in Promoting Ovarian Cancer Cell…

http://dx.doi.org/10.5772/53468

263

Financial. support for the research and the preparation of this book chapter was provided by Internal Funding from Roseman University of Health Sciences (formerly named University of Southern Nevada), Henderson, Nevada, USA, U.S. Department of Defense (DOD) grant W81XWH-07-1-0543 and Start-up Funding from the Nevada Cancer Institute, Las Vegas,

1 Center for Diabetes & Obesity Prevention, Treatment, Research & Education, Cancer Re‐ search Program, and College of Pharmacy (Pharmaceutical Sciences), Roseman University of

3 Cancer Molecular Biology Section, Nevada Cancer Institute, Las Vegas, Nevada, USA

[1] Gershenson. DM. Management of ovarian germ cell tumors. J Clin Oncol. 2007 Jul

[2] Siddik ZH. Cisplatin: mode of cytotoxic action and molecular basis of resistance. On‐

[3] Diaz-Padilla I, Duran I, Clarke BA, Oza AM. Biologic rationale and clinical activity of mTOR inhibitors in gynecological cancer. Cancer Treat Rev. 2012 Oct;38(6):767-75.

ondary tumor formation during ovarian cancer progression.

We hypothesized in ovarian cancer cells, as reported in the lung cancer cells in our recent study [34], that PKG-Iα phosphorylated CREB at Ser133, and the cGMP/PKG-Iα signaling pathway maintains the expression of certain IAPs such as c-IAP1, livin and survivin as well as the anti-apoptotic Bcl-2 family member Mcl-1, leading to decreased activity of caspase-3 and promoting cell survival. In ovarian cancer cells where PKG-Iα is hyperactivated, in‐ creased downstream phosphorylation of CREB at Ser133 and increased IAPs expression may explain the development of resistance to cisplatin-induced apoptosis. Moreover, PKG-Iα siRNA gene knockdown also decreased both basal and EGF-stimulated cell migration in A2780cp ovarian cancer cells, as shown in Figure 3.

VASP phosphorylation at Ser239 has been shown to be a useful indicator of endogenous PKG kinase activity, both in our recent studies [28, 31, 32] and reports from others [61, 63, 64]. In the current study in this book chapter, we show that siRNA gene knockdown of PKG-Iα expression inhibited EGF-stimulated increases in VASP Ser239 phosphorylation and Src/SFK autophosphorylation in A2780cp (cisplatin-resistant, mutated p53) ovarian cancer cells. Therefore, VASP Ser239 phosphorylation may be a useful biomarker in ovarian cancer cells, and hyperactivation of the unique NO/sGC/PKG-Iα signaling pathway may be a novel therapeutic target for regulation of cancer cell migration/invasion.

Also shown in the model of Figure 5 is the role of endothelial cells, which would provide an additional source of endogenous NO within the growing tumor, potentially contributing to the "angiogenic switch", i.e. the increased tumor growth that occurs after the invasion of en‐ dothelial cells into the tumor. Endothelial cells also play another important role in tumor growth by providing new blood vessels needed for the vascularization and blood perfusion of the growing tumor. In endothelial cells, heat shock protein 90 (HSP90) and Akt activate eNOS involving the formation of a HSP90-Akt-Calmodulin (CaM)-eNOS complex, leading to an increase in NO production [108-111]. Interestingly, HSP90 activation of eNOS can be Ca2+-dependent [112] or Ca2+-independent [109, 113].

#### **6. Future experiments**

Our future studies will need to determine: 1) whether PKG-Iα is the only isoform of PKG expressed in other human ovarian cancer cell lines as well as in tumor samples of ovarian cancer patients, 2) the subcellular localization of PKG-Iα (and possibly PKG-Iβ), for exam‐ ple, membrane, nuclear, and/or cytosolic localization, 3) the roles of PKG-Iα, its downstream phosphorylation of CREB at Ser133 (and other transcription factors), expression of the IAPs and anti-apoptotic Bcl-2 family proteins in ovarian cancer cells.

#### **7. Conclusions**

kinase activity was found to cause tyrosine phosphorylation of PKG-Iα, increasing the serine/ threonine kinase activity of PKG-Iα (indicated by phosphorylation of the PKG substrate VASP at Ser239) and its growth-promoting effects in ovarian cancer cells [28]. In human ovarian can‐ cer cells, the c-Src-mediated tyrosine-phosphorylation of the EGF receptor was found to be

We hypothesized in ovarian cancer cells, as reported in the lung cancer cells in our recent study [34], that PKG-Iα phosphorylated CREB at Ser133, and the cGMP/PKG-Iα signaling pathway maintains the expression of certain IAPs such as c-IAP1, livin and survivin as well as the anti-apoptotic Bcl-2 family member Mcl-1, leading to decreased activity of caspase-3 and promoting cell survival. In ovarian cancer cells where PKG-Iα is hyperactivated, in‐ creased downstream phosphorylation of CREB at Ser133 and increased IAPs expression may explain the development of resistance to cisplatin-induced apoptosis. Moreover, PKG-Iα siRNA gene knockdown also decreased both basal and EGF-stimulated cell migration in

VASP phosphorylation at Ser239 has been shown to be a useful indicator of endogenous PKG kinase activity, both in our recent studies [28, 31, 32] and reports from others [61, 63, 64]. In the current study in this book chapter, we show that siRNA gene knockdown of PKG-Iα expression inhibited EGF-stimulated increases in VASP Ser239 phosphorylation and Src/SFK autophosphorylation in A2780cp (cisplatin-resistant, mutated p53) ovarian cancer cells. Therefore, VASP Ser239 phosphorylation may be a useful biomarker in ovarian cancer cells, and hyperactivation of the unique NO/sGC/PKG-Iα signaling pathway may be a novel

Also shown in the model of Figure 5 is the role of endothelial cells, which would provide an additional source of endogenous NO within the growing tumor, potentially contributing to the "angiogenic switch", i.e. the increased tumor growth that occurs after the invasion of en‐ dothelial cells into the tumor. Endothelial cells also play another important role in tumor growth by providing new blood vessels needed for the vascularization and blood perfusion of the growing tumor. In endothelial cells, heat shock protein 90 (HSP90) and Akt activate eNOS involving the formation of a HSP90-Akt-Calmodulin (CaM)-eNOS complex, leading to an increase in NO production [108-111]. Interestingly, HSP90 activation of eNOS can be

Our future studies will need to determine: 1) whether PKG-Iα is the only isoform of PKG expressed in other human ovarian cancer cell lines as well as in tumor samples of ovarian cancer patients, 2) the subcellular localization of PKG-Iα (and possibly PKG-Iβ), for exam‐ ple, membrane, nuclear, and/or cytosolic localization, 3) the roles of PKG-Iα, its downstream phosphorylation of CREB at Ser133 (and other transcription factors), expression of the IAPs

highly dependent on PKG-Iα kinase activity [28].

262 Ovarian Cancer - A Clinical and Translational Update

A2780cp ovarian cancer cells, as shown in Figure 3.

Ca2+-dependent [112] or Ca2+-independent [109, 113].

and anti-apoptotic Bcl-2 family proteins in ovarian cancer cells.

**6. Future experiments**

therapeutic target for regulation of cancer cell migration/invasion.

The NO/cGMP/PKG-Iα pathway and the downstream phosphorylation of the actin-fila‐ ment/focal-adhesion-regulating protein VASP at Ser239 appear to promote migration/inva‐ sion and the downstream phosphorylation of BAD at ser155, CREB at ser133 and c-Src at ser17 appear to promote DNA synthesis, cell proliferation and platinum resistance in ovari‐ an cancer cells. The unique features of this signaling pathway in ovarian cancer cells may provide a novel therapeutic target for disrupting tumor growth and the metastasis and sec‐ ondary tumor formation during ovarian cancer progression.

#### **Acknowledgements**

Financial. support for the research and the preparation of this book chapter was provided by Internal Funding from Roseman University of Health Sciences (formerly named University of Southern Nevada), Henderson, Nevada, USA, U.S. Department of Defense (DOD) grant W81XWH-07-1-0543 and Start-up Funding from the Nevada Cancer Institute, Las Vegas, Nevada, USA, awarded to Dr. Fiscus.

#### **Author details**

Janica C. Wong1,2,3 and Ronald R. Fiscus1,3

1 Center for Diabetes & Obesity Prevention, Treatment, Research & Education, Cancer Re‐ search Program, and College of Pharmacy (Pharmaceutical Sciences), Roseman University of Health Sciences, Henderson, Nevada, and South Jordan, USA

2 Department of Chemistry, University of Nevada, Las Vegas, Nevada, USA

3 Cancer Molecular Biology Section, Nevada Cancer Institute, Las Vegas, Nevada, USA

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**Chapter 13**

.

**The PI3K/Akt/mTOR Pathway in Ovarian Cancer:**

Alexandra Leary, Edouard Auclin,

http://dx.doi.org/10.5772/54170

**1. Introduction**

clinical trials.

Patricia Pautier and Catherine Lhommé

Additional information is available at the end of the chapter

**Biological Rationale and Therapeutic Opportunities**

Ovarian cancer is the most lethal cause of gynecological cancer deaths in the developing world and typically presents at an advanced stage when optimal debulking and platinum based-chemotherapy remain the cornerstone of management. Unfortunately, despite fre‐ quent initial responses to chemotherapy, these tumors almost invariably relapse. Thanks to recent large scale molecular profiling studies in ovarian cancer, such as the integrated ge‐ nomic analyses performed by the Cancer Genome Atlas (TCGA) network, significant head‐ way has been made in our understanding of the molecular pathogenesis of ovarian cancer1

However these advances have failed to translate into meaningful clinical benefit for patients. The only approved novel 'targeted' therapy to date in ovarian cancer is the anti-angiogenic

With the possible exception of the p53 signaling network, the PI3K/Akt/mTOR cascade is probably the most frequently altered signaling pathway in cancer, including ovarian cancer. First generation inhibitors of mTOR have demonstrated anti-tumor activity and are current‐ ly approved for the treatment of renal, pancreatic, breast and some brain cancers. In addi‐ tion, a huge number of PI3K, Akt and second generation mTOR inhibitors are in early

We propose to provide a brief overview of the PI3K/Akt/mTOR signaling network and dis‐ cuss the rationale for targeting this pathway in ovarian cancer. Preclinical data and results of recent clinical trials will be presented. In addition, some of the challenges facing the devel‐ opment of these inhibitors in ovarian cancer will be discussed, such as the need for predic‐ tive markers and quality tumor samples, drug resistance, managing toxicity, as well as trial

> © 2013 Leary et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

antibody, bevacizumab, for which reliable predictive markers still elude us.

### **The PI3K/Akt/mTOR Pathway in Ovarian Cancer: Biological Rationale and Therapeutic Opportunities**

Alexandra Leary, Edouard Auclin, Patricia Pautier and Catherine Lhommé

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54170

#### **1. Introduction**

Ovarian cancer is the most lethal cause of gynecological cancer deaths in the developing world and typically presents at an advanced stage when optimal debulking and platinum based-chemotherapy remain the cornerstone of management. Unfortunately, despite fre‐ quent initial responses to chemotherapy, these tumors almost invariably relapse. Thanks to recent large scale molecular profiling studies in ovarian cancer, such as the integrated ge‐ nomic analyses performed by the Cancer Genome Atlas (TCGA) network, significant head‐ way has been made in our understanding of the molecular pathogenesis of ovarian cancer1 . However these advances have failed to translate into meaningful clinical benefit for patients. The only approved novel 'targeted' therapy to date in ovarian cancer is the anti-angiogenic antibody, bevacizumab, for which reliable predictive markers still elude us.

With the possible exception of the p53 signaling network, the PI3K/Akt/mTOR cascade is probably the most frequently altered signaling pathway in cancer, including ovarian cancer. First generation inhibitors of mTOR have demonstrated anti-tumor activity and are current‐ ly approved for the treatment of renal, pancreatic, breast and some brain cancers. In addi‐ tion, a huge number of PI3K, Akt and second generation mTOR inhibitors are in early clinical trials.

We propose to provide a brief overview of the PI3K/Akt/mTOR signaling network and dis‐ cuss the rationale for targeting this pathway in ovarian cancer. Preclinical data and results of recent clinical trials will be presented. In addition, some of the challenges facing the devel‐ opment of these inhibitors in ovarian cancer will be discussed, such as the need for predic‐ tive markers and quality tumor samples, drug resistance, managing toxicity, as well as trial

© 2013 Leary et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

design considerations in order to optimize the development of novel therapies against the PI3K pathway in ovarian cancer.

less well described but likely regulates cell proliferation and survival in part by Akt activation via phosphorylation at Serine 473[7]. Importantly mTORC1 is sensitive to inhibition by rapa‐ mycin, while mTORC2 is not. In the presence of selective mTORC1 inhibition, mTORC2 can ex‐ ert a positive feedback on Akt[8]. As discussed later, this positive feedback loop may have important implications regarding the emergence of resistance to first generation mTOR inhibi‐

The PI3K/Akt/mTOR Pathway in Ovarian Cancer: Biological Rationale and Therapeutic Opportunities

http://dx.doi.org/10.5772/54170

277

**Figure 1. The PI3K/Akt/mTOR signaling pathway.** This pathway is up-regulated in a significant proportion of ovari‐ an cancers via either (i) direct upstream stimulation (growth factor receptors and their ligands), (ii) indirect activation via cross-talk with the Ras pathway, or (iii) intrinsically via activating genetic alterations in PI3K or Akt, or via loss of

The PI3K/Akt/mTOR pathway is frequently deregulated in ovarian cancer. Array Compara‐ tive Genomic Hybridization (aCGH) studies on 93 ovarian tumors have identified this path‐ way as the most frequently altered in ovarian cancer [9]. Copy gains in the genes encoding both the p110α (*PIK3CA*) and p110β (*PIK3CB*) subunits of PI3K were associated with a poor prognosis in patients with ovarian cancer. Expression levels of both p110α and pAkt were analyzed in over 500 ovarian cancer tumors and associated with decreased survival. Activa‐

**3. Relevance of PI3K/Akt/mTOR signaling in ovarian cancer**

function in the tumor suppressor, PTEN.

tors (rapalogs) that exclusively target mTORC1, with no effect on mTORC2.

#### **2. The PI3K/Akt/mTOR signaling pathway**

The phophatidylinositol 3 Kinase (PI3K) pathway is a complex signaling network coordinat‐ ing a number of direct upstream inputs from growth factors (EGF, heregulin, TGF, and oth‐ ers), tyrosine kinase receptors (IGF1R, EGFR, HER2…) or other membrane receptors such as Met as well as cross-talk with the Ras-Raf-Mek-Erk pathway via indirect input from Ras (Figure 1). PI3K is composed of a p110 catalytic subunit and a p85 regulatory subunit. The p110 subunit of PI3K phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) to the ac‐ tive second messenger, PIP3 which recruits Akt to the plasma membrane, and results in a conformational change and activation of PDK1 and Akt proteins. Akt is a serine threonine kinase that regulates a huge number of downstream targets [2],[3], while the phosphatase and tensin (PTEN) analog protein acts as an endogenous pathway repressor by de-phos‐ phorylating PIP3 back to PIP2. Akt controls critical cellular survival and metabolic processes by influencing some of the following:


In addition to activation via upstream input, the PI3K pathway can be 'intrinsically' activat‐ ed due to i) gain of function mutations or amplifications in the p110 subunit of PI3K (*PIK3CA*), ii) mutations in the p85 subunit (*PIK3R*), iii) mutations or amplifications in one of the Akt isoforms (*AKT1, AKT2, AKT3*), or iv) due to loss of its negative regulator, *PTEN* via inactivating mutations, copy number loss or homozygous deletions.

While mTOR is probably the best described direct target of Akt, the mTOR complex is actually composed of two components, the mTORC1-Raptor complex primary coordinator of transla‐ tional control via 4EBP1 and p70S6K[6]; and the mTORC2-Rictor complex whose function is less well described but likely regulates cell proliferation and survival in part by Akt activation via phosphorylation at Serine 473[7]. Importantly mTORC1 is sensitive to inhibition by rapa‐ mycin, while mTORC2 is not. In the presence of selective mTORC1 inhibition, mTORC2 can ex‐ ert a positive feedback on Akt[8]. As discussed later, this positive feedback loop may have important implications regarding the emergence of resistance to first generation mTOR inhibi‐ tors (rapalogs) that exclusively target mTORC1, with no effect on mTORC2.

design considerations in order to optimize the development of novel therapies against the

The phophatidylinositol 3 Kinase (PI3K) pathway is a complex signaling network coordinat‐ ing a number of direct upstream inputs from growth factors (EGF, heregulin, TGF, and oth‐ ers), tyrosine kinase receptors (IGF1R, EGFR, HER2…) or other membrane receptors such as Met as well as cross-talk with the Ras-Raf-Mek-Erk pathway via indirect input from Ras (Figure 1). PI3K is composed of a p110 catalytic subunit and a p85 regulatory subunit. The p110 subunit of PI3K phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) to the ac‐ tive second messenger, PIP3 which recruits Akt to the plasma membrane, and results in a conformational change and activation of PDK1 and Akt proteins. Akt is a serine threonine kinase that regulates a huge number of downstream targets [2],[3], while the phosphatase and tensin (PTEN) analog protein acts as an endogenous pathway repressor by de-phos‐ phorylating PIP3 back to PIP2. Akt controls critical cellular survival and metabolic processes

**1.** Via downstream regulation of p53, NFκΒ (nuclear factor κΒ) or CREB (cAMP response element-binding protein), Akt promotes the transcription of genes involved in antiapoptotic and proliferative responses such as XIAP (X-linked inhibitor of apoptosis pro‐

**2.** Akt also phosphorylates proteins involved in cell cycle regulation and apoptosis thus

**b.** Phosphorylation of the cyclin-dependent kinase (CDK) inhibitors p21 and p27 commits them to nuclear export and removes their inhibitory effect on cyclin D and cyclin E,

**3.** In addition downstream signaling via mammalian target of rapamycin (mTOR) acti‐ vates two key substrates 4EBP1 and p70S6K resulting in increased translation of target genes involved in angiogenesis (VEGF), or cell cycle progression (cyclin D1, c-Myc)[5].

In addition to activation via upstream input, the PI3K pathway can be 'intrinsically' activat‐ ed due to i) gain of function mutations or amplifications in the p110 subunit of PI3K (*PIK3CA*), ii) mutations in the p85 subunit (*PIK3R*), iii) mutations or amplifications in one of the Akt isoforms (*AKT1, AKT2, AKT3*), or iv) due to loss of its negative regulator, *PTEN* via

While mTOR is probably the best described direct target of Akt, the mTOR complex is actually composed of two components, the mTORC1-Raptor complex primary coordinator of transla‐ tional control via 4EBP1 and p70S6K[6]; and the mTORC2-Rictor complex whose function is

tein), the apoptosis regulating protein Bcl-2, survivin and others[4].

**a.** Phosphorylation of GSK3 inhibits proteosomic degradation of cyclin D1,

promoting cell cycle progression and survival:

**c.** Downregulation of the apoptotic effector, caspase 9.

inactivating mutations, copy number loss or homozygous deletions.

PI3K pathway in ovarian cancer.

276 Ovarian Cancer - A Clinical and Translational Update

by influencing some of the following:

**2. The PI3K/Akt/mTOR signaling pathway**

**Figure 1. The PI3K/Akt/mTOR signaling pathway.** This pathway is up-regulated in a significant proportion of ovari‐ an cancers via either (i) direct upstream stimulation (growth factor receptors and their ligands), (ii) indirect activation via cross-talk with the Ras pathway, or (iii) intrinsically via activating genetic alterations in PI3K or Akt, or via loss of function in the tumor suppressor, PTEN.

#### **3. Relevance of PI3K/Akt/mTOR signaling in ovarian cancer**

The PI3K/Akt/mTOR pathway is frequently deregulated in ovarian cancer. Array Compara‐ tive Genomic Hybridization (aCGH) studies on 93 ovarian tumors have identified this path‐ way as the most frequently altered in ovarian cancer [9]. Copy gains in the genes encoding both the p110α (*PIK3CA*) and p110β (*PIK3CB*) subunits of PI3K were associated with a poor prognosis in patients with ovarian cancer. Expression levels of both p110α and pAkt were analyzed in over 500 ovarian cancer tumors and associated with decreased survival. Activa‐ tion of the pathway as measured by Akt or mTOR phosphorylation levels is almost ubiqui‐ tous in ovarian cancer and an independent negative prognostic marker [10-12].

High grade serous ovarian cancer is exquisitely chemosensitive, with response rates to firstline platinum-based chemotherapy of 75%, but almost invariably relapses with acquired re‐ sistance. The rarer subtypes tend to respond poorly to platinum chemotherapy with response rates of only 15% to 30%. Thus both acquired and de novo chemotherapy resist‐ ance remains a significant clinical challenge in ovarian cancer. Increased phosphorylation of mTOR has been described in cell lines with acquired cisplatin resistance, and Akt signaling has been implicated in primary platinum resistance[12]. Inhibitors of Akt or mTOR were shown to restore chemo-sensitivity in vitro and in xenograft models [26],[27]. These data suggest a potential role for inhibitors of the PI3K pathway in modulating chemotherapy sen‐

The PI3K/Akt/mTOR Pathway in Ovarian Cancer: Biological Rationale and Therapeutic Opportunities

**PI3K activation via upstream membrane RTKs**

> **Amplifications:** *ERBB3* (4%) ERBB2 (3%) IGF1R (4%)

> > **Mutations:** *EGFR* (4-9%) *ERBB2* (1%)

**Amplifications** *ERBB2* (14%)

*ERBB2* (18%)

**Mutations:** *ERBB2* (15%)

**Table 1.** Molecular alterations according to ovarian cancer subtype that could contribute to PI3K pâthway activation either directly (deregulated PI3K members) or indirectly via alterations in upstream RTKs or Ras pathway members.

**PI3K activation via crosstalk with ras**

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279

**Amplifications:** *MAPK* (25%) KRAS (11%) **Deletions:** NF1 (8%)

> **Mutations:** *NF1* (4%) *KRAS* (1-5%) *NRAS* (1%) *BRAF* (1%)

**Mutations:** *KRAS* (40-60%)

> **Mutations:** *KRAS* (40%) *BRAF* (1%)

sitivity and justify their use in combination with conventional cytotoxics.

**Intrinsic PI3K pathway activation**

> **Amplifications**: *PIK3CA* (17-20%) AKT1 (3%) AKT2 (6-12%) AKT3 (8%) RICTOR (6%) RAPTOR (4%) **Deletions:** PTEN (7%)

> > **Mutations:** *PIK3CA* (3%) *PIK3R1* (4%) *PTEN* (1%)

**Deletions** *PTEN* (20%)

**Mutations:** *PIK3CA* (33%)

**Deletions** *PTEN* (20%) **Mutations:** *PIK3CA* (20%) *PTEN* (20%) Mucinous **Amplifications:**

**Ovarian cancer histological subtype**

High grade serous

(70%)

Clear cell

Endometrioid

Low grade serous

Interestingly, the type of PI3K/Akt/mTOR molecular alteration appears to be histological subtype specific (Table 1). There is mounting evidence that ovarian cancer is a highly hetero‐ geneous disease with marked differences in molecular profile, histology, prognosis and che‐ mosensitivity depending on the subtype [1],[13],[14]. The most common subtype (70%) high grade serous ovarian cancer (HGSOC) is characterized by almost universal p53 mutations (95-97% of cases) and marked genomic instability resulting in frequent somatic copy num‐ ber alterations (amplifications or deletions)[13]. In HGSOC, oncogenic mutations are rare, but amplifications of the p110 subunit of PI3K (*PIK3CA)* have been described in 20% of cas‐ es, amplifications of one of the *AKT* isoforms (*AKT 1, AKT2* or *AKT3*) occur in 15% to 20%, while *PTEN* deletions have been described in 5%[15],[16] (Table 1). Finally *RICTOR* or *RAP‐ TOR* amplifications have also been reported [1]. Rare but potentially relevant mutations in HGSOC include activating *PIK3CA* mutations (3%), or loss of function *PTEN* mutations (1%) [17]. Mutations have also been described in the p85α subunit of PI3K (*PIK3R1*, 4%), resulting in loss of its negative regulation on the p110 subunit and constitutive kinase activity[18]. In summary, 40 to 50% of HGSOC may have constitutive PI3K signaling. In a significant pro‐ portion of HGSOC, hyperactive PI3K/Akt/mTOR pathway may also be attributable to up‐ stream deregulations in receptor tyrosine kinases (RTKs) or cross-talk with the Ras/Mek/Mek/Erk pathway. Indeed, amplifications or mutations in RTKs such as *ERBB3*, *ERBB2*, *EGFR* or *IGF1R* have been described with frequencies of 1% to 9% [1],[17]. Similarly, the ras pathway is often altered in HGSOC by amplifications in *KRAS* (11%), *MAPK* (20%), loss of the tumor suppressor *NF1* (8%), or less frequent mutations in *KRAS*, *NRAS*, or *BRAF*.

Whereas individual mutations remain an infrequent event in HGSOC, they are much more prevalent in the rarer subtypes such as low grade serous, mucinous, endometrioid or clear cell ovarian cancer. For example, 20% of endometrioid and 35% of clear cell ovarian tumors display *PIK3CA* mutations[19],[20]. In addition, while *PTEN* loss of function mutations are rare in ovarian cancer in general, they are well documented in up to 20% of endometrioid tumors and *PTEN* deletion occurs in 20% of endometrioid and clear cell ovarian cancers[21]. Low grade mucinous and serous subtypes do not tend to demonstrate intrinsic activation of PI3K effectors, however they frequently exhibit *KRAS* mutations, or amplifications/muta‐ tions in *ERBB2[22],[23]*.

Importantly intrinsic activation of the pathway (via *PIK3CA* mutations and *PTEN* loss) has been shown to initiate ovarian tumors in mice and inhibition of PI3K/mTOR in these models delayed tumor growth and prolonged survival, thus providing critical proof of concept for the pathologic relevance of this pathway in OC and its potential as a therapeutic target[24], [25]. Whether amplifications of pathway members actually activate PI3K signaling and con‐ fer comparable sensitivity to pathway inhibitors remains to be established. Similarly, while cross-talk with Ras may result in PI3K activation, it is unlikely that this also results in PI3K pathway dependence, however as discussed later, alterations in KRAS may be relevant with regards to predicting benefit from dual PI3K-Ras inhibition.

High grade serous ovarian cancer is exquisitely chemosensitive, with response rates to firstline platinum-based chemotherapy of 75%, but almost invariably relapses with acquired re‐ sistance. The rarer subtypes tend to respond poorly to platinum chemotherapy with response rates of only 15% to 30%. Thus both acquired and de novo chemotherapy resist‐ ance remains a significant clinical challenge in ovarian cancer. Increased phosphorylation of mTOR has been described in cell lines with acquired cisplatin resistance, and Akt signaling has been implicated in primary platinum resistance[12]. Inhibitors of Akt or mTOR were shown to restore chemo-sensitivity in vitro and in xenograft models [26],[27]. These data suggest a potential role for inhibitors of the PI3K pathway in modulating chemotherapy sen‐ sitivity and justify their use in combination with conventional cytotoxics.

tion of the pathway as measured by Akt or mTOR phosphorylation levels is almost ubiqui‐

Interestingly, the type of PI3K/Akt/mTOR molecular alteration appears to be histological subtype specific (Table 1). There is mounting evidence that ovarian cancer is a highly hetero‐ geneous disease with marked differences in molecular profile, histology, prognosis and che‐ mosensitivity depending on the subtype [1],[13],[14]. The most common subtype (70%) high grade serous ovarian cancer (HGSOC) is characterized by almost universal p53 mutations (95-97% of cases) and marked genomic instability resulting in frequent somatic copy num‐ ber alterations (amplifications or deletions)[13]. In HGSOC, oncogenic mutations are rare, but amplifications of the p110 subunit of PI3K (*PIK3CA)* have been described in 20% of cas‐ es, amplifications of one of the *AKT* isoforms (*AKT 1, AKT2* or *AKT3*) occur in 15% to 20%, while *PTEN* deletions have been described in 5%[15],[16] (Table 1). Finally *RICTOR* or *RAP‐ TOR* amplifications have also been reported [1]. Rare but potentially relevant mutations in HGSOC include activating *PIK3CA* mutations (3%), or loss of function *PTEN* mutations (1%) [17]. Mutations have also been described in the p85α subunit of PI3K (*PIK3R1*, 4%), resulting in loss of its negative regulation on the p110 subunit and constitutive kinase activity[18]. In summary, 40 to 50% of HGSOC may have constitutive PI3K signaling. In a significant pro‐ portion of HGSOC, hyperactive PI3K/Akt/mTOR pathway may also be attributable to up‐ stream deregulations in receptor tyrosine kinases (RTKs) or cross-talk with the Ras/Mek/Mek/Erk pathway. Indeed, amplifications or mutations in RTKs such as *ERBB3*, *ERBB2*, *EGFR* or *IGF1R* have been described with frequencies of 1% to 9% [1],[17]. Similarly, the ras pathway is often altered in HGSOC by amplifications in *KRAS* (11%), *MAPK* (20%), loss of the tumor suppressor *NF1* (8%), or less frequent mutations in *KRAS*, *NRAS*, or *BRAF*.

Whereas individual mutations remain an infrequent event in HGSOC, they are much more prevalent in the rarer subtypes such as low grade serous, mucinous, endometrioid or clear cell ovarian cancer. For example, 20% of endometrioid and 35% of clear cell ovarian tumors display *PIK3CA* mutations[19],[20]. In addition, while *PTEN* loss of function mutations are rare in ovarian cancer in general, they are well documented in up to 20% of endometrioid tumors and *PTEN* deletion occurs in 20% of endometrioid and clear cell ovarian cancers[21]. Low grade mucinous and serous subtypes do not tend to demonstrate intrinsic activation of PI3K effectors, however they frequently exhibit *KRAS* mutations, or amplifications/muta‐

Importantly intrinsic activation of the pathway (via *PIK3CA* mutations and *PTEN* loss) has been shown to initiate ovarian tumors in mice and inhibition of PI3K/mTOR in these models delayed tumor growth and prolonged survival, thus providing critical proof of concept for the pathologic relevance of this pathway in OC and its potential as a therapeutic target[24], [25]. Whether amplifications of pathway members actually activate PI3K signaling and con‐ fer comparable sensitivity to pathway inhibitors remains to be established. Similarly, while cross-talk with Ras may result in PI3K activation, it is unlikely that this also results in PI3K pathway dependence, however as discussed later, alterations in KRAS may be relevant with

regards to predicting benefit from dual PI3K-Ras inhibition.

tions in *ERBB2[22],[23]*.

tous in ovarian cancer and an independent negative prognostic marker [10-12].

278 Ovarian Cancer - A Clinical and Translational Update


**Table 1.** Molecular alterations according to ovarian cancer subtype that could contribute to PI3K pâthway activation either directly (deregulated PI3K members) or indirectly via alterations in upstream RTKs or Ras pathway members.

### **4. Results of clinical trials targeting the PI3K/Akt/mTOR pathway in ovarian cancer**

combinations. A phase I study of weekly topotecan (1mg/m2 days 1, 8 and 15) and temsiroli‐ mus 25mg days 1, 8, 15 and 22 on a 28 day schedule was conducted in 15 patients with gyne‐ cological malignancies including 7 patients with ovarian cancer. Dose limiting toxicities were myelosuppression and although efficacy was not a primary objective, 8 of 11 patients had stable disease at first evaluation and one patient with clear cell histology was still pro‐

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281

A phase Ib dose escalation study of temsirolimus (T) and pegylated liposomal doxorubicin (PLD) in advanced breast and gynaecological malignancies identified T 15mg and PLD 40mg/m2 as the maximum tolerated dose (MTD)[31]. The most frequent grade 3-4 adverse events were fatigue (5%), nausea (16%), mucositis (21%), rash (11%) and hand-foot syn‐ drome (21%). The mean PFS was 4.9 months and the authors concluded that the combina‐

Two other phase I studies of rapalogs in combination with chemotherapy (temsirolimus plus carboplatin/paclitaxel[32] and everolimus plus weekly paclitaxel[33]) have been con‐ ducted with grade 3-4 neutropenia being the major DLT (at 89% and 56%, respectively) as well as fatigue and mucositis. These studies included a small number of patients with ad‐ vanced ovarian cancer and responses were described (3 of 6 patients with ovarian cancer had a PR to temsirolimus plus carboplatin and paclitaxel). However given the small num‐ bers and the combination with chemotherapy, no robust conclusions may be drawn regard‐

These early studies have begun to establish the feasibility and safety of mTORi-cytotoxic combinations, randomized trials will be required to investigate efficacy. In the interim, a number of non-randomized phase I and II studies are ongoing (Table 4). Given the heteroge‐ neity of ovarian cancer, non-randomized phase II studies may require a degree of patient se‐ lection by molecular alteration or even histology in order to enrich the trial for potential responders and make the patient population more uniform with regards to natural disease course and chemosensitivity. Indeed studies recruiting patients with both high and low grade tumors with marked differences in tumor growth rates and responsiveness to chemo‐ therapy may mask any benefit from the addition of the mTOR inhibitor. For example, a phase II trial of temsirolimus plus carboplatin and paclitaxel as adjuvant treatment is ongo‐

ing for patients with stage III or IV clear cell ovarian cancer (NCT01196429).

**4.3. mTOR inhibitors in combination with anti-angiogenics in ovarian cancer (Table 2)**

Finally, given the activity of VEGF inhibitors in ovarian cancer and the fact that downstream mTOR targets include angiogenic genes, there is A biological rationale for using mTOR and VEGF inhibitors in combination. A phase II trial of temsirolimus and bevacizumab in ovari‐ an cancer has been conducted[34]. Thirty one (31) patients were evaluable for toxicity and 25 for efficacy. Adverse events included fatigue, mucositis, hypertension and neutropenia. In addition one grade 4 rash and 6% colonic perforations (2/31) were reported. While the con‐ firmed PR rate is only 12% in the first 25 evaluable patients (all in platinum-resistant pa‐ tients), the 6 months PFS rate of 56% (14/25) met efficacy criteria to justify progression to second stage accrual. Updated results are awaited. It is noteworthy that the study only en‐

gression free at 6 months[30].

tion warranted further study.

ing the added value of the mTOR inhibitor.

The frequent PI3K/Akt alterations demonstrated in vivo in tumors from patients with ovari‐ an cancer, combined with the evidence for dependence on this oncogenic pathway in pre‐ clinical models provide a robust biological rationale for investigating the benefit of targeting PI3K, Akt or mTOR in ovarian cancer. However as detailed throughout this chapter, the in‐ trinsic complexity of this signaling network may limit the anti-tumor potential of inhibiting a single effector along the pathway.

#### **4.1. mTOR inhibitor monotherapy in ovarian cancer (Table 2)**

The first inhibitors of the pathway to enter the clinic were rapamycin analogs that bind to the FK506 binding protein-12 of the MTORC1 complex and prevent mTOR activity. Rapamycin was used for years as an immunosuppressant to prevent rejection in solid organ transplants and hematological malignancies; its toxicity profile is therefore well described with main side effects consisting of edema, hypertension, renal toxicity, hematologic toxicity, and hypertrigly‐ ceridemia and hypercholesterolemia. In addition, rarer but potentially more concerning side effects included interstitial lung disease, risk of secondary lymphoma, and reactivation of la‐ tent infections[28]. Rapamycin analogs with less immunosuppressive properties, such as tem‐ sirolimus, everolimus and ridaforolimus have shown activity in a number of tumor types.

A phase II trial of temsirolimus at a flat dose of 25mg IV weekly in patients with ovarian cancer progressing after 1-3 previous regimens met its first stage response and PFS criteria at interim analysis with three responses and seven PFS at 6 months and pursued accrual through the sec‐ ond stage[29]. At final analysis, with 54 evaluable patients, grade 3-4 toxicities were as expect‐ ed for mTOR inhibitors, mainly gastrointestinal (10%), metabolic (15%), and study drug was discontinued in 6% for interstitial pneumonitis. Unfortunately, objective responses were only seen in 9.3% (5/54) and 6 months PFS was 24% thus the study failed to meet its efficacy end‐ point. Exploratory analyses were conducted in order to identify potential predictive markers. Phosphorylated-Akt, p-mTOR, p-p70-S6K, and cyclinD1 were measured in archival tumor samples as surrogates for activation of the PI3K pathway; only cyclinD1 levels were weakly correlated with PFS>6 months (r=0.28). The authors concluded that observed activity was in‐ sufficient to justify a phase III trial of temsirolimus in unselected patients with ovarian cancer. As discussed later in the chapter; these negative results may be explained by i) the lack of pa‐ tient selection, ii) the cytostatic rather than cytotoxic effect of mTOR inhibitors (mTORi) and iii) the fact that these agents may require combinations with chemotherapy or other targeted agents to achieve a robust anti-tumor effect. The trial just fell short of its PFS efficacy endpoint (>24% PFS at 6 months), had the study limited enrollment to clear cell and endometrioid histol‐ ogies known to show frequent PI3K alterations, the results may have been different.

#### **4.2. mTOR inhibitors in combination with chemotherapy in ovarian cancer (Table 2)**

Given the implication of mTOR and Akt in chemo-resistance and the preclinical studies sug‐ gesting an additive benefit with chemotherapy, studies have investigated mTORi-cytotoxic combinations. A phase I study of weekly topotecan (1mg/m2 days 1, 8 and 15) and temsiroli‐ mus 25mg days 1, 8, 15 and 22 on a 28 day schedule was conducted in 15 patients with gyne‐ cological malignancies including 7 patients with ovarian cancer. Dose limiting toxicities were myelosuppression and although efficacy was not a primary objective, 8 of 11 patients had stable disease at first evaluation and one patient with clear cell histology was still pro‐ gression free at 6 months[30].

**4. Results of clinical trials targeting the PI3K/Akt/mTOR pathway in**

The frequent PI3K/Akt alterations demonstrated in vivo in tumors from patients with ovari‐ an cancer, combined with the evidence for dependence on this oncogenic pathway in pre‐ clinical models provide a robust biological rationale for investigating the benefit of targeting PI3K, Akt or mTOR in ovarian cancer. However as detailed throughout this chapter, the in‐ trinsic complexity of this signaling network may limit the anti-tumor potential of inhibiting

The first inhibitors of the pathway to enter the clinic were rapamycin analogs that bind to the FK506 binding protein-12 of the MTORC1 complex and prevent mTOR activity. Rapamycin was used for years as an immunosuppressant to prevent rejection in solid organ transplants and hematological malignancies; its toxicity profile is therefore well described with main side effects consisting of edema, hypertension, renal toxicity, hematologic toxicity, and hypertrigly‐ ceridemia and hypercholesterolemia. In addition, rarer but potentially more concerning side effects included interstitial lung disease, risk of secondary lymphoma, and reactivation of la‐ tent infections[28]. Rapamycin analogs with less immunosuppressive properties, such as tem‐ sirolimus, everolimus and ridaforolimus have shown activity in a number of tumor types.

A phase II trial of temsirolimus at a flat dose of 25mg IV weekly in patients with ovarian cancer progressing after 1-3 previous regimens met its first stage response and PFS criteria at interim analysis with three responses and seven PFS at 6 months and pursued accrual through the sec‐ ond stage[29]. At final analysis, with 54 evaluable patients, grade 3-4 toxicities were as expect‐ ed for mTOR inhibitors, mainly gastrointestinal (10%), metabolic (15%), and study drug was discontinued in 6% for interstitial pneumonitis. Unfortunately, objective responses were only seen in 9.3% (5/54) and 6 months PFS was 24% thus the study failed to meet its efficacy end‐ point. Exploratory analyses were conducted in order to identify potential predictive markers. Phosphorylated-Akt, p-mTOR, p-p70-S6K, and cyclinD1 were measured in archival tumor samples as surrogates for activation of the PI3K pathway; only cyclinD1 levels were weakly correlated with PFS>6 months (r=0.28). The authors concluded that observed activity was in‐ sufficient to justify a phase III trial of temsirolimus in unselected patients with ovarian cancer. As discussed later in the chapter; these negative results may be explained by i) the lack of pa‐ tient selection, ii) the cytostatic rather than cytotoxic effect of mTOR inhibitors (mTORi) and iii) the fact that these agents may require combinations with chemotherapy or other targeted agents to achieve a robust anti-tumor effect. The trial just fell short of its PFS efficacy endpoint (>24% PFS at 6 months), had the study limited enrollment to clear cell and endometrioid histol‐

ogies known to show frequent PI3K alterations, the results may have been different.

**4.2. mTOR inhibitors in combination with chemotherapy in ovarian cancer (Table 2)**

Given the implication of mTOR and Akt in chemo-resistance and the preclinical studies sug‐ gesting an additive benefit with chemotherapy, studies have investigated mTORi-cytotoxic

**ovarian cancer**

a single effector along the pathway.

280 Ovarian Cancer - A Clinical and Translational Update

**4.1. mTOR inhibitor monotherapy in ovarian cancer (Table 2)**

A phase Ib dose escalation study of temsirolimus (T) and pegylated liposomal doxorubicin (PLD) in advanced breast and gynaecological malignancies identified T 15mg and PLD 40mg/m2 as the maximum tolerated dose (MTD)[31]. The most frequent grade 3-4 adverse events were fatigue (5%), nausea (16%), mucositis (21%), rash (11%) and hand-foot syn‐ drome (21%). The mean PFS was 4.9 months and the authors concluded that the combina‐ tion warranted further study.

Two other phase I studies of rapalogs in combination with chemotherapy (temsirolimus plus carboplatin/paclitaxel[32] and everolimus plus weekly paclitaxel[33]) have been con‐ ducted with grade 3-4 neutropenia being the major DLT (at 89% and 56%, respectively) as well as fatigue and mucositis. These studies included a small number of patients with ad‐ vanced ovarian cancer and responses were described (3 of 6 patients with ovarian cancer had a PR to temsirolimus plus carboplatin and paclitaxel). However given the small num‐ bers and the combination with chemotherapy, no robust conclusions may be drawn regard‐ ing the added value of the mTOR inhibitor.

These early studies have begun to establish the feasibility and safety of mTORi-cytotoxic combinations, randomized trials will be required to investigate efficacy. In the interim, a number of non-randomized phase I and II studies are ongoing (Table 4). Given the heteroge‐ neity of ovarian cancer, non-randomized phase II studies may require a degree of patient se‐ lection by molecular alteration or even histology in order to enrich the trial for potential responders and make the patient population more uniform with regards to natural disease course and chemosensitivity. Indeed studies recruiting patients with both high and low grade tumors with marked differences in tumor growth rates and responsiveness to chemo‐ therapy may mask any benefit from the addition of the mTOR inhibitor. For example, a phase II trial of temsirolimus plus carboplatin and paclitaxel as adjuvant treatment is ongo‐ ing for patients with stage III or IV clear cell ovarian cancer (NCT01196429).

#### **4.3. mTOR inhibitors in combination with anti-angiogenics in ovarian cancer (Table 2)**

Finally, given the activity of VEGF inhibitors in ovarian cancer and the fact that downstream mTOR targets include angiogenic genes, there is A biological rationale for using mTOR and VEGF inhibitors in combination. A phase II trial of temsirolimus and bevacizumab in ovari‐ an cancer has been conducted[34]. Thirty one (31) patients were evaluable for toxicity and 25 for efficacy. Adverse events included fatigue, mucositis, hypertension and neutropenia. In addition one grade 4 rash and 6% colonic perforations (2/31) were reported. While the con‐ firmed PR rate is only 12% in the first 25 evaluable patients (all in platinum-resistant pa‐ tients), the 6 months PFS rate of 56% (14/25) met efficacy criteria to justify progression to second stage accrual. Updated results are awaited. It is noteworthy that the study only en‐ rolled patients who had not been exposed to anti-angiogenics; the previously reported RR of 15-21% in early trials of bevacizumab monotherapy among heavily pretreated patients with ovarian cancer raises the possibility that temsirolimus may be adding little anti-tumor effect to bevacizumab alone[35],[36]. A randomized phase II study is ongoing comparing bevaci‐ zumab alone to bevacizumab and everolimus in patients with recurrent ovarian cancer (NCT00886691, Table 4). Patients will be stratified according to their platinum-free interval or prior treatment with bevacizumab. This study should provide valuable insight into the potential additive benefit of this combinatorial strategy.

While the evidence for clinical activity of mTOR inhibitors in ovarian cancer remains quite lim‐ ited, especially compared to endometrial cancer where efficacy has been more encouraging, a number of phase II trials of mTOR inhibitors alone or in combination with conventional cyto‐ toxics or targeted therapies are currently ongoing. These should help clarify the role mTOR in‐

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283

Targeting Akt upstream from mTOR may produce a more effective knock-down of signal transduction and a number of Akt inhibitors have therefore been generated. These include ATP-competitive inhibitors, allosteric inhibitors, peptide-based inhibitors and lipid-based inhibitors (reviewed in Stronach et al[37]). Akt inhibitors are still in early stages of clinical development and two compounds have been specifically tested in ovarian cancer (Table 3).

The most mature inhibitor in clinical development is the lipid-based inhibitor, perifosine, it interferes with the cell membrane recruitment of Akt (thus preventing activation). However early data in phase I and II trials in other tumor types were disappointing with frequent gas‐ trointestinal toxicity and a lack of meaningful activity[38]-[41]. Given the suggestion that the narrow therapeutic window of perifosine may limit its clinical usefulness, combination trials with conventional cytotoxics have been conducted in order to improve the therapeutic in‐ dex. Preclinical studies have shown that perifosine inhibited ovarian cancer cell prolifera‐ tion, motility and angiogenesis and potentiated paclitaxel sensitivity in vitro and in vivo[42], [43]. On this basis, a phase I trial of perifosine and docetaxel in platinum and taxane resist‐ ant ovarian cancer was conducted[42]. Perifosine was given at a loading dose of 100mg ev‐ ery 6 hours for 4 doses followed by a daily dose according to dose level (50, 100 or 150mg daily) in combination with docetaxel 75mg/m2 day 1 every 3 weeks. Twenty one patients were enrolled including 11 at the MTD level of perifosine 150mg. No DLTs were observed, frequent adverse events included nausea, vomiting, anorexia, constipation and fatigue. With regards to efficacy at the MTD (N=11), there was one PR in an endometrioid ovarian cancer with a loss of function *PTEN* mutation (R130Q) and one SD maintained for 4 months in a PI3K mutated clear cell tumor. Two other patients without apparent PI3K alterations ach‐ ieved SD while two patients with KRAS mutations progressed quickly. The investigators al‐ so performed pharmacodynamic studies using reverse phase protein array (RPPA) to detect changes in total and phosphorylated markers in pre-treatment versus day 7 tumor biopsies and functional imaging studies using FDG-PET scans. Bcl2 and ERK2 levels were increased by treatment suggesting that the low response rate may be in part explained by perifosine induced increases in alternate signalling pathways. However FDG-PET responses at one week correlated with inhibition of S6 phosphorylation raising the possibility that FDG-PET

GSK795 is an oral ATP-competitive pan-Akt inhibitor in early stages of development and a small phase I pharmacodynamic and pharmacokinetic study was conducted in order to characterize the relationship between AKT inhibition by GSK795 and downstream effects in patients with advanced platinum resistant ovarian cancer[44]. Twelve patients were enrol‐ led. The only toxicities were grade 2 anorexia (18%) and vomiting (18%). FDG metabolism

hibitors may have in the management of patients with ovarian cancer (Table 4).

may serve as an early surrogate indicator of Akt inhibition.

**4.4. Akt inhibitors**


**Abbreviations:** N: number of patients; IV: intravenous; D: day; Q: every; G3-4: grade 3-4; RR: response rate; PFS: pro‐ gression-free survival; SD: stable disease; MTD: maximum tolerated dose; PLD: pegylated liposomal doxorubicin; NA: information not available; carbo: carboplatin; pac: paclitaxel; PO: per os.

**Table 2.** Completed clinical trials of mTOR inhibitors in ovarian cancer

While the evidence for clinical activity of mTOR inhibitors in ovarian cancer remains quite lim‐ ited, especially compared to endometrial cancer where efficacy has been more encouraging, a number of phase II trials of mTOR inhibitors alone or in combination with conventional cyto‐ toxics or targeted therapies are currently ongoing. These should help clarify the role mTOR in‐ hibitors may have in the management of patients with ovarian cancer (Table 4).

#### **4.4. Akt inhibitors**

rolled patients who had not been exposed to anti-angiogenics; the previously reported RR of 15-21% in early trials of bevacizumab monotherapy among heavily pretreated patients with ovarian cancer raises the possibility that temsirolimus may be adding little anti-tumor effect to bevacizumab alone[35],[36]. A randomized phase II study is ongoing comparing bevaci‐ zumab alone to bevacizumab and everolimus in patients with recurrent ovarian cancer (NCT00886691, Table 4). Patients will be stratified according to their platinum-free interval or prior treatment with bevacizumab. This study should provide valuable insight into the

**enrolled**

**N, ovarian cancer**

54 54 G3-4 GI (10%),

15 7 G3-4 neutropenia and

20 NA G3-4 fatigue (5%),

39 6 G3-4 neutropenia

16 3 G3 neutropenia, anemia,

31 31

**Abbreviations:** N: number of patients; IV: intravenous; D: day; Q: every; G3-4: grade 3-4; RR: response rate; PFS: pro‐ gression-free survival; SD: stable disease; MTD: maximum tolerated dose; PLD: pegylated liposomal doxorubicin; NA:

evaluable for toxicity and 25 evaluable for efficacy (89%),

thrombocytopenia (21%), pulmonary (5%)

thrombocytopenia, mucositis, fatigue

G3-4 fatigue (13%), mucositis (13%), HTN (6%), neutropenia (10%), rash (3%), colonic perforation (6%)

**Selected toxicities Efficacy**

RR=9% 6 month PFS=24%

RR=0 One SD for 6 months

NA

NA

RR=12% 6month PFS 56%

RR= 50% (3/6) SD=50% (3/6)

metabolic (15%), pulmonary (6%)

thrombocytopenia

nausea (16%), mucositis (21%), vomiting (16%), rash (11%), hand-foot syndrome (21%)

potential additive benefit of this combinatorial strategy.

8, 15, 22 Q28 days

8, 15, 22 + topotecan 1mg/m2 IV D1, 8, 15 Q28

Ib MTD= temsirolimus IV 15mg D1, 8, 15, 22 + PLD IV 40mg/m2 D1 Q28 days

I MTD= temsirolimus IV 25mg D1 and 8 + carbo AUC5 IV D1 + Pac IV 175mg/m2 D1

Pac 80mg/m2 D 1, 8, 15 Q

8, 15, 22 + Bev 10mg/kg D1 and 15 Q 28 days

information not available; carbo: carboplatin; pac: paclitaxel; PO: per os.

**Table 2.** Completed clinical trials of mTOR inhibitors in ovarian cancer

Q 21 days

28 days

Campone et al I Everolimus PO 30mg daily +

Morgan et al II Temsirolimus IV 25mg D 1,

**Reference Phase Treatment N, total**

Behbakht et al II Temsirolimus, 25mg IV D1,

282 Ovarian Cancer - A Clinical and Translational Update

Temkin et al I Temsirolimus IV 25mg D 1,

Boers-Sonderen et al

Kollsmannberg er et al

days

Targeting Akt upstream from mTOR may produce a more effective knock-down of signal transduction and a number of Akt inhibitors have therefore been generated. These include ATP-competitive inhibitors, allosteric inhibitors, peptide-based inhibitors and lipid-based inhibitors (reviewed in Stronach et al[37]). Akt inhibitors are still in early stages of clinical development and two compounds have been specifically tested in ovarian cancer (Table 3).

The most mature inhibitor in clinical development is the lipid-based inhibitor, perifosine, it interferes with the cell membrane recruitment of Akt (thus preventing activation). However early data in phase I and II trials in other tumor types were disappointing with frequent gas‐ trointestinal toxicity and a lack of meaningful activity[38]-[41]. Given the suggestion that the narrow therapeutic window of perifosine may limit its clinical usefulness, combination trials with conventional cytotoxics have been conducted in order to improve the therapeutic in‐ dex. Preclinical studies have shown that perifosine inhibited ovarian cancer cell prolifera‐ tion, motility and angiogenesis and potentiated paclitaxel sensitivity in vitro and in vivo[42], [43]. On this basis, a phase I trial of perifosine and docetaxel in platinum and taxane resist‐ ant ovarian cancer was conducted[42]. Perifosine was given at a loading dose of 100mg ev‐ ery 6 hours for 4 doses followed by a daily dose according to dose level (50, 100 or 150mg daily) in combination with docetaxel 75mg/m2 day 1 every 3 weeks. Twenty one patients were enrolled including 11 at the MTD level of perifosine 150mg. No DLTs were observed, frequent adverse events included nausea, vomiting, anorexia, constipation and fatigue. With regards to efficacy at the MTD (N=11), there was one PR in an endometrioid ovarian cancer with a loss of function *PTEN* mutation (R130Q) and one SD maintained for 4 months in a PI3K mutated clear cell tumor. Two other patients without apparent PI3K alterations ach‐ ieved SD while two patients with KRAS mutations progressed quickly. The investigators al‐ so performed pharmacodynamic studies using reverse phase protein array (RPPA) to detect changes in total and phosphorylated markers in pre-treatment versus day 7 tumor biopsies and functional imaging studies using FDG-PET scans. Bcl2 and ERK2 levels were increased by treatment suggesting that the low response rate may be in part explained by perifosine induced increases in alternate signalling pathways. However FDG-PET responses at one week correlated with inhibition of S6 phosphorylation raising the possibility that FDG-PET may serve as an early surrogate indicator of Akt inhibition.

GSK795 is an oral ATP-competitive pan-Akt inhibitor in early stages of development and a small phase I pharmacodynamic and pharmacokinetic study was conducted in order to characterize the relationship between AKT inhibition by GSK795 and downstream effects in patients with advanced platinum resistant ovarian cancer[44]. Twelve patients were enrol‐ led. The only toxicities were grade 2 anorexia (18%) and vomiting (18%). FDG metabolism decreased in the majority of tumors but there was no dose response relationship. Among 5 patients treated at the higher dose levels, paired pre- and post-treatment tumor biopsies demonstrated downregulation in pAkt and in the tumor proliferative marker, Ki67. Two pa‐ tients have achieved >6 months PFS with objective tumor regressions of 26% and 11%, re‐ spectively.

cancer patient, and 7 patients had stable disease for more than 8 months. Five of these 7 pa‐ tients had either PTEN loss or PI3K mutation. GDC0941 is an oral selective class I PI3K in‐ hibitor that showed evidence of clinical activity in 3 patients enrolled in a phase I trial, including one ovarian cancer (PTEN negative) patient who remained on study for 5 months with a FDG-PET response, >50% decrease in pS6 staining in paired biopsies, and 80% de‐ crease in CA-125[48]. XL147 is another selective PI3K inhibitor which was well tolerated in a phase I trial with rash as the main DLT. An associated trial of XL147 in combination with carboplatin and paclitaxel demonstrated that the combination was feasible with no evidence of PK interactions or overlapping toxicities and dose expansion cohorts are ongoing in ovar‐

The PI3K/Akt/mTOR Pathway in Ovarian Cancer: Biological Rationale and Therapeutic Opportunities

Despite a strong preclinical rationale, clinical trials of novel agents targeting the PI3K/Akt/ mTOR pathway in ovarian cancer have been disappointing. Given the complexity and re‐ dundancy of the PI3K signaling network, combined targeting may be required. The fact that all the trials conducted to date enrolled an unselected patient population may have diluted objective activity in a subset. It is therefore crucial that efforts are made to uncover resist‐ ance mechanisms, develop rationale combinatorial strategies, identify predictive biomark‐

Compensatory feedback loops may allow escape from blockade of a single effector of the pathway. Early on, paradoxical increases in pAkt were identified in preclinical models and

suppress MTORC1 but do not affect the other subunit of mTOR, MTORC2. MTORC2 is a positive regulator of Akt, and selective inhibition of MTORC1 results in compensatory in‐ crease in Akt phosphorylation at Serine 473[50]. Rapalog-induced rebound Akt activation has been proposed as one of the mechanisms accounting for resistance to first generation in‐ hibitors in the clinic. In addition, although the function and downstream effectors of MTORC2 are less well described, it is reasonable to expect that complete abrogation of the whole mTOR complex may be required to achieve a robust anti-tumor effect. As a result, mTORC1/mTORC2 dual inhibitors have been developed such as DS3078a, INK128,

Another postulated compensatory escape route from mTOR inhibition is via insulin growth factor 1 receptor (IGF1R, see Figure 2)[52]. Insulin receptor substrate-1 is normally under basal negative regulation via phosphorylation by mTOR; mTOR inhibition prevents IRS-1 phosphorylation thus allowing IRS-1 to complex with IGF1R and promote Akt signaling[53]

thereby generating another positive feedback loop accounting for resistance.

. As illustrated in Figure 2, rapalogs

http://dx.doi.org/10.5772/54170

285

**5. Challenges of PI3K/Akt/mTOR pathway inhibitors**

ian cancer[49].

**5.1. Resistance**

ers, and explore novel trial designs.

*5.1.1. Feedback loops via MTORC2 or IRS1*

in tumors from patients treated with mTOR inhibitors8

AZD8055, OSI027 and AZD2014 (reviewed in [51]).

In addition to the aforementioned inhibitors, Akt isoform specific inhibitors are being developed, however the distinct functions of each of these isoforms and their rele‐ vance to different tumor types or individual tumor genetic background is still poorly understood. Studies of AKT isoform knockouts provide some insight into their relative roles: AKT1 loss is associated with impaired fetal development and increased fetal mortality; AKT2 loss leads to diabetes and AKT3 loss results in defective central nerv‐ ous system development[45].


**Table 3.** Completed clinical trials of Akt inhibitors in ovarian cancer

#### **4.5. PI3K inhibitors**

The PI3K inhibitors, LY290002 and wortmannin have been used for years as tools in preclin‐ ical experiments to demonstrate the biological relevance of PI3K and explore its potential as a therapeutic target in cancer. However, the micromolar IC50 (50% inhibitory concentration) and off-target effects of these agents have limited their clinical applicability. Less toxic PI3K inhibitors are just entering phase II stages of clinical development (reviewed in Kurtz et al[46]). BKM120 is an oral selective PI3K inhibitor with an IC50 for the PI3K kinase of 35nM. A dose escalation phase I trial has shown that the drug is well tolerated at the MTD of 100mg once a day with rash, hyperglycemia, diarrhea and mood alterations in over a third of patients[47]. BKM120 demonstrated dose dependent inhibition of FDG activity and downregulation in p-S6 in skin biopsies. The only response was in a KRAS mutated breast cancer patient, and 7 patients had stable disease for more than 8 months. Five of these 7 pa‐ tients had either PTEN loss or PI3K mutation. GDC0941 is an oral selective class I PI3K in‐ hibitor that showed evidence of clinical activity in 3 patients enrolled in a phase I trial, including one ovarian cancer (PTEN negative) patient who remained on study for 5 months with a FDG-PET response, >50% decrease in pS6 staining in paired biopsies, and 80% de‐ crease in CA-125[48]. XL147 is another selective PI3K inhibitor which was well tolerated in a phase I trial with rash as the main DLT. An associated trial of XL147 in combination with carboplatin and paclitaxel demonstrated that the combination was feasible with no evidence of PK interactions or overlapping toxicities and dose expansion cohorts are ongoing in ovar‐ ian cancer[49].

#### **5. Challenges of PI3K/Akt/mTOR pathway inhibitors**

Despite a strong preclinical rationale, clinical trials of novel agents targeting the PI3K/Akt/ mTOR pathway in ovarian cancer have been disappointing. Given the complexity and re‐ dundancy of the PI3K signaling network, combined targeting may be required. The fact that all the trials conducted to date enrolled an unselected patient population may have diluted objective activity in a subset. It is therefore crucial that efforts are made to uncover resist‐ ance mechanisms, develop rationale combinatorial strategies, identify predictive biomark‐ ers, and explore novel trial designs.

#### **5.1. Resistance**

decreased in the majority of tumors but there was no dose response relationship. Among 5 patients treated at the higher dose levels, paired pre- and post-treatment tumor biopsies demonstrated downregulation in pAkt and in the tumor proliferative marker, Ki67. Two pa‐ tients have achieved >6 months PFS with objective tumor regressions of 26% and 11%, re‐

In addition to the aforementioned inhibitors, Akt isoform specific inhibitors are being developed, however the distinct functions of each of these isoforms and their rele‐ vance to different tumor types or individual tumor genetic background is still poorly understood. Studies of AKT isoform knockouts provide some insight into their relative roles: AKT1 loss is associated with impaired fetal development and increased fetal mortality; AKT2 loss leads to diabetes and AKT3 loss results in defective central nerv‐

**enrolled**

The PI3K inhibitors, LY290002 and wortmannin have been used for years as tools in preclin‐ ical experiments to demonstrate the biological relevance of PI3K and explore its potential as a therapeutic target in cancer. However, the micromolar IC50 (50% inhibitory concentration) and off-target effects of these agents have limited their clinical applicability. Less toxic PI3K inhibitors are just entering phase II stages of clinical development (reviewed in Kurtz et al[46]). BKM120 is an oral selective PI3K inhibitor with an IC50 for the PI3K kinase of 35nM. A dose escalation phase I trial has shown that the drug is well tolerated at the MTD of 100mg once a day with rash, hyperglycemia, diarrhea and mood alterations in over a third of patients[47]. BKM120 demonstrated dose dependent inhibition of FDG activity and downregulation in p-S6 in skin biopsies. The only response was in a KRAS mutated breast

**N, ovarian cancer**

21 21 Nausea, vomiting,

12 12 G2 anorexia (18%),

**Selected toxicities Efficacy**

At MTD (N=11) PR in 1 PTEN null, SD 3/11.

16% SD for 6 mo (2/12) with tumor shrinkage of 26% and 11%

anorexia, fatigue

vomiting (18%)

spectively.

ous system development[45].

284 Ovarian Cancer - A Clinical and Translational Update

**Reference Phase Treatment N, total**

150mg/day + docetaxel, 75mg IV D1 Q21 days

Fu et al I MTD Perifosine orally

Gungor et al I GSK795 25, 50 or 75mg

**4.5. PI3K inhibitors**

orqlly/day

**Table 3.** Completed clinical trials of Akt inhibitors in ovarian cancer

#### *5.1.1. Feedback loops via MTORC2 or IRS1*

Compensatory feedback loops may allow escape from blockade of a single effector of the pathway. Early on, paradoxical increases in pAkt were identified in preclinical models and in tumors from patients treated with mTOR inhibitors8 . As illustrated in Figure 2, rapalogs suppress MTORC1 but do not affect the other subunit of mTOR, MTORC2. MTORC2 is a positive regulator of Akt, and selective inhibition of MTORC1 results in compensatory in‐ crease in Akt phosphorylation at Serine 473[50]. Rapalog-induced rebound Akt activation has been proposed as one of the mechanisms accounting for resistance to first generation in‐ hibitors in the clinic. In addition, although the function and downstream effectors of MTORC2 are less well described, it is reasonable to expect that complete abrogation of the whole mTOR complex may be required to achieve a robust anti-tumor effect. As a result, mTORC1/mTORC2 dual inhibitors have been developed such as DS3078a, INK128, AZD8055, OSI027 and AZD2014 (reviewed in [51]).

Another postulated compensatory escape route from mTOR inhibition is via insulin growth factor 1 receptor (IGF1R, see Figure 2)[52]. Insulin receptor substrate-1 is normally under basal negative regulation via phosphorylation by mTOR; mTOR inhibition prevents IRS-1 phosphorylation thus allowing IRS-1 to complex with IGF1R and promote Akt signaling[53] thereby generating another positive feedback loop accounting for resistance.

*5.1.3. Dysfunctional apoptotic machinery*

*5.1.4. Cell cycle dependent kinase (cdk) inhibitors*

mor biopsies[42].

PI3K/Akt inhibition[58].

**5.2. Combinatorial strategies**

(horizontal combinations).

*5.2.1. Vertical combinations*

With membrane growth factor receptor inhibitors

are ongoing in ovarian cancer (Table 4).

Even in tumor types such as renal cell or pancreatic neuroendocrine cancers where mTOR inhibitors have demonstrated sufficient clinical benefit to justify FDA approval, objective tu‐ mor responses are sporadic[56]. Some researchers have hypothesized that tumor shrinkage in response to mTOR inhibitors requires a functional apoptotic machinery. Majumder et al demonstrated that rapamycin-resistant SKOV3 ovarian cells have an activated PI3K path‐ way but upregulated levels of the anti-apoptotic protein, bcl2, and bcl2 knock-down using siRNA restored rapamycin sensitivity[57]. In line with this preclinical data, the Phase I trial of the Akt inhibitor perifosine reported compensatory increases in bcl2 in post treatment tu‐

The PI3K/Akt/mTOR Pathway in Ovarian Cancer: Biological Rationale and Therapeutic Opportunities

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287

One of the major anti-tumor effects of PI3K blockade is to activate the cdk inhibitors p27 and p21, allow their nuclear translocation where they interact with, and inhibit cdks, thereby promoting cell cycle arrest. p27-null cells are resistant to rapamycin in vitro, some therefore postulate that tumors that have very low levels of p27 may therefore be less responsive to

Given the presence of redundant pathways and the adaptive capacity of cancer cells, drug combinations are increasingly being investigated in an effort to abrogate both primary and acquired resistance to PI3K pathway inhibitors. Different approaches include targeting the same pathway at different levels (vertical combinations) or aiming for different pathways

Activation of the PI3K pathway can be attributable to upstream activation via membrane re‐ ceptor kinases, and preclinical data suggest that concurrent inhibition of mTOR and EGFR may result in synergistic anti-tumor effect. Studies are investigating the benefit of dual mTOR/EGFR blockade[59]. A completed phase I trial showed that the combination of evero‐ limus, bevacizumab and panitumumab was well tolerated, and three patients with ovarian cancer achieved prolonged disease control for 11 to >40 months[59]. In addition, mTOR in‐ hibition may induce IRS1 expression and promote Akt activation via IGF1R thus attenuating the anti-tumor effects of rapalogs[60]. The addition of IFG1R antibodies to mTOR inhibitors has been shown to improve growth inhibition in vitro[52]. Studies investigating concurrent IGF1R/mTOR targeting have shown that treatment is feasible with an acceptable toxicity profile and encouraging activity in other tumor types[61] and studies using this approach

**Figure 2. Proposed mechanisms accounting for resistance to inhibitors of the PI3K pathway.** (1) Selective block‐ ade of MTORC1 by rapalogs increases MTORC2 and results in positive feedback activation of pAkt. (2) Inhibition of mTOR removes the basal inhibition of IRS1, now free to bind to and activate IGF1R and promote PI£K activation. (3) In the presence of constitutive activation of KRAS, abrogation of the PI3K pathway alone does not inhibit cancer cell growth. (4) A dysfunctional apoptotic pathway (high bcl2, high survivin...) may lead to resistance to the pro-apoptotic effects of PI3K apthway inhibitors. (5) One downstream effect of Akt inhibition is cell cyle arrest via increase in the cdk inhibitors, p21 or p27; p27 low tumors may be resistant to PI3K pathway inhibitor induced cell cycles arrest.

#### *5.1.2. The Ras pathway: KRAS/BRAF mutations and compensatory increases in Erk signaling*

Interactions with parallel pathways may also allow escape from PI3K inhibition. Akt has been shown to be phosphorylated via cross-talk with Ras. Thus, in KRAS mutant tumors primarily driven by a constitutively upregulated Ras pathway, PI3k pathway inhibitors alone are unlikely to be effective. This hypothesis is supported by studies demonstrating that KRAS or BRAF mutated tumors are insensitive to mTOR inhibitors. Using a panel of cell lines including ovarian cancer, PI3K mutated tumors were shown to be sensitive, while dual PI3K and KRAS or BRAF mutated tumors were resistant to everolimus[54]. Important‐ ly, they also demonstrated that knock-down of the KRAS mutation in these cells restored ev‐ erolimus sensitivity in vitro and in vivo. In the presence of KRAS or BRAF mutations, tumors may exhibit 'oncogenic addiction' to an alternate survival pathway, e.g. Ras-Raf-Mek-Erk. This illustrates the fact that sensitivity to PI3K transduction inhibitors may require not only pathway activation but also demonstration of pathway dependence.

In addition to reactivating Akt, rapalogs have been reported to cause treatment induced in‐ creases in Mek/Erk signalling. In mice models and human tumors, everolimus increased Erk1/2 activation in post treatment tumor samples, suggesting the existence of crosstalk be‐ tween the PI3K/mTOR and Mek/Erk signal transduction cascades[55]. Selective targeting of one pathway may simply result in compensatory upregulation in the other, and vice versa.

#### *5.1.3. Dysfunctional apoptotic machinery*

Even in tumor types such as renal cell or pancreatic neuroendocrine cancers where mTOR inhibitors have demonstrated sufficient clinical benefit to justify FDA approval, objective tu‐ mor responses are sporadic[56]. Some researchers have hypothesized that tumor shrinkage in response to mTOR inhibitors requires a functional apoptotic machinery. Majumder et al demonstrated that rapamycin-resistant SKOV3 ovarian cells have an activated PI3K path‐ way but upregulated levels of the anti-apoptotic protein, bcl2, and bcl2 knock-down using siRNA restored rapamycin sensitivity[57]. In line with this preclinical data, the Phase I trial of the Akt inhibitor perifosine reported compensatory increases in bcl2 in post treatment tu‐ mor biopsies[42].

#### *5.1.4. Cell cycle dependent kinase (cdk) inhibitors*

One of the major anti-tumor effects of PI3K blockade is to activate the cdk inhibitors p27 and p21, allow their nuclear translocation where they interact with, and inhibit cdks, thereby promoting cell cycle arrest. p27-null cells are resistant to rapamycin in vitro, some therefore postulate that tumors that have very low levels of p27 may therefore be less responsive to PI3K/Akt inhibition[58].

#### **5.2. Combinatorial strategies**

**Figure 2. Proposed mechanisms accounting for resistance to inhibitors of the PI3K pathway.** (1) Selective block‐ ade of MTORC1 by rapalogs increases MTORC2 and results in positive feedback activation of pAkt. (2) Inhibition of mTOR removes the basal inhibition of IRS1, now free to bind to and activate IGF1R and promote PI£K activation. (3) In the presence of constitutive activation of KRAS, abrogation of the PI3K pathway alone does not inhibit cancer cell growth. (4) A dysfunctional apoptotic pathway (high bcl2, high survivin...) may lead to resistance to the pro-apoptotic effects of PI3K apthway inhibitors. (5) One downstream effect of Akt inhibition is cell cyle arrest via increase in the cdk

inhibitors, p21 or p27; p27 low tumors may be resistant to PI3K pathway inhibitor induced cell cycles arrest.

286 Ovarian Cancer - A Clinical and Translational Update

*5.1.2. The Ras pathway: KRAS/BRAF mutations and compensatory increases in Erk signaling*

not only pathway activation but also demonstration of pathway dependence.

Interactions with parallel pathways may also allow escape from PI3K inhibition. Akt has been shown to be phosphorylated via cross-talk with Ras. Thus, in KRAS mutant tumors primarily driven by a constitutively upregulated Ras pathway, PI3k pathway inhibitors alone are unlikely to be effective. This hypothesis is supported by studies demonstrating that KRAS or BRAF mutated tumors are insensitive to mTOR inhibitors. Using a panel of cell lines including ovarian cancer, PI3K mutated tumors were shown to be sensitive, while dual PI3K and KRAS or BRAF mutated tumors were resistant to everolimus[54]. Important‐ ly, they also demonstrated that knock-down of the KRAS mutation in these cells restored ev‐ erolimus sensitivity in vitro and in vivo. In the presence of KRAS or BRAF mutations, tumors may exhibit 'oncogenic addiction' to an alternate survival pathway, e.g. Ras-Raf-Mek-Erk. This illustrates the fact that sensitivity to PI3K transduction inhibitors may require

In addition to reactivating Akt, rapalogs have been reported to cause treatment induced in‐ creases in Mek/Erk signalling. In mice models and human tumors, everolimus increased Erk1/2 activation in post treatment tumor samples, suggesting the existence of crosstalk be‐ tween the PI3K/mTOR and Mek/Erk signal transduction cascades[55]. Selective targeting of one pathway may simply result in compensatory upregulation in the other, and vice versa.

Given the presence of redundant pathways and the adaptive capacity of cancer cells, drug combinations are increasingly being investigated in an effort to abrogate both primary and acquired resistance to PI3K pathway inhibitors. Different approaches include targeting the same pathway at different levels (vertical combinations) or aiming for different pathways (horizontal combinations).

#### *5.2.1. Vertical combinations*

#### With membrane growth factor receptor inhibitors

Activation of the PI3K pathway can be attributable to upstream activation via membrane re‐ ceptor kinases, and preclinical data suggest that concurrent inhibition of mTOR and EGFR may result in synergistic anti-tumor effect. Studies are investigating the benefit of dual mTOR/EGFR blockade[59]. A completed phase I trial showed that the combination of evero‐ limus, bevacizumab and panitumumab was well tolerated, and three patients with ovarian cancer achieved prolonged disease control for 11 to >40 months[59]. In addition, mTOR in‐ hibition may induce IRS1 expression and promote Akt activation via IGF1R thus attenuating the anti-tumor effects of rapalogs[60]. The addition of IFG1R antibodies to mTOR inhibitors has been shown to improve growth inhibition in vitro[52]. Studies investigating concurrent IGF1R/mTOR targeting have shown that treatment is feasible with an acceptable toxicity profile and encouraging activity in other tumor types[61] and studies using this approach are ongoing in ovarian cancer (Table 4).


**Treatment type Phase Experimental**

antiangiogenic therapy

mTOR or Akt inhibitor + IGF1R inhibitor

mTOR inhibitor in combination with Notch pathway inhibitor

**treatment**

II Temsirolimus + bevacizumab

I Temsirolimus + Cediranib (VEGFR 2 inhibitor)

II Everolimus +/ bevacizumab randomised trial

IB MK-2206 (Akt inhibitor) or ridaforolimus + IGF1R Ab (dalotuzumab), non randomized study

I Ridaforolimus + MK-0752

**Prior treatment Selection**

Previously treated

<2 prior line of CT for recurrent disease

Platinum-based

CT. Stratification according to platinum resistant vs. not, measurable disease vs. not and prior bevacizumab vs.

not

tor ; IGF1R : insulin-like growth factor receptor ; AUC : area under the curve.

**Table 4.** Ongoing trials of PI3K pathway inhibitors in ovarian cancer

Combined PI3K-mTOR or Akt-mTOR inhibition

Previously treated. Platinum resistance required for MK-2206 arm **criteria (biomarker vs allcomers)**

**Secondary endpoints Clinical**

All comers RR, PFS at 6 months, OS, duration of response, TTP. No specific biomarker objectives specified but

The PI3K/Akt/mTOR Pathway in Ovarian Cancer: Biological Rationale and Therapeutic Opportunities

blood and tumor collected on all

All comers Number of participants with dose limiting toxicities,

<3 prior CT lines All comers Number of participants with dose limiting toxicities,

**Abbreviations:** PARP : poly-ADP-ribose polymerase ; CT : chemotherapy ; MTD : maximum tolerated dose, PK : phar‐ macokinetic ; PD : pharmacodynamic ; BRCA : breast cancer susceptibility gene ; IHC : immunohisotchemistry ; FDG/ PET : fluorodeoxyglucose positron emission tomography ; RR : response rate ; PFS : progression-free survival ; OS : over‐ all survival ; carbo : carboplatin ; pac : paclitaxel ; NY-ESO-1 : cancer-testis antigen-1 ; LAGE-1 : cancer-testis antigen-2 ; PLD : pegylated liposomal doxorubicin ; TTP : time to progression ; VEGFR : vascular endothelial growth factor recep‐

As previously discussed, positive feedback loops generated by selective mTOR inhibition may result in paradoxical activation of Akt via mTORC2 and account for early resistance.

number of participants whose best response is a partial response (PR) or complete response (CR)

AUC for the ridaforolimus + MK-0752 doublet

All comers MTD , response rate, clinical benefit NCT01065662

All comers PFS, tolerability, OS, RR, CA-125 response. NCT00886691

**trial.gov identifier** 289

http://dx.doi.org/10.5772/54170

NCT01010126

NCT01243762

NCT01295632

The PI3K/Akt/mTOR Pathway in Ovarian Cancer: Biological Rationale and Therapeutic Opportunities http://dx.doi.org/10.5772/54170 289


**Abbreviations:** PARP : poly-ADP-ribose polymerase ; CT : chemotherapy ; MTD : maximum tolerated dose, PK : phar‐ macokinetic ; PD : pharmacodynamic ; BRCA : breast cancer susceptibility gene ; IHC : immunohisotchemistry ; FDG/ PET : fluorodeoxyglucose positron emission tomography ; RR : response rate ; PFS : progression-free survival ; OS : over‐ all survival ; carbo : carboplatin ; pac : paclitaxel ; NY-ESO-1 : cancer-testis antigen-1 ; LAGE-1 : cancer-testis antigen-2 ; PLD : pegylated liposomal doxorubicin ; TTP : time to progression ; VEGFR : vascular endothelial growth factor recep‐ tor ; IGF1R : insulin-like growth factor receptor ; AUC : area under the curve.

**Table 4.** Ongoing trials of PI3K pathway inhibitors in ovarian cancer

#### Combined PI3K-mTOR or Akt-mTOR inhibition

**Treatment type Phase Experimental**

PI3K inhibitor I BKM120 +

**treatment**

288 Ovarian Cancer - A Clinical and Translational Update

Olaparib (PARP inhibitor)

II MK-2206 Platinum

I Perifosine + docetaxel

I/II GSK2110183 + carbo+pac

I Sirolimus +

II Temsirolimus + carbo + pac

I Everolimus + PLD + carbo

I Ridaforolimus + carbo + pac

> Temsirolimus + carbo + pac followed by maintenance temsirolimus

II Adjuvant

II Everolimus + bevacizumab

ALVAC(2)-NY-ESO-1 vaccine

II Temsirolimus Taxane based

1st generation MTOR inhibitor

1st generation mTOR inhibitor in combination with **Prior treatment Selection**

First line platinium-based

resistant

Platinum resistant, "/>2 prior lines of CT

Not specified Tumor

treatment, <3 prior CT

Refractory to standard treatment

One prior platinum/ taxane-CT

Previously treated

First line Clear cell

histology only

CT

**criteria (biomarker vs allcomers)**

AKT inhibitor I GSK2141795 Not specified All comers PK and PD by FDG/PET NCT01266954

*P13K* or *AKT* mutation or low PTEN expression

expression of NY-ESO-1 or LAGE-1

**Secondary endpoints Clinical**

markers of PI3K inhibition, determination of BRCA1 IHC, BRCA1 promoter hypermethylation and BRCA1/2 somatic mutation status

RR, PFS and OS, toxicities of MK-2206, explore the association between select biomarkers and response to MK-2206, to explore the development of feedback loop activation and target inhibition

All comers Phase I : safety and tolerability NCT01653912

Safety, effectiveness of sirolimus on enhancing vaccine efficacy, antibody titers, NY-ESO-1 specific CD8+ and CD4+ frequency and function, PFS.

antigen 125 (for ovarian cancer), overall survival, safety and toxicity, quality of life, rate and duration

All comers MTD, toxicity, RR, PK. NCT00408655

PFS at 12 months, median PFS, OS, toxicity and RR.

All comers MTD for the combination, safety, PK, efficacy. PD

with MK-2206.

Phase II : overall RR

of stable diseases

tumor activity

Not specified All comers Tumor response NCT00431054

All comers PFS, rate and duration of stable diseases, cancer

All comers MTD for the combination, safety/tolerability, anti-

<4 prior CT lines All comers MTD, preliminary efficacity, toxicity NCT01256268

mTOR signaling pathway by IHC.

All comers PFS at 6 months, complete response + partial response + stable disease

**trial.gov identifier**

NCT01623349

NCT01283035

NCT01536054

NCT01460979

NCT01281514

NCT01196429

NCT01031381

As previously discussed, positive feedback loops generated by selective mTOR inhibition may result in paradoxical activation of Akt via mTORC2 and account for early resistance. Dual MTORC1 and MTORC2 inhibitors have therefore been developed and shown to result in greater anti-tumor activity than rapalogs in preclinical studies[62]. Another strategy in‐ volves co-targeting mTOR as well as upstream PI3K in order to overcome the positive feed‐ back loops via Akt. In addition, simultaneous targeting of several effectors of the PI3K pathway may improve the likelihood of completely shutting down the signaling cascade. A combination of everolimus and the PI3K inhibitor, PI-103 blocked rebound rapalog induced Akt activation and resulted in greater cell cycle arrest than either treatment alone in ovarian cancer cells[63]. NVP-BEZ235 is a novel agent that is both an ATP-competitive PI3K inhibi‐ tor and an inhibitor of both mTORC1 and mTORC2. Studies in ovarian cancer cell lines and mouse models have suggested that this drug caused cell cycle arrest and apoptosis, and pro‐ longed survival of mice with established ovarian tumors[64]. A phase I trial of ridaforolimus with the Akt inhibitor MK2206 is ongoing and a dose expansion cohort in ovarian cancer is planned (NCT01295632). Other studies are exploring the benefit of inhibiting further down‐ stream effectors such as p70S6 in combination with everolimus (NCT01115803).

cancer. Given the putative anti-angiogenic effects of PI3K pathway inhibitors and the known activity of the VEGF antibody, bevacizumab in ovarian cancer, there is a rationale for target‐ ing multiple angiogenic regulators at once in an effort to shut down angiogenesis complete‐ ly. In fact, clear cell ovarian cancers with their reported angiogenic signature and increased HIF1α signaling[68] may be particularly suited to a therapeutic strategy combining tradi‐

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291

In light of the heterogeneity of ovarian cancer, predictive as well as pharmacodynamic (demonstrating target downregulation) biomarkers are desperately needed in order to select patients most likely to respond. In addition biomarkers would be useful to identify the sub‐ set of patients who may benefit from specific combinations. One question is whether sensi‐

The main intrinsic effectors of the pathway that have been studied in preclinical and clinical models have been PTEN loss, and PIK3CA activating mutations. Early studies in cell lines including ovarian cancer demonstrated greater anti-proliferative activity of PI3K pathway inhibitors in PTEN-null or PIK3CA mutated cells[69]-[71], Di Nicolantonio et al, showed in cell lines and in 43 patient tumor samples that PIK3CA mutations sensitized cancer cells to everolimus, but co-existing KRAS or BRAF mutations predicted resistance[54]. More recent clinical and preclinical studies have reported contradictory correlations between PI3K muta‐ tions or PTEN loss and response to inhibitors[72],[73]; in particular, a significant number of PI3K mutated tumors fail to respond, while a proportion of tumors lacking PI3K and PTEN alterations respond. This is in contrast to the much stronger association between activating mutations and response to other targeted agents such as EGFR, BRAF or ALK inhibitors. Studies in tumor types with frequent PTEN mutations, such as melanoma have not demon‐ strated significant responses to mTOR inhibitors suggesting that patient selection on the ba‐ sis of PTEN loss alone may not identify responders[74]. In a pooled analysis of 3 trials of mTOR inhibitors in endometrial cancers, MacKay et al found no correlation between PIK3CA mutation or PTEN loss and response[75]. However a recent report by Janku and colleagues suggested that PI3K mutations did preferentially identify responders[76]. They conducted mutational analyses on 140 patients with breast and gynecological malignancies (including 60 with ovarian cancer) enrolled in phase I trials of PI3K/Akt/mTOR inhibitors. They demonstrated that the response rate was higher among patients with PIK3CA mutated tumors (RR=30% versus 10%). However these results should be interpreted in light of the fact that all responders were included in a trial of temsirolimus, bevacizumab and liposomal doxorubicin. Given the known activity of bevacizumab and liposomal doxorubicin in ovari‐ an cancer and the fact that half the responding patients had never been previously exposed to liposomal doxorubicin, mutations may simply correlate with prognosis, or with an im‐

tivity can be predicted on the basis of activation status of pathway members.

*5.3.1. Constitutive PI3K activity: PIK3CA mutations and PTEN loss of function*

tional anti-angiogenics with PI3K pathway inhibitors.

proved response to treatment in general.

**5.3. Biomarkers**

#### *5.2.2. Horizontal combinations*

#### With Mek inhibitors

Given the evidence that oncogenic activation of the ras pathway may be associated with re‐ sistance to mTOR inhibitors even in the presence of PI3K oncogenic mutations, targeting both PI3K and Ras pathways simultaneously is worthy of investigation. In a mouse model of ovarian cancer driven by PTEN loss and KRAS mutation, simultaneous blockade of both PI3K and Mek signalling using pharmacological inhibitors resulted in significant tumor re‐ gressions and prolonged survival compared to monotherapy[65]. A phase I study compar‐ ing the tolerability and efficacy of dual PI3K and Mek targeting to either treatment alone showed that the combination significantly increased the risk of Grade 3-4 toxicity from 18% to 54% (p=0.001), but all patients with alterations in the PI3K pathway and a KRAS or BRAF mutation had tumor regressions with dual targeting[66].

#### With chemotherapy

One of the earliest explored strategy has been the combination of novel inhibitors with che‐ motherapy. There has been the theoretical concern that the cytostatic effects of these drugs may in fact antagonize the cell cycle dependent effects of chemotherapy. Preclinical studies in ovarian cancer have indeed suggested that PI3K inhibitor-induced G1 arrest undermined the cytotoxic effects of agents such as cisplatin, paclitaxel, gemcitabine and topotecan that are primarily effective in the S or G2 phase of the cell cycle[67]. However preliminary data from non-randomized studies of mTOR inhibitors in combination with chemotherapy have reported objective response rates comparable to those expected for chemotherapy alone, thus providing indirect evidence for a lack of antagonism. Randomized studies will be re‐ quired to rule out any antagonism between PI3K inhibitors and conventional cytotoxics.

#### With anti-angiogenics

Pro-angiogenic factors such as HIF1α and VEGF are downregulated by inhibition of PI3K signaling. This may explain the activity of mTOR inhibitors in HIF1α-driven clear cell renal cancer. Given the putative anti-angiogenic effects of PI3K pathway inhibitors and the known activity of the VEGF antibody, bevacizumab in ovarian cancer, there is a rationale for target‐ ing multiple angiogenic regulators at once in an effort to shut down angiogenesis complete‐ ly. In fact, clear cell ovarian cancers with their reported angiogenic signature and increased HIF1α signaling[68] may be particularly suited to a therapeutic strategy combining tradi‐ tional anti-angiogenics with PI3K pathway inhibitors.

#### **5.3. Biomarkers**

Dual MTORC1 and MTORC2 inhibitors have therefore been developed and shown to result in greater anti-tumor activity than rapalogs in preclinical studies[62]. Another strategy in‐ volves co-targeting mTOR as well as upstream PI3K in order to overcome the positive feed‐ back loops via Akt. In addition, simultaneous targeting of several effectors of the PI3K pathway may improve the likelihood of completely shutting down the signaling cascade. A combination of everolimus and the PI3K inhibitor, PI-103 blocked rebound rapalog induced Akt activation and resulted in greater cell cycle arrest than either treatment alone in ovarian cancer cells[63]. NVP-BEZ235 is a novel agent that is both an ATP-competitive PI3K inhibi‐ tor and an inhibitor of both mTORC1 and mTORC2. Studies in ovarian cancer cell lines and mouse models have suggested that this drug caused cell cycle arrest and apoptosis, and pro‐ longed survival of mice with established ovarian tumors[64]. A phase I trial of ridaforolimus with the Akt inhibitor MK2206 is ongoing and a dose expansion cohort in ovarian cancer is planned (NCT01295632). Other studies are exploring the benefit of inhibiting further down‐

stream effectors such as p70S6 in combination with everolimus (NCT01115803).

mutation had tumor regressions with dual targeting[66].

Given the evidence that oncogenic activation of the ras pathway may be associated with re‐ sistance to mTOR inhibitors even in the presence of PI3K oncogenic mutations, targeting both PI3K and Ras pathways simultaneously is worthy of investigation. In a mouse model of ovarian cancer driven by PTEN loss and KRAS mutation, simultaneous blockade of both PI3K and Mek signalling using pharmacological inhibitors resulted in significant tumor re‐ gressions and prolonged survival compared to monotherapy[65]. A phase I study compar‐ ing the tolerability and efficacy of dual PI3K and Mek targeting to either treatment alone showed that the combination significantly increased the risk of Grade 3-4 toxicity from 18% to 54% (p=0.001), but all patients with alterations in the PI3K pathway and a KRAS or BRAF

One of the earliest explored strategy has been the combination of novel inhibitors with che‐ motherapy. There has been the theoretical concern that the cytostatic effects of these drugs may in fact antagonize the cell cycle dependent effects of chemotherapy. Preclinical studies in ovarian cancer have indeed suggested that PI3K inhibitor-induced G1 arrest undermined the cytotoxic effects of agents such as cisplatin, paclitaxel, gemcitabine and topotecan that are primarily effective in the S or G2 phase of the cell cycle[67]. However preliminary data from non-randomized studies of mTOR inhibitors in combination with chemotherapy have reported objective response rates comparable to those expected for chemotherapy alone, thus providing indirect evidence for a lack of antagonism. Randomized studies will be re‐ quired to rule out any antagonism between PI3K inhibitors and conventional cytotoxics.

Pro-angiogenic factors such as HIF1α and VEGF are downregulated by inhibition of PI3K signaling. This may explain the activity of mTOR inhibitors in HIF1α-driven clear cell renal

*5.2.2. Horizontal combinations*

290 Ovarian Cancer - A Clinical and Translational Update

With Mek inhibitors

With chemotherapy

With anti-angiogenics

In light of the heterogeneity of ovarian cancer, predictive as well as pharmacodynamic (demonstrating target downregulation) biomarkers are desperately needed in order to select patients most likely to respond. In addition biomarkers would be useful to identify the sub‐ set of patients who may benefit from specific combinations. One question is whether sensi‐ tivity can be predicted on the basis of activation status of pathway members.

#### *5.3.1. Constitutive PI3K activity: PIK3CA mutations and PTEN loss of function*

The main intrinsic effectors of the pathway that have been studied in preclinical and clinical models have been PTEN loss, and PIK3CA activating mutations. Early studies in cell lines including ovarian cancer demonstrated greater anti-proliferative activity of PI3K pathway inhibitors in PTEN-null or PIK3CA mutated cells[69]-[71], Di Nicolantonio et al, showed in cell lines and in 43 patient tumor samples that PIK3CA mutations sensitized cancer cells to everolimus, but co-existing KRAS or BRAF mutations predicted resistance[54]. More recent clinical and preclinical studies have reported contradictory correlations between PI3K muta‐ tions or PTEN loss and response to inhibitors[72],[73]; in particular, a significant number of PI3K mutated tumors fail to respond, while a proportion of tumors lacking PI3K and PTEN alterations respond. This is in contrast to the much stronger association between activating mutations and response to other targeted agents such as EGFR, BRAF or ALK inhibitors. Studies in tumor types with frequent PTEN mutations, such as melanoma have not demon‐ strated significant responses to mTOR inhibitors suggesting that patient selection on the ba‐ sis of PTEN loss alone may not identify responders[74]. In a pooled analysis of 3 trials of mTOR inhibitors in endometrial cancers, MacKay et al found no correlation between PIK3CA mutation or PTEN loss and response[75]. However a recent report by Janku and colleagues suggested that PI3K mutations did preferentially identify responders[76]. They conducted mutational analyses on 140 patients with breast and gynecological malignancies (including 60 with ovarian cancer) enrolled in phase I trials of PI3K/Akt/mTOR inhibitors. They demonstrated that the response rate was higher among patients with PIK3CA mutated tumors (RR=30% versus 10%). However these results should be interpreted in light of the fact that all responders were included in a trial of temsirolimus, bevacizumab and liposomal doxorubicin. Given the known activity of bevacizumab and liposomal doxorubicin in ovari‐ an cancer and the fact that half the responding patients had never been previously exposed to liposomal doxorubicin, mutations may simply correlate with prognosis, or with an im‐ proved response to treatment in general.

In conclusion, if trials of PI3K/mTOR inhibitors had limited enrolment to PTEN null or PI3K mutated tumors a significant proportion of responding patients would have been missed. In light of the imperfect association between PI3K mutations or PTEN loss and response to PI3K pathway inhibitors, most ongoing trials are enrolling an unselected patient population; unfortunately, most of these studies do not appear to be collecting archival tumor samples for detailed molecular analyses (Table 4).

**6. Practical issues: Samples and trial design**

As the data to date suggest that there is insufficient evidence to select patients for trials of PI3K inhibitors on the basis of specific molecular alterations, it is imperative that fu‐ ture trials enrolling unselected patient populations include parallel biological studies in an effort to uncover candidate biomarkers. Biological assays must be reproducible, ro‐ bust and require access to high quality tumor samples. As such, pre-analytical variables must be controlled for as much as possible by following standardized sample collection, fixation, processing and storage procedures. When dealing with paraffin-embedded tis‐ sue, markers of the PI3K Akt pathway may be particularly susceptible to artefactual loss[80]. In fact, the optimal fixative for in depth genomic analyses is unlikely to be for‐ malin, and may therefore require a shift in routine practice from paraffin to fresh frozen

The PI3K/Akt/mTOR Pathway in Ovarian Cancer: Biological Rationale and Therapeutic Opportunities

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It is likely that clonal evolution and treatment selection pressure will lead to important genomic and/or phenotypic modifications in the tumor in the interval between diagnosis and relapse. An increasing number of phase I and II trials are therefore requesting op‐ tional biopsies of metastatic disease and the vast majority of patients are willing to con‐ sent this procedure. A study of patients enrolled in phase I trials at our institution revealed that 84% of patients who were proposed optional tumor biopsies consented to the procedure, including sequential pre- and post-treatment biopsies[85]. All procedures were performed using an 18-gauge needle under ultrasound or computed tomography scanning and were associated with low minor complication rates (9/145 tumor biopsies). In 70% of the cases the biopsy met quality criteria for ancillary molecular (RNA and DNA) analyses. Access to samples from relapsed disease is likely to be particularly rele‐ vant to high grade ovarian cancer, where the initial disease is exquisitely chemosensitive and repeat profiling of the chemoresistant recurrence may reveal a completely different

The molecular characterization of ovarian cancer clones surviving after chemotherapy could identify targets for novel agents designed to eradicate chemoresistant residual dis‐ ease. As discussed above, the combination of PI3K/Akt/mTOR inhibitors with chemother‐ apy may not be optimal because of the risk of cumulative toxicities as well as the theoretical risk that these inhibitors may antagonize the cytotoxic effects of chemothera‐ py. A more attractive approach may be sequential, where primarily chemosensitive ovar‐ ian cancer is treated with chemotherapy followed by PI3K inhibitors if indicated by the

**6.1. Access to quality ovarian cancer samples**

or RNAlater for sample storage.

*6.2.1. At relapse*

molecular profile.

*6.2.2. Residual disease post-chemotherapy*

**6.2. Access to post-treatment samples**

#### *5.3.2. pAkt and stathmin*

The level of phosphorylated Akt has been identified as a read-out for activation of the PI3K pathway and thus a potential biomarker for responsiveness to PI3K inhibitors. An in vitro and in silico study using a panel of cell lines and xenograft models treated with PI3K path‐ way inhibitors showed that pAkt correlated with efficacy, and KRAS or BRAF mutations with resistance; neither PTEN loss nor PIK3CA mutations correlated with response[77]. Udai et al analyzed PI3K signaling output in patient tumor samples by measuring phos‐ phorylation of 3 effectors downstream of PI3K, ie pAkt, p p70S6K and pGSK3beta[78]. No correlation was found between the presence of genomic alterations in PI3K or PTEN and ac‐ tivation of the pathway as measured by phosphorylated downstream targets. In a study of 17 well-characterized ovarian cancer cell lines, the majority failed to respond to Akt inhibi‐ tors despite Akt phosphorylation[79]. A high level of pAkt may not only reflect PI3K path‐ way intrinsic activation, but also result from cross-talk with Ras or other upstream signals.

In addition to being a non-specific measure of PI3K signal transduction, pAkt is a labile phosphorylated tumor marker, its stability is affected by pre-analytical factors such as tissue acquisition, ischemic time and fixation method[80],[81], In an effort to identify more stable biomarkers, Saal et al developed a gene expression signature of PI3K pathway activation and Stathmin, a regulator of microtubule dynamics was an accurate marker of the gene sig‐ nature. Stathmin can be easily measured by immunohistochemistry and is increasingly be‐ ing used as a surrogate marker for activation of the PI3K pathway[82].

#### *5.3.3. KRAS/BRAF*

As previously discussed a number of preclinical studies have demonstrated that KRAS and BRAF mutations confer resistance to inhibitors of the PI3K pathway[54],[77]. Intringingly, in a pooled molecular analysis of patients treated with PI3K/Akt/mTOR inhibitors in phase I trials, Janku et al reported 2 objective responses in patients with co-existing PI3K and KRAS or BRAF mutation[76]. Genomic analyses of tumors and cell lines has established that a sub‐ set of ovarian cancers have co-existing Ras and PI3K/Akt amplifications or mutations. This easily identifiable subset may benefit from coordinated inhibition of both pathways, and a trial combining a Mek inhibitor with a PI3K/mTOR inhibitor in ovarian cancer patients har‐ boring KRAS/BRAF and PI3K/Akt genomic alterations is warranted.

#### **6. Practical issues: Samples and trial design**

#### **6.1. Access to quality ovarian cancer samples**

As the data to date suggest that there is insufficient evidence to select patients for trials of PI3K inhibitors on the basis of specific molecular alterations, it is imperative that fu‐ ture trials enrolling unselected patient populations include parallel biological studies in an effort to uncover candidate biomarkers. Biological assays must be reproducible, ro‐ bust and require access to high quality tumor samples. As such, pre-analytical variables must be controlled for as much as possible by following standardized sample collection, fixation, processing and storage procedures. When dealing with paraffin-embedded tis‐ sue, markers of the PI3K Akt pathway may be particularly susceptible to artefactual loss[80]. In fact, the optimal fixative for in depth genomic analyses is unlikely to be for‐ malin, and may therefore require a shift in routine practice from paraffin to fresh frozen or RNAlater for sample storage.

#### **6.2. Access to post-treatment samples**

#### *6.2.1. At relapse*

In conclusion, if trials of PI3K/mTOR inhibitors had limited enrolment to PTEN null or PI3K mutated tumors a significant proportion of responding patients would have been missed. In light of the imperfect association between PI3K mutations or PTEN loss and response to PI3K pathway inhibitors, most ongoing trials are enrolling an unselected patient population; unfortunately, most of these studies do not appear to be collecting archival tumor samples

The level of phosphorylated Akt has been identified as a read-out for activation of the PI3K pathway and thus a potential biomarker for responsiveness to PI3K inhibitors. An in vitro and in silico study using a panel of cell lines and xenograft models treated with PI3K path‐ way inhibitors showed that pAkt correlated with efficacy, and KRAS or BRAF mutations with resistance; neither PTEN loss nor PIK3CA mutations correlated with response[77]. Udai et al analyzed PI3K signaling output in patient tumor samples by measuring phos‐ phorylation of 3 effectors downstream of PI3K, ie pAkt, p p70S6K and pGSK3beta[78]. No correlation was found between the presence of genomic alterations in PI3K or PTEN and ac‐ tivation of the pathway as measured by phosphorylated downstream targets. In a study of 17 well-characterized ovarian cancer cell lines, the majority failed to respond to Akt inhibi‐ tors despite Akt phosphorylation[79]. A high level of pAkt may not only reflect PI3K path‐ way intrinsic activation, but also result from cross-talk with Ras or other upstream signals.

In addition to being a non-specific measure of PI3K signal transduction, pAkt is a labile phosphorylated tumor marker, its stability is affected by pre-analytical factors such as tissue acquisition, ischemic time and fixation method[80],[81], In an effort to identify more stable biomarkers, Saal et al developed a gene expression signature of PI3K pathway activation and Stathmin, a regulator of microtubule dynamics was an accurate marker of the gene sig‐ nature. Stathmin can be easily measured by immunohistochemistry and is increasingly be‐

As previously discussed a number of preclinical studies have demonstrated that KRAS and BRAF mutations confer resistance to inhibitors of the PI3K pathway[54],[77]. Intringingly, in a pooled molecular analysis of patients treated with PI3K/Akt/mTOR inhibitors in phase I trials, Janku et al reported 2 objective responses in patients with co-existing PI3K and KRAS or BRAF mutation[76]. Genomic analyses of tumors and cell lines has established that a sub‐ set of ovarian cancers have co-existing Ras and PI3K/Akt amplifications or mutations. This easily identifiable subset may benefit from coordinated inhibition of both pathways, and a trial combining a Mek inhibitor with a PI3K/mTOR inhibitor in ovarian cancer patients har‐

ing used as a surrogate marker for activation of the PI3K pathway[82].

boring KRAS/BRAF and PI3K/Akt genomic alterations is warranted.

for detailed molecular analyses (Table 4).

292 Ovarian Cancer - A Clinical and Translational Update

*5.3.2. pAkt and stathmin*

*5.3.3. KRAS/BRAF*

It is likely that clonal evolution and treatment selection pressure will lead to important genomic and/or phenotypic modifications in the tumor in the interval between diagnosis and relapse. An increasing number of phase I and II trials are therefore requesting op‐ tional biopsies of metastatic disease and the vast majority of patients are willing to con‐ sent this procedure. A study of patients enrolled in phase I trials at our institution revealed that 84% of patients who were proposed optional tumor biopsies consented to the procedure, including sequential pre- and post-treatment biopsies[85]. All procedures were performed using an 18-gauge needle under ultrasound or computed tomography scanning and were associated with low minor complication rates (9/145 tumor biopsies). In 70% of the cases the biopsy met quality criteria for ancillary molecular (RNA and DNA) analyses. Access to samples from relapsed disease is likely to be particularly rele‐ vant to high grade ovarian cancer, where the initial disease is exquisitely chemosensitive and repeat profiling of the chemoresistant recurrence may reveal a completely different molecular profile.

#### *6.2.2. Residual disease post-chemotherapy*

The molecular characterization of ovarian cancer clones surviving after chemotherapy could identify targets for novel agents designed to eradicate chemoresistant residual dis‐ ease. As discussed above, the combination of PI3K/Akt/mTOR inhibitors with chemother‐ apy may not be optimal because of the risk of cumulative toxicities as well as the theoretical risk that these inhibitors may antagonize the cytotoxic effects of chemothera‐ py. A more attractive approach may be sequential, where primarily chemosensitive ovar‐ ian cancer is treated with chemotherapy followed by PI3K inhibitors if indicated by the molecular profile of the residual resistant clones. Although recent trials using such an approach with erlotinib or olaparib after response to platinum based treatment were dis‐ appointing, neither trial selected the maintenance treatment on the basis of the profile of residual disease.

∙ Randomized placebo controlled phase II trials instead of single arm phase II.

natural disease course may be difficult to make.

∙ Metabolic response on functional imaging by FDG/PET.

conventional cytotoxics.

novel therapies

**7. Conclusion**

will likely be essential.

∙ Randomized discontinuation design: After an initial run-in phase where all patients receive the experimental agent, patients with stable disease are randomized to placebo versus continued drug. This model may be particularly suited to slower growing Type I ovarian cancers where the distinction between treatment induced disease stabilization and

The PI3K/Akt/mTOR Pathway in Ovarian Cancer: Biological Rationale and Therapeutic Opportunities

http://dx.doi.org/10.5772/54170

295

∙ When evaluating tumor response on imaging, percentage tumor shrinkage as a continuous variable could be used, rather than categorical RECIST where an arbitrary cut-off of 30% decrease to define response may be more suited to

**Table 6.** Suggested modifications to the traditional trial design adapted to testing PI3K pathway inhibitors and other

The PI3K pathway is emerging as an important and viable therapeutic target. However evidence for efficacy in ovarian cancer remains limited and predictive biomarkers to identify the patients most likely to benefit from this approach are desperately needed. Given the complexicity of the PI3K pathway and its cross-talk with other signaling net‐ works, inhibiting a single member of the pathway may be insufficient to abrogate onco‐ genic signaling and result in meaningful tumor control. A number of resistance mechanisms to PI3K pathway inhibitors have been identified. Primary resistance may be attributable to co-existing KRAS or BRAF mutations; therefore concurrent PI3K and Mek inhibition in dual PI3K/KRAS mutated ovarian cancer may be worthy of investigation. In addition, treatment induced compensatory increases in alternate pathways (via IGF1R, MTORC2/Akt and others) may allow escape from selective mTOR targeting; response could be improved by appropriately designed combinatorial strategies. This suggests that abrogating adaptive escape pathways will require truly individualized treatment, select‐ ed on the basis of on-treatment tumor biopsies to identify the culprit compensatory path‐ ways. A number of trials are ongoing exploring the benefit of combinations, unfortunately few are including correlative biological studies. Finally, for decades, ovari‐ an cancer was treated as a uniform disease, a greater understanding of the biology of ep‐ ithelial ovarian tumors has encouraged the initiation of a few histology-specific trials. The successful transition of novel PI3K pathway inhibitors from bench to the bedside of patients with ovarian cancer will depend on a greater integration of translation research in trial development. Efforts must be made to include comprehensive molecular profiling both at baseline and sequentially throughout the disease course, and studies investigat‐ ing the usefulness of novel surrogate tumor markers such as ascites or circulating DNA

∙ Using each patient as internal control for evidence of drug activity: the ratio of time to progression (TTP) on experimental drug to TTP on last treatment (TTPn+1/TTPn), where TTPn+1/TTPn ≥1.3 would suggest drug activity[89];

#### **6.3. Surrogate tissue**

Any effort to sample relapsed disease in ovarian cancer patients invariably faces the chal‐ lenge of access to tumor. Recurrences tend to be limited to the abdominal cavity with diffuse carcinomatosis which can be difficult to biopsy safely. This is a critical need for more easily accessible surrogate tumor samples which would allow for serial tumor sampling through‐ out the disease course, to identify both predictive and pharmacodynamic markers. Possibili‐ ties include circulating tumor cells, ascites and circulating DNA.

Serial sampling of circulating tumor cells (CTCs) has been shown to provide useful prognos‐ tic and/or predictive information in a number of tumor types such as breast and prostate cancer[86],[87]. In the temsirolimus trial, CTCs were detected in 45% of patients before cycle 1 and found to correlate weakly with progressive disease, however no significant change in CTC levels were observed with treatment[29].

Udai et al demonstrated the feasibility of profiling the PI3K pathway from ascites in patients with advanced ovarian cancer: they successfully measured PI3K and PTEN mutations, am‐ plifications and losses as well as PI3K signaling output in ascitic samples by ELISA for phos‐ phorylated proteins[78]. Finally, cancer mutations have been identified by deep sequencing of circulating plasma DNA from patients with advanced ovarian cancer, providing another example of a non-invasive "liquid biopsy"[88].

2) Sequential biopsies for post-treatment/resistant tumor molecular profiling.

3) Studies investigating the feasibility and translational research value of surrogate tissue samples: ascites, circulating tumor cells, circulating DNA

**Table 5.** Sample-related considerations to enhance the development of PI3K pathway inhibitors in ovarian cancer

#### **6.4. Novel trial designs**

Conventional endpoints such as RECIST response may not be appropriate for inhibitors of the PI3K pathway that may result in disease stabilization rather than objective tumor shrink‐ age. Single arm phase II trials offer little data regarding activity of a novel drug: patient numbers are small, heterogeneous and comparisons with historical controls are intrinsically unreliable. A number of subtle deviations from traditional trial designs could help improve the likelihood that novel PI3K inhibitors make a successful transition from preclinical testing through early and late phase trials. Various strategies are outlined in table 6.

<sup>1)</sup> Standardized quality ovarian cancer sample collection protocols at diagnosis and surgery optimized for comprehensive molecular studies.

∙ Randomized placebo controlled phase II trials instead of single arm phase II.

∙ Randomized discontinuation design: After an initial run-in phase where all patients receive the experimental agent, patients with stable disease are randomized to placebo versus continued drug. This model may be particularly suited to slower growing Type I ovarian cancers where the distinction between treatment induced disease stabilization and natural disease course may be difficult to make.

∙ When evaluating tumor response on imaging, percentage tumor shrinkage as a continuous variable could be used, rather than categorical RECIST where an arbitrary cut-off of 30% decrease to define response may be more suited to conventional cytotoxics.

∙ Metabolic response on functional imaging by FDG/PET.

∙ Using each patient as internal control for evidence of drug activity: the ratio of time to progression (TTP) on experimental drug to TTP on last treatment (TTPn+1/TTPn), where TTPn+1/TTPn ≥1.3 would suggest drug activity[89];

**Table 6.** Suggested modifications to the traditional trial design adapted to testing PI3K pathway inhibitors and other novel therapies

#### **7. Conclusion**

molecular profile of the residual resistant clones. Although recent trials using such an approach with erlotinib or olaparib after response to platinum based treatment were dis‐ appointing, neither trial selected the maintenance treatment on the basis of the profile of

Any effort to sample relapsed disease in ovarian cancer patients invariably faces the chal‐ lenge of access to tumor. Recurrences tend to be limited to the abdominal cavity with diffuse carcinomatosis which can be difficult to biopsy safely. This is a critical need for more easily accessible surrogate tumor samples which would allow for serial tumor sampling through‐ out the disease course, to identify both predictive and pharmacodynamic markers. Possibili‐

Serial sampling of circulating tumor cells (CTCs) has been shown to provide useful prognos‐ tic and/or predictive information in a number of tumor types such as breast and prostate cancer[86],[87]. In the temsirolimus trial, CTCs were detected in 45% of patients before cycle 1 and found to correlate weakly with progressive disease, however no significant change in

Udai et al demonstrated the feasibility of profiling the PI3K pathway from ascites in patients with advanced ovarian cancer: they successfully measured PI3K and PTEN mutations, am‐ plifications and losses as well as PI3K signaling output in ascitic samples by ELISA for phos‐ phorylated proteins[78]. Finally, cancer mutations have been identified by deep sequencing of circulating plasma DNA from patients with advanced ovarian cancer, providing another

1) Standardized quality ovarian cancer sample collection protocols at diagnosis and surgery optimized for

3) Studies investigating the feasibility and translational research value of surrogate tissue samples: ascites, circulating

**Table 5.** Sample-related considerations to enhance the development of PI3K pathway inhibitors in ovarian cancer

Conventional endpoints such as RECIST response may not be appropriate for inhibitors of the PI3K pathway that may result in disease stabilization rather than objective tumor shrink‐ age. Single arm phase II trials offer little data regarding activity of a novel drug: patient numbers are small, heterogeneous and comparisons with historical controls are intrinsically unreliable. A number of subtle deviations from traditional trial designs could help improve the likelihood that novel PI3K inhibitors make a successful transition from preclinical testing

through early and late phase trials. Various strategies are outlined in table 6.

ties include circulating tumor cells, ascites and circulating DNA.

CTC levels were observed with treatment[29].

example of a non-invasive "liquid biopsy"[88].

2) Sequential biopsies for post-treatment/resistant tumor molecular profiling.

comprehensive molecular studies.

tumor cells, circulating DNA

**6.4. Novel trial designs**

residual disease.

**6.3. Surrogate tissue**

294 Ovarian Cancer - A Clinical and Translational Update

The PI3K pathway is emerging as an important and viable therapeutic target. However evidence for efficacy in ovarian cancer remains limited and predictive biomarkers to identify the patients most likely to benefit from this approach are desperately needed. Given the complexicity of the PI3K pathway and its cross-talk with other signaling net‐ works, inhibiting a single member of the pathway may be insufficient to abrogate onco‐ genic signaling and result in meaningful tumor control. A number of resistance mechanisms to PI3K pathway inhibitors have been identified. Primary resistance may be attributable to co-existing KRAS or BRAF mutations; therefore concurrent PI3K and Mek inhibition in dual PI3K/KRAS mutated ovarian cancer may be worthy of investigation. In addition, treatment induced compensatory increases in alternate pathways (via IGF1R, MTORC2/Akt and others) may allow escape from selective mTOR targeting; response could be improved by appropriately designed combinatorial strategies. This suggests that abrogating adaptive escape pathways will require truly individualized treatment, select‐ ed on the basis of on-treatment tumor biopsies to identify the culprit compensatory path‐ ways. A number of trials are ongoing exploring the benefit of combinations, unfortunately few are including correlative biological studies. Finally, for decades, ovari‐ an cancer was treated as a uniform disease, a greater understanding of the biology of ep‐ ithelial ovarian tumors has encouraged the initiation of a few histology-specific trials. The successful transition of novel PI3K pathway inhibitors from bench to the bedside of patients with ovarian cancer will depend on a greater integration of translation research in trial development. Efforts must be made to include comprehensive molecular profiling both at baseline and sequentially throughout the disease course, and studies investigat‐ ing the usefulness of novel surrogate tumor markers such as ascites or circulating DNA will likely be essential.

#### **Author details**

Alexandra Leary, Edouard Auclin, Patricia Pautier and Catherine Lhommé

\*Address all correspondence to: alexandra.leary@igr.fr

Department of Medicine, Gynecological Oncology, Institut Gustave Roussy, Villejuif, France

[12] Mabuchi S, Kawase C, Altomare DA, et al: mTOR is a promising therapeutic target both in cisplatin-sensitive and cisplatin-resistant clear cell carcinoma of the ovary.

The PI3K/Akt/mTOR Pathway in Ovarian Cancer: Biological Rationale and Therapeutic Opportunities

http://dx.doi.org/10.5772/54170

297

[13] Kurman RJ, Shih Ie M: Molecular pathogenesis and extraovarian origin of epithelial

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[16] Shayesteh L, Lu Y, Kuo WL, et al: PIK3CA is implicated as an oncogene in ovarian

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**Author details**

296 Ovarian Cancer - A Clinical and Translational Update

**References**

Alexandra Leary, Edouard Auclin, Patricia Pautier and Catherine Lhommé

Department of Medicine, Gynecological Oncology, Institut Gustave Roussy, Villejuif, France

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**Chapter 14**

**PARP Inhibitors in Ovarian Cancer**

Jeanine Staples and Annekathryn Goodman

Additional information is available at the end of the chapter

Targeted therapy in cancer has led to intensive searches for the molecular pathways of ma‐ lignant transformation [1]. In gynecologic malignancies, BRCA 1 and BRCA 2 gene muta‐ tions on chromosomes 17 and 13 respectively were identified in the 1990s [2]. Since then, intensive investigation of the pathways for these genes has led to a wealth of information about molecular pathways [3]. The understanding of these molecular pathways has led in turn to the development of novel targeted therapies for women with identified gene muta‐ tions [4]. This review reviews the current knowledge of the subset of women with BRCA gene mutations. The characteristics of BRCA deficiency including genetic background and current chemotherapeutic options including mechanisms of resistance are discussed as well. Finally, the data on emerging targeted therapeutic strategies with poly-ADP-ribose-poly‐

Hereditary Breast and Ovarian Cancer Syndrome (HBOCS) is characterized by a few distinct features: earlier age of cancer onset, higher incidence of bilateral disease, higher incidence of other cancers, and inheritance in an autosomal dominant pattern [5]. A number of genes, as well as associated syndromes, have been implicated in HBOCS, but none more so than the BRCA genes. Individuals with impaired BRCA protein function have a 50-85% lifetime risk of developing breast cancer and 10-40% lifetime risk of developing ovarian cancer [6]. Al‐ though deleterious mutations in either of the 2 BRCA genes significantly increases one's risk for breast and ovarian cancer, mutations in these genes account for only about 5-10% of all breast and ovarian cancer cases [7]. Because of genetic testing, the families of the subset of women with BRCA associated cancers can be tested and early intervention is a possibility [8]. These include tamoxifen therapy, bilateral prophylactic oophorectomy, prophylactic

and reproduction in any medium, provided the original work is properly cited.

© 2013 Staples and Goodman; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

merase (PARP) inhibitors and specific PARP clinical trials are reviewed.

http://dx.doi.org/10.5772/52888

**2. BRCA genetic background**

**1. Introduction**


### **PARP Inhibitors in Ovarian Cancer**

Jeanine Staples and Annekathryn Goodman

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52888

#### **1. Introduction**

[79] Hanrahan AJ, Schultz N, Westfal ML, et al: Genomic complexity and AKT depend‐

[80] Pinhel IF, Macneill FA, Hills MJ, et al: Extreme loss of immunoreactive p-Akt and p-Erk1/2 during routine fixation of primary breast cancer. Breast Cancer Res 12:R76,

[81] Holzer TR, Fulford AD, Arkins AM, et al: Ischemic time impacts biological integrity of phospho-proteins in PI3K/Akt, Erk/MAPK, and p38 MAPK signaling networks.

[82] Saal LH, Johansson P, Holm K, et al: Poor prognosis in carcinoma is associated with a gene expression signature of aberrant PTEN tumor suppressor pathway activity.

[83] Iacovelli R, Palazzo A, Mezi S, et al: Incidence and risk of pulmonary toxicity in pa‐ tients treated with mTOR inhibitors for malignancy. A meta-analysis of published

[84] Lopez-Fauqued M, Gil R, Grueso J, et al: The dual PI3K/mTOR inhibitor PI-103 pro‐ motes immunosuppression, in vivo tumor growth and increases survival of sorafe‐

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[86] Cristofanilli M, Budd GT, Ellis MJ, et al: Circulating tumor cells, disease progression,

[87] Goodman OB, Jr., Fink LM, Symanowski JT, et al: Circulating tumor cells in patients with castration-resistant prostate cancer baseline values and correlation with prog‐

[88] Forshew T, Murtaza M, Parkinson C, et al: Noninvasive identification and monitor‐ ing of cancer mutations by targeted deep sequencing of plasma DNA. Sci Transl Med

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and survival in metastatic breast cancer. N Engl J Med 351:781-91, 2004

nostic factors. Cancer Epidemiol Biomarkers Prev 18:1904-13, 2009

ence in serous ovarian cancer. Cancer Discov 2:56-67, 2012

nib-treated melanoma cells. Int J Cancer 126:1549-61, 2010

2010

Anticancer Res 31:2073-81, 2011

302 Ovarian Cancer - A Clinical and Translational Update

trials. Acta Oncol, 2012

4:136ra68, 2012

cancers. J Clin Oncol 28:4877-83, 2010

Proc Natl Acad Sci U S A 104:7564-9, 2007

Targeted therapy in cancer has led to intensive searches for the molecular pathways of ma‐ lignant transformation [1]. In gynecologic malignancies, BRCA 1 and BRCA 2 gene muta‐ tions on chromosomes 17 and 13 respectively were identified in the 1990s [2]. Since then, intensive investigation of the pathways for these genes has led to a wealth of information about molecular pathways [3]. The understanding of these molecular pathways has led in turn to the development of novel targeted therapies for women with identified gene muta‐ tions [4]. This review reviews the current knowledge of the subset of women with BRCA gene mutations. The characteristics of BRCA deficiency including genetic background and current chemotherapeutic options including mechanisms of resistance are discussed as well. Finally, the data on emerging targeted therapeutic strategies with poly-ADP-ribose-poly‐ merase (PARP) inhibitors and specific PARP clinical trials are reviewed.

#### **2. BRCA genetic background**

Hereditary Breast and Ovarian Cancer Syndrome (HBOCS) is characterized by a few distinct features: earlier age of cancer onset, higher incidence of bilateral disease, higher incidence of other cancers, and inheritance in an autosomal dominant pattern [5]. A number of genes, as well as associated syndromes, have been implicated in HBOCS, but none more so than the BRCA genes. Individuals with impaired BRCA protein function have a 50-85% lifetime risk of developing breast cancer and 10-40% lifetime risk of developing ovarian cancer [6]. Al‐ though deleterious mutations in either of the 2 BRCA genes significantly increases one's risk for breast and ovarian cancer, mutations in these genes account for only about 5-10% of all breast and ovarian cancer cases [7]. Because of genetic testing, the families of the subset of women with BRCA associated cancers can be tested and early intervention is a possibility [8]. These include tamoxifen therapy, bilateral prophylactic oophorectomy, prophylactic

© 2013 Staples and Goodman; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

contralateral mastectomy and combinations of these strategies. Such interventions have been shown to offer substantial life expectancy gain for young women with BRCA associat‐ ed early stage cancer. Currently, treatment of BRCA-associated breast and/or ovarian cancer is no different than that for the general population. Survival data of BRCA positive patients with ovarian cancer suggests that they have prolonged disease free intervals and overall longer survival than their wild-type counterparts [9].

(See Figure 1) 1) DNA damage triggers activation of the ataxia-telangiectasia mutated ser‐ ine-protein kinase as well as the ataxia-telangiectasia and Rad3 related protein kinase [14]. 2) Once activated, these enzymes phosphorylate and activate a number of intracellular items, including the BRCA1 protein, Checkpoint kinase 2 (which also activates BRCA1), BRCA2, the Fanconi anemia protein complex, H2AX histones involved in forming nuclear foci cru‐ cial for DNA repair, and a few others [15]. 3) The H2AX histones co-localize and join with other proteins to form nuclear foci at the sites of DNA damage. These other proteins include a tumor binding protein 53BP1, the nuclear factor with BRCT domains protein 1 NFBD1, checkpoint kinase 2, and Nijmegen breakage syndrome 1 protein Nibrin. 4) Nibrin is part of the MRN complex, which also includes Rad50 and MreII. The MRN complex is important in initial processing of double stranded DNA breaks. It directs the cellular process to continue via homologous recombination or non-homologous end joining. BRCA1 plays a part in reg‐ ulating the MRN complex by inhibiting MreII [16]. This action depresses the NHEJ pathway and stimulates the Rad51 pathway favoring homologous recombination. 5) BRCA1 also acti‐ vates BRCA1 interacting protein C-terminal helicase 1 (BRIP1), which unwinds DNA strands near sites of damage allowing other repair machinery to access the damaged sites. The gene that encodes BRIP1 may also act as an oncogene in ovarian cancer. 6) Activation of Rad51 allows it to form a complex with BRCA2 and Fanconi anemia complementation group D2 protein, FANCD2 [17]. This complex is a key player in homologous recombination repair as it is involved in searching for homology as well as strand pairing, while causing Sphase and G2 arrest [18]. 7) Certain homologous recombination repair (HRR) promoting ac‐ tivities of BRCA1 are amplified when it forms a complex with BRCA1 associated RING domain protein 1, BARD1 [19]. This complex may also play a role in mismatch repair via

PARP Inhibitors in Ovarian Cancer http://dx.doi.org/10.5772/52888 305

Inheritance of a damaging mutation in either BRCA gene can cause disruption of the smoothly regulated replication process of the body's cells, potentially leading to cancer. Though the mechanism is poorly understood, mutations in the BRCA genes show preferen‐ tial deleterious consequences in breast and ovarian tissues. In the general population, the lifetime prevalence of breast cancer is 12% and ovarian cancer 1.4% [21]. A mutation in the BRCA1 gene puts an individual at a 50-85% lifetime risk of developing breast cancer and a 15-40% risk of developing ovarian cancer. BRCA2 mutation carriers have a 50-85% lifetime risk of developing breast cancer and a 10-20% risk of developing epithelial ovarian cancer. In addition, BRCA mutations are associated with bilateral disease, cancer at a younger age (BRCA1), autosomal dominant inheritance pattern, increased risk of male breast cancer (BRCA2), and increased risk of cancer in other organs [22,23]. Specifically, BRCA positive in‐ dividuals are at increased risk of epithelial ovarian cancer (EOC), which behaves differently than EOC seen in the general population. Both BRCA1 and 2 carriers have an improved prognosis when compared to their wild-type counterparts. A pooled analysis of 26 observa‐ tional studies on the survival of women with ovarian cancer, including 1213 EOC cases

downstream action [20].

**3. Associated breast and ovarian cancer**

The Breast Cancer Susceptibility genes (BRCA 1 and 2) are considered tumor suppressors, whose job it is to maintain appropriate cell growth, ultimately by upholding genomic stability [10]. No single unified theory exists regarding the action of the BRCA genes. While disruption of either of the BRCA genes demonstrates similar pathophysiological manifestations, they are indeed unique. They lie on 2 separate chromosomes, each have unique primary sequences, and ultimately carry out their responsibilities via discrete (proposed) mechanisms. Furthermore, their unique mechanisms of action give them distinct characteristics that coincide with dis‐ tinct risks and prognoses with gene disturbance. Both BRCA genes share a relationship with the gene RAD51, which encodes a protein responsible for assisting in repair of DNA double strand breaks via homologous recombination. When there is a double strand break in the ge‐ netic sequence, *sequenceX* of a specific chromatid, homologous recombination provides a means for the exchange of the same genetic sequence, *sequenceX*, from the healthy homolo‐ gous sister chromatid to the damaged one. Rad51 is one of the most important players in HRR. It assists in the search for homology and strand pairing, and its responsibilities are ultimately complemented by the proteins encoded by the 2 BRCA genes [11]. Studies suggest that BRCA2 regulates the intracellular transport and function of RAD51, as well as the enzymatic activity of the RAD51 protein [12]. The relationship between BRCA1 and RAD51 is less clear. There is evi‐ dence that BRCA1 physically associates with proteins other than RAD51, creating a complex likely responsible for the creation of resected single-stranded DNA at double strand repair sites [13]. Further data supports BRCA1's role in activation of DNA damage checkpoints. There is also evidence that supports BRCA1's role in altering chromatin structure upon DNA damage to allow easier access for repair.

**Figure 1.** Role of BRCA proteins in Homologous Recombination Repair

(See Figure 1) 1) DNA damage triggers activation of the ataxia-telangiectasia mutated ser‐ ine-protein kinase as well as the ataxia-telangiectasia and Rad3 related protein kinase [14]. 2) Once activated, these enzymes phosphorylate and activate a number of intracellular items, including the BRCA1 protein, Checkpoint kinase 2 (which also activates BRCA1), BRCA2, the Fanconi anemia protein complex, H2AX histones involved in forming nuclear foci cru‐ cial for DNA repair, and a few others [15]. 3) The H2AX histones co-localize and join with other proteins to form nuclear foci at the sites of DNA damage. These other proteins include a tumor binding protein 53BP1, the nuclear factor with BRCT domains protein 1 NFBD1, checkpoint kinase 2, and Nijmegen breakage syndrome 1 protein Nibrin. 4) Nibrin is part of the MRN complex, which also includes Rad50 and MreII. The MRN complex is important in initial processing of double stranded DNA breaks. It directs the cellular process to continue via homologous recombination or non-homologous end joining. BRCA1 plays a part in reg‐ ulating the MRN complex by inhibiting MreII [16]. This action depresses the NHEJ pathway and stimulates the Rad51 pathway favoring homologous recombination. 5) BRCA1 also acti‐ vates BRCA1 interacting protein C-terminal helicase 1 (BRIP1), which unwinds DNA strands near sites of damage allowing other repair machinery to access the damaged sites. The gene that encodes BRIP1 may also act as an oncogene in ovarian cancer. 6) Activation of Rad51 allows it to form a complex with BRCA2 and Fanconi anemia complementation group D2 protein, FANCD2 [17]. This complex is a key player in homologous recombination repair as it is involved in searching for homology as well as strand pairing, while causing Sphase and G2 arrest [18]. 7) Certain homologous recombination repair (HRR) promoting ac‐ tivities of BRCA1 are amplified when it forms a complex with BRCA1 associated RING domain protein 1, BARD1 [19]. This complex may also play a role in mismatch repair via downstream action [20].

#### **3. Associated breast and ovarian cancer**

contralateral mastectomy and combinations of these strategies. Such interventions have been shown to offer substantial life expectancy gain for young women with BRCA associat‐ ed early stage cancer. Currently, treatment of BRCA-associated breast and/or ovarian cancer is no different than that for the general population. Survival data of BRCA positive patients with ovarian cancer suggests that they have prolonged disease free intervals and overall

The Breast Cancer Susceptibility genes (BRCA 1 and 2) are considered tumor suppressors, whose job it is to maintain appropriate cell growth, ultimately by upholding genomic stability [10]. No single unified theory exists regarding the action of the BRCA genes. While disruption of either of the BRCA genes demonstrates similar pathophysiological manifestations, they are indeed unique. They lie on 2 separate chromosomes, each have unique primary sequences, and ultimately carry out their responsibilities via discrete (proposed) mechanisms. Furthermore, their unique mechanisms of action give them distinct characteristics that coincide with dis‐ tinct risks and prognoses with gene disturbance. Both BRCA genes share a relationship with the gene RAD51, which encodes a protein responsible for assisting in repair of DNA double strand breaks via homologous recombination. When there is a double strand break in the ge‐ netic sequence, *sequenceX* of a specific chromatid, homologous recombination provides a means for the exchange of the same genetic sequence, *sequenceX*, from the healthy homolo‐ gous sister chromatid to the damaged one. Rad51 is one of the most important players in HRR. It assists in the search for homology and strand pairing, and its responsibilities are ultimately complemented by the proteins encoded by the 2 BRCA genes [11]. Studies suggest that BRCA2 regulates the intracellular transport and function of RAD51, as well as the enzymatic activity of the RAD51 protein [12]. The relationship between BRCA1 and RAD51 is less clear. There is evi‐ dence that BRCA1 physically associates with proteins other than RAD51, creating a complex likely responsible for the creation of resected single-stranded DNA at double strand repair sites [13]. Further data supports BRCA1's role in activation of DNA damage checkpoints. There is also evidence that supports BRCA1's role in altering chromatin structure upon DNA

longer survival than their wild-type counterparts [9].

304 Ovarian Cancer - A Clinical and Translational Update

damage to allow easier access for repair.

**Figure 1.** Role of BRCA proteins in Homologous Recombination Repair

Inheritance of a damaging mutation in either BRCA gene can cause disruption of the smoothly regulated replication process of the body's cells, potentially leading to cancer. Though the mechanism is poorly understood, mutations in the BRCA genes show preferen‐ tial deleterious consequences in breast and ovarian tissues. In the general population, the lifetime prevalence of breast cancer is 12% and ovarian cancer 1.4% [21]. A mutation in the BRCA1 gene puts an individual at a 50-85% lifetime risk of developing breast cancer and a 15-40% risk of developing ovarian cancer. BRCA2 mutation carriers have a 50-85% lifetime risk of developing breast cancer and a 10-20% risk of developing epithelial ovarian cancer. In addition, BRCA mutations are associated with bilateral disease, cancer at a younger age (BRCA1), autosomal dominant inheritance pattern, increased risk of male breast cancer (BRCA2), and increased risk of cancer in other organs [22,23]. Specifically, BRCA positive in‐ dividuals are at increased risk of epithelial ovarian cancer (EOC), which behaves differently than EOC seen in the general population. Both BRCA1 and 2 carriers have an improved prognosis when compared to their wild-type counterparts. A pooled analysis of 26 observa‐ tional studies on the survival of women with ovarian cancer, including 1213 EOC cases showed that among patients with invasive EOC, having a BRCA mutation was associated with improved 5-year overall survival, with BRCA2 carriers having the best prognosis [9]. See Table 1.

cepted that a deleterious BRCA mutation adversely affects homologous recombination. While the BRCA genes indeed play a very important role in homologous recombination, they are not the only key players. A defect or hiccup in any of the other genes involved in homologous recombination would in theory, affect this process. This describes the notion of BRCAness: Individuals with structurally healthy BRCA genes that are functionally incapa‐ ble of carrying out homologous recombination due to a defect elsewhere that hinders the en‐ tire process. In fact, about 40-50% of ovarian cancer cases have shown to have some defect in homologous recombination, with a large number of these being associated with BRCA-relat‐

PARP Inhibitors in Ovarian Cancer http://dx.doi.org/10.5772/52888 307

**Figure 2.** illustrates the molecular profile of serous ovarian cancer. BRCA1 germline, BRCA2 germline, BRCA1 somatic, BRCA2 somatic, BRCA1 hypermethylation, EMSY amplification and PTEN loss have all been shown to lead to impaired ho‐ mologous recombination, comprising about half of all cases [29]. Sporadic ovarian cancer mutations that affect homolo‐ gous recombination are clinically significant because they tend to behave like the BRCA cancers. This special sub-group of epithelial ovarian cancer patients show similar therapeutic response and prognosis as their BRCA mutant counterparts which includes improved sensitivity to platinum as well as improved 5 year survival [30]. A few studies have attempted to reveal the mechanism behind BRCAness on a genetic level. There is some data to suggest that transcriptional or post-tran‐ scriptional repression of the BRCA1 gene is responsible for such sporadic tumors. For example, hypermethylation of a pro‐ motor region upstream from BRCA1 gene that leads to inactivation of BRCA1 protein, causes impaired homologous

It should be noted that the specific target of the PARP inhibitor class is not only BRCA mu‐ tant cancers; however, it encompasses all that fall under the umbrella of impaired homolo‐ gous recombination. So in theory, PARP inhibitors should show efficacy in a number of

sporadic cancers as well, specifically those that elicit "BRCAness" characteristics [32].

recombination leading to a sporadic "BRCAness" with an intact yet silenced BRCA1 gene [31].

ed defects. See Figure 2.


**Table 1.** Characteristics of BRCA 1/2 associated Epithelial Ovarian Cancer (EOC) [9,24,25]

BRCA1 associated breast cancers tend to be of the triple-negative type which is significant because it lacks the 3 biomarkers most commonly implicated in breast cancer, the estrogen and progesterone receptors and the human epidermal growth factor receptor 2 (Her2/neu) [26]. The presence of these biomarkers helps in the guidance of treatment options. Estrogen/ progesterone positive breast cancers are typically treated with hormonal therapy (in con‐ junction with surgery/radiation), and Her2/neu positive breast cancers are treated with an agent that specifically targets the Her2 receptor, such as Herceptin. When these 3 biomark‐ ers are negative in a newly diagnosed breast cancer, the treatment approach becomes more complicated and prognosisis poor. On the other hand, BRCA2 associated breast cancer is typically hormonal in nature [22]. Current evidence suggests no difference in overall prog‐ nosis of breast cancers in BRCA carriers compared to sporadic breast cancers, but BRCA de‐ ficiency does appear to be predict chemo-sensitivity [27].

#### **4. BRCAness**

The term "BRCAness" describes a subset of women with sporadic EOC who display similar phenotypic characteristics to those with a hereditary BRCA mutation [28]. It is widely ac‐ cepted that a deleterious BRCA mutation adversely affects homologous recombination. While the BRCA genes indeed play a very important role in homologous recombination, they are not the only key players. A defect or hiccup in any of the other genes involved in homologous recombination would in theory, affect this process. This describes the notion of BRCAness: Individuals with structurally healthy BRCA genes that are functionally incapa‐ ble of carrying out homologous recombination due to a defect elsewhere that hinders the en‐ tire process. In fact, about 40-50% of ovarian cancer cases have shown to have some defect in homologous recombination, with a large number of these being associated with BRCA-relat‐ ed defects. See Figure 2.

showed that among patients with invasive EOC, having a BRCA mutation was associated with improved 5-year overall survival, with BRCA2 carriers having the best prognosis [9].

Inheritance Auto-Dominant Auto-Dominant Sporadic Prevalence of EOC 39% 22% 1.4% Mean age of onset 54 62 63

Tumor: Stage @ surgery Advanced Advanced Advanced

Recurrence-Free interval 14 months 14 months 7 months 5-year Survival 44% 52% 36%

> hepatobiliary, renal, testicular, leukemia

**Table 1.** Characteristics of BRCA 1/2 associated Epithelial Ovarian Cancer (EOC) [9,24,25]

ficiency does appear to be predict chemo-sensitivity [27].

**4. BRCAness**

Other malignancies Breast, gastric

306 Ovarian Cancer - A Clinical and Translational Update

Treatment Surgery + Chemo Surgery + Chemo Surgery + Chemo

Histolology Serous Serous Serous Differentiation Mod-Poor Mod-Poor Mod-poor

BRCA1 associated breast cancers tend to be of the triple-negative type which is significant because it lacks the 3 biomarkers most commonly implicated in breast cancer, the estrogen and progesterone receptors and the human epidermal growth factor receptor 2 (Her2/neu) [26]. The presence of these biomarkers helps in the guidance of treatment options. Estrogen/ progesterone positive breast cancers are typically treated with hormonal therapy (in con‐ junction with surgery/radiation), and Her2/neu positive breast cancers are treated with an agent that specifically targets the Her2 receptor, such as Herceptin. When these 3 biomark‐ ers are negative in a newly diagnosed breast cancer, the treatment approach becomes more complicated and prognosisis poor. On the other hand, BRCA2 associated breast cancer is typically hormonal in nature [22]. Current evidence suggests no difference in overall prog‐ nosis of breast cancers in BRCA carriers compared to sporadic breast cancers, but BRCA de‐

The term "BRCAness" describes a subset of women with sporadic EOC who display similar phenotypic characteristics to those with a hereditary BRCA mutation [28]. It is widely ac‐

**BRCA 1 carriers BRCA 2 carriers Non-Carriers**

Breast (including higher incidence of male breast cancer), prostate, pancreatic

See Table 1.

**Figure 2.** illustrates the molecular profile of serous ovarian cancer. BRCA1 germline, BRCA2 germline, BRCA1 somatic, BRCA2 somatic, BRCA1 hypermethylation, EMSY amplification and PTEN loss have all been shown to lead to impaired ho‐ mologous recombination, comprising about half of all cases [29]. Sporadic ovarian cancer mutations that affect homolo‐ gous recombination are clinically significant because they tend to behave like the BRCA cancers. This special sub-group of epithelial ovarian cancer patients show similar therapeutic response and prognosis as their BRCA mutant counterparts which includes improved sensitivity to platinum as well as improved 5 year survival [30]. A few studies have attempted to reveal the mechanism behind BRCAness on a genetic level. There is some data to suggest that transcriptional or post-tran‐ scriptional repression of the BRCA1 gene is responsible for such sporadic tumors. For example, hypermethylation of a pro‐ motor region upstream from BRCA1 gene that leads to inactivation of BRCA1 protein, causes impaired homologous recombination leading to a sporadic "BRCAness" with an intact yet silenced BRCA1 gene [31].

It should be noted that the specific target of the PARP inhibitor class is not only BRCA mu‐ tant cancers; however, it encompasses all that fall under the umbrella of impaired homolo‐ gous recombination. So in theory, PARP inhibitors should show efficacy in a number of sporadic cancers as well, specifically those that elicit "BRCAness" characteristics [32].

#### **5. Mechanisms of resistance**

Currently, there is no difference in management of EOC between BRCA positive patients and BRCA wild type patients. Maximal surgical cytoreduction, with or without neoadjuvant chemotherapy, is the standard initial approach, excluding individuals who are not good sur‐ gical candidates or whose disease precludes optimal cytoreduction [34]. The typical chemo‐ therapeutic regimen is a taxane/platinum based combination therapy. The standard postoperative approach would be 6 cycles (21 days each) of IV paclitaxal with carboplatin [35]

ly studied. The PARP enzyme regulates cellular responses to DNA damage, and plays an important role in the repair of single-stranded breaks by excision repair [41]. See Figure 3.

PARP Inhibitors in Ovarian Cancer http://dx.doi.org/10.5772/52888 309

The base excision repair (BER) pathway recognizes and removes damaged or inappropriate bases [42]. Damage specific glycosylases recognize the presence of a faulty base and imme‐ diately removes it. This leads to the formation of a potentially cytotoxic apurinic or apirimi‐ dinic (AP) site. Such sites are then processed by an AP endonuclease, which creates a strand break in the DNA. The enzyme Poly (ADP-ribose) polymerase binds to the strand break and relaxes the chromatin structure to allow for easier access of the BER machinery. Then, PARP transfers ADP-ribose units from NAD+ to nuclear target proteins, histones, and itself. This forms long and branched polymers of poly (ADP-ribose) on the PARP enzyme, that act as a signaling mechanism to recruit the BER machinery, including adaptor factor XECC1, PCNA, RFC, ligase III and DNA polymerase β. The BER complex assembles at the site of damage and facilitates repair in a coordinated fashion. Once complete, the complex disperses [43].

When PARP is inhibited, common single-strand breaks are converted into double-stranded breaks during DNA replication. In normal wild type cells, homologous recombination is the most common mechanism of repair in these double-stranded breaks which provides a safety net in maintaining genomic stability. In the presence of a deleterious BRCA gene, homolo‐ gous recombination is impaired. These cells and the tumors that they form have increased susceptibility to PARP inhibition, which leads to decreased chromosomal stability and, ulti‐

**Figure 3.** Role of Poly (ADP-ribose) polymerase in Base Excision Repair

mately, cell death. See Figure 4.

The 5-year survival and prognosis for *advanced* stage ovarian cancer, in BRCA or wild-type carriers ranges between 10 and 25 percent. Such poor prognosis is due to an incredibly high recurrence rate of advanced ovarian cancer. The extremely high rate of recurrence is due to the development of drug resistance [36]. Initially, 75% of patients with advanced disease show response to chemotherapy, while the remaining 25% show intrinsic resistance. How‐ ever, most of the population with initial chemosensitivity will show relapse within 2 years of initial treatment. Patients who have a shorter interval from their last course of chemother‐ apy to relapse will show decreased response with future courses [37]. For women who re‐ lapse within 6 months of treatment completion, there is a less than 10% chance of responding to any future therapies. For women whose disease recurs more than 6 to 12 months after initial therapy, chemotherapy includes platinum-based, multi-agent regimens. These women are considered partially platinum sensitive (relapse within 6-12 months) or platinum sensitive (relapse greater than 12 months after initial treatment). For patients who are platinum resistant (relapse within 6 months) or platinum refractory (no initial response), sequential single agents, such as pegylated liposomal doxorubicin hydrochloride or topote‐ can are suggested [38]. The slightly superior prognosis of BRCA patients compared to spora‐ dic EOC is likely related to their greater sensitivity to platinum based therapy [33]. However, despite initial chemo-sensitivity of BRCA carriers, they will ultimately develop to platinum resistant recurrent ovarian cancer.

PARP Inhibitors are not unlike the standard therapies for ovarian cancer, in that they too have shown evidence of resistance. Theoretically, their maximum potential is seen in cells with impaired homologous recombination. Researchers have hypothesized that resistance to PARP Inhibitors is through DNA repair mechanisms that actually correct the homologous recombination process. Through compensatory mutations, the initial mutational reading frame is corrected to a reading frame that actually produces a wild type BRCA protein [39]. A restoration in BRCA function would further contribute to platinum resistance and create PARP inhibitor resistance. This will need further investigations.

#### **6. PARP inhibitors**

PARP proteins are involved in a number of cellular processes, including DNA replication, transcriptional regulation, and DNA damage repair [40].Of the numerous PARP proteins detected, PARP1 and PARP2, which are associated with DNA stability, have been intensive‐ ly studied. The PARP enzyme regulates cellular responses to DNA damage, and plays an important role in the repair of single-stranded breaks by excision repair [41]. See Figure 3.

**Figure 3.** Role of Poly (ADP-ribose) polymerase in Base Excision Repair

**5. Mechanisms of resistance**

308 Ovarian Cancer - A Clinical and Translational Update

platinum resistant recurrent ovarian cancer.

**6. PARP inhibitors**

PARP inhibitor resistance. This will need further investigations.

Currently, there is no difference in management of EOC between BRCA positive patients and BRCA wild type patients. Maximal surgical cytoreduction, with or without neoadjuvant chemotherapy, is the standard initial approach, excluding individuals who are not good sur‐ gical candidates or whose disease precludes optimal cytoreduction [34]. The typical chemo‐ therapeutic regimen is a taxane/platinum based combination therapy. The standard postoperative approach would be 6 cycles (21 days each) of IV paclitaxal with carboplatin [35]

The 5-year survival and prognosis for *advanced* stage ovarian cancer, in BRCA or wild-type carriers ranges between 10 and 25 percent. Such poor prognosis is due to an incredibly high recurrence rate of advanced ovarian cancer. The extremely high rate of recurrence is due to the development of drug resistance [36]. Initially, 75% of patients with advanced disease show response to chemotherapy, while the remaining 25% show intrinsic resistance. How‐ ever, most of the population with initial chemosensitivity will show relapse within 2 years of initial treatment. Patients who have a shorter interval from their last course of chemother‐ apy to relapse will show decreased response with future courses [37]. For women who re‐ lapse within 6 months of treatment completion, there is a less than 10% chance of responding to any future therapies. For women whose disease recurs more than 6 to 12 months after initial therapy, chemotherapy includes platinum-based, multi-agent regimens. These women are considered partially platinum sensitive (relapse within 6-12 months) or platinum sensitive (relapse greater than 12 months after initial treatment). For patients who are platinum resistant (relapse within 6 months) or platinum refractory (no initial response), sequential single agents, such as pegylated liposomal doxorubicin hydrochloride or topote‐ can are suggested [38]. The slightly superior prognosis of BRCA patients compared to spora‐ dic EOC is likely related to their greater sensitivity to platinum based therapy [33]. However, despite initial chemo-sensitivity of BRCA carriers, they will ultimately develop to

PARP Inhibitors are not unlike the standard therapies for ovarian cancer, in that they too have shown evidence of resistance. Theoretically, their maximum potential is seen in cells with impaired homologous recombination. Researchers have hypothesized that resistance to PARP Inhibitors is through DNA repair mechanisms that actually correct the homologous recombination process. Through compensatory mutations, the initial mutational reading frame is corrected to a reading frame that actually produces a wild type BRCA protein [39]. A restoration in BRCA function would further contribute to platinum resistance and create

PARP proteins are involved in a number of cellular processes, including DNA replication, transcriptional regulation, and DNA damage repair [40].Of the numerous PARP proteins detected, PARP1 and PARP2, which are associated with DNA stability, have been intensive‐

The base excision repair (BER) pathway recognizes and removes damaged or inappropriate bases [42]. Damage specific glycosylases recognize the presence of a faulty base and imme‐ diately removes it. This leads to the formation of a potentially cytotoxic apurinic or apirimi‐ dinic (AP) site. Such sites are then processed by an AP endonuclease, which creates a strand break in the DNA. The enzyme Poly (ADP-ribose) polymerase binds to the strand break and relaxes the chromatin structure to allow for easier access of the BER machinery. Then, PARP transfers ADP-ribose units from NAD+ to nuclear target proteins, histones, and itself. This forms long and branched polymers of poly (ADP-ribose) on the PARP enzyme, that act as a signaling mechanism to recruit the BER machinery, including adaptor factor XECC1, PCNA, RFC, ligase III and DNA polymerase β. The BER complex assembles at the site of damage and facilitates repair in a coordinated fashion. Once complete, the complex disperses [43].

When PARP is inhibited, common single-strand breaks are converted into double-stranded breaks during DNA replication. In normal wild type cells, homologous recombination is the most common mechanism of repair in these double-stranded breaks which provides a safety net in maintaining genomic stability. In the presence of a deleterious BRCA gene, homolo‐ gous recombination is impaired. These cells and the tumors that they form have increased susceptibility to PARP inhibition, which leads to decreased chromosomal stability and, ulti‐ mately, cell death. See Figure 4.

formation of the poly-ADP-ribose polymer in intact cells, but Iniparib exhibited little or no ability to inhibit poly(ADP-ribose) polymer formation in situ. Other experiments revealed Ini‐ parib's inability to sensitize cells to cisplatin, gemcitabine and paclitaxel [51]. Iniparib's actual role may be modification of cysteine-containing proteins in tumor cells. If this is in fact the case, failure of the Iniparib phase III trial should not be used to guide further decisions about other PARP inhibitors. Keeping that in mind, Iniparib is continuing to be studied in a number of ovarian cancer trials, some showing promise. A phase II trial examining Iniparib in combina‐ tion with gemcitabine/carboplatin in patients with platinum-resistant recurrent ovarian can‐ cer has to date shown a 25% overall response rate consisting of 8 out of 32 confirmed responses [52]. Progression free survival of 6.4 months (95% CI, 3.0-NE) was demonstrated in an early analysis. The PFS and ORR are significantly improved when compared to a previous study of pegylated liposomal doxorubicin in platinum-resistant recurrent ovarian cancer with an ORR of 11.7% and mean progression free survival of 3.1 months [53]. Once again, these patients were not stratified based on their BRCA carrier status. A similar study looked at iniparib in combination with gemcitabine/carboplatin in patients with platinum-sensitive recurrent ovar‐ ian cancer [54]. This study showed an ORR of 65%, consisting of 26 out of 40 patients, as well as a median progression free survival of 9.5 months. Interestingly, there was no indication of a re‐

**Trial Interests Side Effects**

Nausea, vomiting, fatigue, taste-

PARP Inhibitors in Ovarian Cancer http://dx.doi.org/10.5772/52888 311

Thrombocytopenia and neutropenia

Fatigue, reversible pneumonitis,

alteration, anorexia

myelosuppression, thrombocytopenia

Myelosuppression, fatigue

Iniparib IV Ovarian cancer, uterine carcinosarcoma, NSCLCFatigue, nausea, diarrhea, dizziness

population, other advanced solid tumors

advanced or metastatic breast cancer,

hematologic malignancies, advanced melanoma, advanced glioblastoma

INO-1001 IV Melanoma, p53 deficient cancer cells Myelosuppression, transaminitis

advanced ovarian cancer

lationship between BRCA status and response.

Olaparib Oral Breast and ovarian cancer, the BRCA

Rucaparib IV BRCA associated breast cancer, locally

MK-4827 Oral Advanced solid tumors, ovarian cancer,

AZD-2461 Oral Single agent for refractory solid tumors CEP-9722 Oral Single agent for advanced solid tumors E7016 Oral Advanced solid tumors, melanoma,

**Table 2.** PARP Inhibitors in Clinical Development

glioblastoma BMN-673 Oral Advanced solid tumors and hematologic malignancies

Veliparib Oral TNBC and BRCA deficient breast and ovarian cancer

**Administration**

**Agent Route of**

**Figure 4.** Synthetic Lethality of PARP Inhibition

PARP inhibitors are also hypothesized to increase cytotoxicity of chemotherapeutic agents. Cells treated with chemotherapy, especially platinum based agents, ultimately attain resist‐ ance by altering DNA repair processes [44]. It is hypothesized that through PARP inhibition, cells with faulty BRCA function could be prevented from repairing chemotherapy -induced DNA damage [45,46]. Clinical trials have examined PARP inhibitors as single agents in pa‐ tients with impaired homologous recombination, as well as in combination with chemo-ra‐ diation therapy.

#### **7. Clinical trials**

Table 2 shows a list of PARP inhibitors that are currently in clinical development [4]. A full list of studies involving all tumor types can be found through the National Institute of Health website, www.clinicaltrials.gov.

Iniparib is one of the first drugs looked at in the PARP inhibitor class. A phase II trial of inipar‐ ib in women with advanced ovarian or breast cancer compared the chemotherapeutic regi‐ men carboplatin/gemcitabine with and without iniparib. The iniparib arm showed an improved rate of clinical benefit (56% vs 34%, P=0.01), increased rate of overall response (52% vs 32%, P=0.02), prolonged median progression free survival (5.9 movs 3.6 mo, P=0.01), and in‐ creased median overall survival (12.3 mo vs 7.7 mo, P=0.01) [47]. However, a phase III trial of Iniparib for women with triple negative breast cancer proved to be disappointing [48,49]. This trial, which randomized 519 women with triple negative breast cancer to carboplatin with gemcitabine versus carboplatin, gemcitabine plus iniparib demonstrated an increase in pro‐ gression-free survival among the iniparib arm (5.1 vs 4.1 months, with P=0.027), but did not reach statistical significance. Patients were not stratified based on BRCA status, specific triple negative breast cancer subtype, or level of expression of PARP proteins. More recent studies suggest that Iniparib may not actually inhibit the PARP enzyme in vitro. One in particular compared Iniparib to Olaparib and Veliparib [50]. Olaparib and Veliparib proved to inhibit formation of the poly-ADP-ribose polymer in intact cells, but Iniparib exhibited little or no ability to inhibit poly(ADP-ribose) polymer formation in situ. Other experiments revealed Ini‐ parib's inability to sensitize cells to cisplatin, gemcitabine and paclitaxel [51]. Iniparib's actual role may be modification of cysteine-containing proteins in tumor cells. If this is in fact the case, failure of the Iniparib phase III trial should not be used to guide further decisions about other PARP inhibitors. Keeping that in mind, Iniparib is continuing to be studied in a number of ovarian cancer trials, some showing promise. A phase II trial examining Iniparib in combina‐ tion with gemcitabine/carboplatin in patients with platinum-resistant recurrent ovarian can‐ cer has to date shown a 25% overall response rate consisting of 8 out of 32 confirmed responses [52]. Progression free survival of 6.4 months (95% CI, 3.0-NE) was demonstrated in an early analysis. The PFS and ORR are significantly improved when compared to a previous study of pegylated liposomal doxorubicin in platinum-resistant recurrent ovarian cancer with an ORR of 11.7% and mean progression free survival of 3.1 months [53]. Once again, these patients were not stratified based on their BRCA carrier status. A similar study looked at iniparib in combination with gemcitabine/carboplatin in patients with platinum-sensitive recurrent ovar‐ ian cancer [54]. This study showed an ORR of 65%, consisting of 26 out of 40 patients, as well as a median progression free survival of 9.5 months. Interestingly, there was no indication of a re‐ lationship between BRCA status and response.


**Table 2.** PARP Inhibitors in Clinical Development

**Figure 4.** Synthetic Lethality of PARP Inhibition

310 Ovarian Cancer - A Clinical and Translational Update

Health website, www.clinicaltrials.gov.

diation therapy.

**7. Clinical trials**

PARP inhibitors are also hypothesized to increase cytotoxicity of chemotherapeutic agents. Cells treated with chemotherapy, especially platinum based agents, ultimately attain resist‐ ance by altering DNA repair processes [44]. It is hypothesized that through PARP inhibition, cells with faulty BRCA function could be prevented from repairing chemotherapy -induced DNA damage [45,46]. Clinical trials have examined PARP inhibitors as single agents in pa‐ tients with impaired homologous recombination, as well as in combination with chemo-ra‐

Table 2 shows a list of PARP inhibitors that are currently in clinical development [4]. A full list of studies involving all tumor types can be found through the National Institute of

Iniparib is one of the first drugs looked at in the PARP inhibitor class. A phase II trial of inipar‐ ib in women with advanced ovarian or breast cancer compared the chemotherapeutic regi‐ men carboplatin/gemcitabine with and without iniparib. The iniparib arm showed an improved rate of clinical benefit (56% vs 34%, P=0.01), increased rate of overall response (52% vs 32%, P=0.02), prolonged median progression free survival (5.9 movs 3.6 mo, P=0.01), and in‐ creased median overall survival (12.3 mo vs 7.7 mo, P=0.01) [47]. However, a phase III trial of Iniparib for women with triple negative breast cancer proved to be disappointing [48,49]. This trial, which randomized 519 women with triple negative breast cancer to carboplatin with gemcitabine versus carboplatin, gemcitabine plus iniparib demonstrated an increase in pro‐ gression-free survival among the iniparib arm (5.1 vs 4.1 months, with P=0.027), but did not reach statistical significance. Patients were not stratified based on BRCA status, specific triple negative breast cancer subtype, or level of expression of PARP proteins. More recent studies suggest that Iniparib may not actually inhibit the PARP enzyme in vitro. One in particular compared Iniparib to Olaparib and Veliparib [50]. Olaparib and Veliparib proved to inhibit Olaparib, on the other hand, is a considered a bona-fide PARP inhibitor and has shown promising results in phase II trials in the treatment of BRCA-deficient advanced ovarian cancer. A recent study looked at oral Olaparib as a single agent, and its effect on BRCA posi‐ tive vs. BRCA wild-type patients in women with ovarian and breast cancer [55]. Women with advanced high grade serous and/or undifferentiated ovarian carcinoma or triple-nega‐ tive breast cancer were enrolled and received olaparib 400 mg twice a day. 91 patients were enrolled in this particular study, 65 with ovarian cancer and 26 with breast cancer. 63 of the 65 ovarian cancer cohorts had target lesions and were evaluable for objective response. Among these 63 patients, 41% of BRCA carriers showed confirmed objective response (7 out of 17 with 95% CI) and a surprising 24% of BRCA wild-type patients showed confirmed ob‐ jective response (11 of 46 with 95% CI). The 24% response of BRCA wild type patients sug‐ gests that Olaparib, if not all PARP inhibitors, may provide significant benefit for all patients with ovarian cancer, and not only those with selective BRCA mutation. As mentioned above, around 40-50% of ovarian cancers, in the absence of a mutation of the BRCA gene, can affect the functional aspect of the BRCA proteins and ultimately homologous recombi‐ nation. It is for this reason that PARP inhibition likely shows benefit in not just the BRCA mutated population, but a larger population that umbrellas deficient homologous repair. However, for this particular study phase III trials are no longer scheduled to commence be‐ cause the interim analysis of survival did not show the desired benefit in relation to the ben‐ efit in progression free survival. Also, there were no confirmed objective responses reported in the breast cancer patients.

sensitive patients, a 41.7% response rate in platinum-resistant patients and 15.4% response

PARP Inhibitors in Ovarian Cancer http://dx.doi.org/10.5772/52888 313

A randomized phase II trial in BRCA deficient advanced ovarian cancer (platinum interval<12 months) enrolled 97 women and looked at olaparib dosing, 200mg vs 400mg, and compared its efficacy to pegylated liposomal doxorubicin [60]. Of the 32 low dose olaparib cohorts (200mg BID), 38 % showed objective response with a median progression free survival of 6.5 months. Of the 32 high dose (400mg BID) cohorts, 59% showed objective response with a median PFS of 8.8 months. These were compared to 33 women who received the standard pegylated liposo‐ mal doxorubicin, with an objective response of 38% and median PFS of 7.1 months, results that were not clinically significant. This study, however, does not necessarily rule out Olaparib's action in BRCA deficient patients. Recent studies suggest that women with BRCA associated ovarian cancer may demonstrate increased sensitivity to Doxil than previously reported in un‐ selected cases [61]. One study in particular showed a 57% response rate to PLD among BRCA deficient patients, compared to 20% response rate among those with sporadic EOC. This re‐ sponse was associated with significantly improved progression-free and overall survival. Fur‐ thermore, the initial study was actually potentially comparing 2 drugs with particular benefit in BRCA patients and though there is no clinically significant difference among them, they

Another PARP inhibitor under investigation is Veliparib. A phase II trial of Veliparib in combination with cyclophosphamide compared to single-agent cyclophosphamide is cur‐ rently ongoing [62]. This study examines Veliparib's activity against advanced solid tumors and lymphomas. Preliminary results show promising activity in the BRCA subset. Of the 35 patients enrolled, 7 have shown partial response and 6 have stable disease in the veliparib arm. In another study veliparib with or without carboplatin was evaluated in patients with stage III and IV BRCA-associated breast cancer [63]. Of the 22 patients enrolled, only 12 were eligible for evaluation. Complete response was seen in 2 patients and partial response

A phase I study showed activity of veliparib and temozolomide in combination against metastatic breast cancer [64]. Of the 41 patients enrolled, complete response was seen in 1 patient, partial response in 2, stable disease in 7 and disease progression in 14. BRCA muta‐ tion analysis is currently underway. Another study is currently looking at veliparib in com‐ bination with doxorubicin and cyclophosphamide for the treatment of breast cancer and other solid tumors [65]. Of the 18 patients enrolled, 14 have breast cancer (including 5 with BRCA mutations), 3 have ovarian cancer, and 1 other solid tumor. There has been objective anti-tumor activity seen in the BRCA mutation carriers. With this particular regimen, dosing was limited by myelosuppresion. Furthermore, although combination therapy has shown to enhance chemotherapeutic effects, myelosuppression appears to be enhanced as well. More than 50 clinical trials examining Veliparib are currently ongoing, looking at gynecologic can‐ cers, solid tumors, lymphomas, brain tumors, GI and prostate cancer. Most of these are cur‐ rently recruiting, with only a few in Stage II. It will be very exciting to see the end results of these trials, specifically for our purposes, those involving BRCA analysis. Very few updates

rate in platinum-refractory patients.

each may show clinical significance when standing alone.

in 6 patients, with a clinical benefit of 75% seen.

have been given, as most of these are in the beginning stages.

A very similar 2-part study looked at single agent Olaparib, and compared doses (100mg vs. 400mg) in the treatment of advanced breast and ovarian cancer in BRCA deficient individu‐ als [56,57]. Among the 57 ovarian cancer patients, the overall response rate of olaparib 100mg BID was 12.5% with a clinical benefit rate of 16.7%. For the 400mg BID arm, ORR was 33% with a clinical benefit rate of 57.6%. Further stratification showed a response in both platinum-sensitive individuals (38% ORR) and platinum-resistant individuals (30%). Among the 54 breast cancer patients, the overall response of olaparib 100mg BID was 25% with a progression free survival of 3.8months. For the 400mg BID arm, ORR was 42% with PFS of 5.7 months.

Olaparib is also being looked at as maintenance therapy. A phase II trial studied Olaparib as a maintenance therapy in relapsed serous ovarian cancer. The 265 enrolled patients had re‐ ceived at least 2 previous platinum based chemo regimens with eventual relapse [58]. Early analysis has shown a 65% reduced risk of progression in the Olaparib arm, improving pro‐ gression free survival by 3.6 months (8.4 mo vs. 4.8). Patients were stratified based on BRCA status, age, race, Jewish ethnicity, prior response to platinum regimen and relapse time, and each subgroup showed an improved progression free survival in the Olaparib arm. Overall survival data has not been analyzed.

Another study looking at differences in response to olaparib based on platinum-response status [59]. This trial looked at oral olaparib as a single agent against advanced ovarian can‐ cer in 50 women with BRCA mutations. Results showed a 61.5% response rate in platinumsensitive patients, a 41.7% response rate in platinum-resistant patients and 15.4% response rate in platinum-refractory patients.

Olaparib, on the other hand, is a considered a bona-fide PARP inhibitor and has shown promising results in phase II trials in the treatment of BRCA-deficient advanced ovarian cancer. A recent study looked at oral Olaparib as a single agent, and its effect on BRCA posi‐ tive vs. BRCA wild-type patients in women with ovarian and breast cancer [55]. Women with advanced high grade serous and/or undifferentiated ovarian carcinoma or triple-nega‐ tive breast cancer were enrolled and received olaparib 400 mg twice a day. 91 patients were enrolled in this particular study, 65 with ovarian cancer and 26 with breast cancer. 63 of the 65 ovarian cancer cohorts had target lesions and were evaluable for objective response. Among these 63 patients, 41% of BRCA carriers showed confirmed objective response (7 out of 17 with 95% CI) and a surprising 24% of BRCA wild-type patients showed confirmed ob‐ jective response (11 of 46 with 95% CI). The 24% response of BRCA wild type patients sug‐ gests that Olaparib, if not all PARP inhibitors, may provide significant benefit for all patients with ovarian cancer, and not only those with selective BRCA mutation. As mentioned above, around 40-50% of ovarian cancers, in the absence of a mutation of the BRCA gene, can affect the functional aspect of the BRCA proteins and ultimately homologous recombi‐ nation. It is for this reason that PARP inhibition likely shows benefit in not just the BRCA mutated population, but a larger population that umbrellas deficient homologous repair. However, for this particular study phase III trials are no longer scheduled to commence be‐ cause the interim analysis of survival did not show the desired benefit in relation to the ben‐ efit in progression free survival. Also, there were no confirmed objective responses reported

A very similar 2-part study looked at single agent Olaparib, and compared doses (100mg vs. 400mg) in the treatment of advanced breast and ovarian cancer in BRCA deficient individu‐ als [56,57]. Among the 57 ovarian cancer patients, the overall response rate of olaparib 100mg BID was 12.5% with a clinical benefit rate of 16.7%. For the 400mg BID arm, ORR was 33% with a clinical benefit rate of 57.6%. Further stratification showed a response in both platinum-sensitive individuals (38% ORR) and platinum-resistant individuals (30%). Among the 54 breast cancer patients, the overall response of olaparib 100mg BID was 25% with a progression free survival of 3.8months. For the 400mg BID arm, ORR was 42% with

Olaparib is also being looked at as maintenance therapy. A phase II trial studied Olaparib as a maintenance therapy in relapsed serous ovarian cancer. The 265 enrolled patients had re‐ ceived at least 2 previous platinum based chemo regimens with eventual relapse [58]. Early analysis has shown a 65% reduced risk of progression in the Olaparib arm, improving pro‐ gression free survival by 3.6 months (8.4 mo vs. 4.8). Patients were stratified based on BRCA status, age, race, Jewish ethnicity, prior response to platinum regimen and relapse time, and each subgroup showed an improved progression free survival in the Olaparib arm. Overall

Another study looking at differences in response to olaparib based on platinum-response status [59]. This trial looked at oral olaparib as a single agent against advanced ovarian can‐ cer in 50 women with BRCA mutations. Results showed a 61.5% response rate in platinum-

in the breast cancer patients.

312 Ovarian Cancer - A Clinical and Translational Update

PFS of 5.7 months.

survival data has not been analyzed.

A randomized phase II trial in BRCA deficient advanced ovarian cancer (platinum interval<12 months) enrolled 97 women and looked at olaparib dosing, 200mg vs 400mg, and compared its efficacy to pegylated liposomal doxorubicin [60]. Of the 32 low dose olaparib cohorts (200mg BID), 38 % showed objective response with a median progression free survival of 6.5 months. Of the 32 high dose (400mg BID) cohorts, 59% showed objective response with a median PFS of 8.8 months. These were compared to 33 women who received the standard pegylated liposo‐ mal doxorubicin, with an objective response of 38% and median PFS of 7.1 months, results that were not clinically significant. This study, however, does not necessarily rule out Olaparib's action in BRCA deficient patients. Recent studies suggest that women with BRCA associated ovarian cancer may demonstrate increased sensitivity to Doxil than previously reported in un‐ selected cases [61]. One study in particular showed a 57% response rate to PLD among BRCA deficient patients, compared to 20% response rate among those with sporadic EOC. This re‐ sponse was associated with significantly improved progression-free and overall survival. Fur‐ thermore, the initial study was actually potentially comparing 2 drugs with particular benefit in BRCA patients and though there is no clinically significant difference among them, they each may show clinical significance when standing alone.

Another PARP inhibitor under investigation is Veliparib. A phase II trial of Veliparib in combination with cyclophosphamide compared to single-agent cyclophosphamide is cur‐ rently ongoing [62]. This study examines Veliparib's activity against advanced solid tumors and lymphomas. Preliminary results show promising activity in the BRCA subset. Of the 35 patients enrolled, 7 have shown partial response and 6 have stable disease in the veliparib arm. In another study veliparib with or without carboplatin was evaluated in patients with stage III and IV BRCA-associated breast cancer [63]. Of the 22 patients enrolled, only 12 were eligible for evaluation. Complete response was seen in 2 patients and partial response in 6 patients, with a clinical benefit of 75% seen.

A phase I study showed activity of veliparib and temozolomide in combination against metastatic breast cancer [64]. Of the 41 patients enrolled, complete response was seen in 1 patient, partial response in 2, stable disease in 7 and disease progression in 14. BRCA muta‐ tion analysis is currently underway. Another study is currently looking at veliparib in com‐ bination with doxorubicin and cyclophosphamide for the treatment of breast cancer and other solid tumors [65]. Of the 18 patients enrolled, 14 have breast cancer (including 5 with BRCA mutations), 3 have ovarian cancer, and 1 other solid tumor. There has been objective anti-tumor activity seen in the BRCA mutation carriers. With this particular regimen, dosing was limited by myelosuppresion. Furthermore, although combination therapy has shown to enhance chemotherapeutic effects, myelosuppression appears to be enhanced as well. More than 50 clinical trials examining Veliparib are currently ongoing, looking at gynecologic can‐ cers, solid tumors, lymphomas, brain tumors, GI and prostate cancer. Most of these are cur‐ rently recruiting, with only a few in Stage II. It will be very exciting to see the end results of these trials, specifically for our purposes, those involving BRCA analysis. Very few updates have been given, as most of these are in the beginning stages.


**Agent Cancer Summary Prelim/Conclusions**

cancer

57 patients with ovarian

265 enrolled with at least 2 previous platinum based chemo regimens

97 women enrolled, study compared efficacy/safety of

22 patients enrolled, 12 eligible for evaluation

18 patients enrolled, 14 with breast cancer (5 Brca+), 3 with ovarian cancer and 1 other

S.A.- single agent; PFS-progression free survival; ORR-overall response rate; COR-confirmed objective response; BRCA-

olaparib vs. PLD


PARP Inhibitors in Ovarian Cancer http://dx.doi.org/10.5772/52888 315






partial response in 2 patients, stable disease in 7, progression in 14






brca mutation carriers -3/5 BRCA+ TNBC with partial

veliparib tolerated well

response

cancer pt -MTD 100mgBID

individuals

35 patients -combination of Cyclophosphamide/



(30%) disease

Olaparib arm

PFS 8.8mo

7.1mo

41 patients -complete response in 1 patient,

Advanced ovarian cancer in BRCA deficient individuals

Relapsed ovarian cancer, platinumsensitive

BRCA deficient advanced ovarian cancer with platinum interval <12 mo.

Metastatic triple negative breast cancer

Stage IV breast cancer

Breast cancer and other solid tumors

Refractory solid tumors and lymphomas

Olaparib S.A. [57]

Olaparib SA [58]

Olaparib SA vs.

Veliparib with Temozolomide

Veliparib with Carboplatin [63]

Veliparib with Doxo/ Cyclophospha mide [65]

Veliparib with Cyclophospho mide [62]

WT-wild type; BRCA-m-mutant

**Table 3.** Clinical Trials [47-65]

PLD [60]

[64]


S.A.- single agent; PFS-progression free survival; ORR-overall response rate; COR-confirmed objective response; BRCA-WT-wild type; BRCA-m-mutant

**Table 3.** Clinical Trials [47-65]

**Agent Cancer Summary Prelim/Conclusions**

C/G + Iniparib

C/G + Iniparib

enzyme in vitro

disease

status

to platinum

Ovarian cancer 50 patients enrolled and

Single arm study -41 patients with dx of recurrent platinum sensitive

ovarian cancer

91 patients (65 with ovarian ca, 26 with breast ca), stratified based on BRCA

stratified based on response

48 patients with dx of epithelial ovarian carcinoma, follopian tube cancer, or primary peritoneal carcinoma with platinum-resistant

NA Study looked at ability of



Iniparib to inhibit the PARP












5.7months -ORR 25% with 100mgBID,



arm vs. 34%

(12.3 mo vs 7.7 mo)

inhibit PARP in situ.

of 11.7% w/ PLD)

3.1 mo w/ PLD)


patients showed COR

breast cancer patients

PFS 3.8months

54 patients with breast cancer -ORR 42% with 400mgBID, PFS

(5.1 vs. 4.1mo)

status

Metastatic TNBC Phase 2

314 Ovarian Cancer - A Clinical and Translational Update

Metastatic TNBC Phase 3

Platinum resistant recurrent ovarian

Platinum sensitive recurrent ovarian

Ovarian and breast

Advanced breast cancer in BRCA deficient individuals

cancer

cancer

cancer

Iniparib + Carbo/Gem [47]

Iniparib + Carbo/Gem [48,49]

Iniparib [50]

Iniparib + Carbo/Gem [52]

Iniparib + C/G

Olaparib S.A. [55]

Olaparib S.A. [59]

Olaparib S.A. [56]

[54]

BMN-673 is the newest PARP inhibitor to be developed and is to date the most potent and selective PARP inhibitor. It has been shown to be up to 700-fold more active in vitro in BRCA deficient cell lines when compared to olaparib [66]. Phase I trials have yet to begin. Rucaparib is another new PARP inhibitor being looked into. It is currently the focus of 3 dif‐ ferent clinical trials; examining its activity in combination with several different chemothera‐ peutic regimens, efficacy in BRCA-associated breast cancer, and treatment of patients with locally advanced or metastatic breast and advanced ovarian cancer [4]. There a few other PARP inhibitors that are currently being evaluated for efficacy and tolerability, and will likely acquire more interest in the near future. Table 3 summarizes the findings of the PARP inhibitors discussed above.

**Author details**

Jeanine Staples1

**References**

Gynecology, Boston, USA

2001; 411:366-374

1995; 11:428-433.

8:562-594.

302:643.

Lancet 1997; 349:1505-1510.

JAMA, 2000; 283(5):617-24.

and Annekathryn Goodman2\*

2 Harvard Medical School, Massachusetts General Hospital, Department of Obstetrics and

PARP Inhibitors in Ovarian Cancer http://dx.doi.org/10.5772/52888 317

[1] Hoeijmakers JH. Genome maintenance mechanisms for preventing cancer. Nature.

[2] Gayther SA, Warren W, Mazoyer S, Russell PA, Harrington PA, Chiano M, Seal S, Hamoudi R, van Rensburg EJ, Dunning AM, Love R, Evans G, Easton D, Clayton D, Stratton MR, Ponder BA. Germline mutations of the BRCA1 gene in breast and ovari‐ an cancer families provide evidence for a genotype-phenotype correlation. Nat genet,

[3] Venkitaraman, Ashok. Functions of BRCA1 and BRCA2 in the biological response to

[4] Underhill C, Toulmonde M, Bonnefoi H. A review of PARP inhibitors: from bench to

[5] Daly MB, Axilbund JE, Buys S, Crawford B, Farrell CD, Friedman S, Garber JE, Goo‐ rha S, Gruber SB, Hampel H, Kaklamani V, Kohlmann W, Kurian A, Litton J,Marcom PK, Nussbaum R, Offit K, Pal T, Pasche B, Pilarski R, Reiser G, Shannon KM, Smith JR, Swisher E, Weitzel JN; National Comprehensive Cancer Network. Genetic/fami‐ lial high-risk assessment: breast and ovarian. J Natl Compr Canc Netw 2010;

[6] King MC, Marks JH, Mandell JB. New York Breast Cancer Study Group. Breast and ovarian cancer risks due to inherited mutations in BRCA1 and BRCA2. Science 2003;

[7] Breast Cancer Linkage Consortium. Pathology of familial breast cancer: differences between breast cancers in carriers of BRCA1 or BRCA2 mutations and sporadic cases.

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\*Address all correspondence to: agoodman@partners.org

DNA damage. J Cell Sci 2001; 114: 3591-3598.

bedside. Annals Oncology, 2011; 22(2):268-279.

1 Boston University School of Medicine, Boston, USA

Phase I and II trials demonstrated PARP inhibitor's favorable side effect profile; potentially including fatigue, nausea, vomiting, and mood disturbance. Also, myelosuppression has been noted in higher doses, especially when in combination with chemotherapy. These ad‐ verse effects are generally mild, especially in comparison with current chemotherapeutic regimens.

#### **8. Conclusion**

PARP inhibitors have been increasingly studied. This new group of drugs has been shown in multiple phase I and II trials to be efficacious with favorable side effect profiles. The pre‐ clinical data which suggested the effect of PARP inhibitors in BRCA deficient cells did not take into account the much larger population of tumors deficient in homologous recombina‐ tion, with physically healthy yet functionally impaired BRCA genes. This new understand‐ ing has further raised the bar for the potential of this new class. PARP inhibitors have been shown to increase progression-free-survival and overall response rate in a number of trials. While there have indeed been some setbacks in the development of these new drugs, some of the major concerns have been addressed. Though Iniparib's phase III trial failure was ini‐ tially viewed as a major disappointment, later studies proved that Iniparib does not have the same efficacy as its counterparts Olaparib/Veliparib, and ultimately may have no role in PARP inhibition of single-strand break repair. Also, the discovery that Olaparib did not show a clinically significant difference when compared to pegylated liposomal doxorubicin, led to the detection of PLD as a particularly potent agent against BRCA deficient cells. So rather than dismissing Olaparib's effect as not clinically significant, this particular study en‐ couraged further investigation of PLD in this special population. Furthermore, the efficacy of PARP inhibition is evident, but the ideal population that could benefit most from this new class has yet to be determined. Future trials involving PARP inhibitors should undergo extremely strict stratification, as it is crucial to reveal precisely which population stands to benefit. Other concerns that need to be further investigated include mechanisms of resist‐ ance, use as frontline vs. maintenance vs. recurrent disease therapy, use in mono-therapy vs. combination with chemo or combination with other targeted therapies.

#### **Author details**

BMN-673 is the newest PARP inhibitor to be developed and is to date the most potent and selective PARP inhibitor. It has been shown to be up to 700-fold more active in vitro in BRCA deficient cell lines when compared to olaparib [66]. Phase I trials have yet to begin. Rucaparib is another new PARP inhibitor being looked into. It is currently the focus of 3 dif‐ ferent clinical trials; examining its activity in combination with several different chemothera‐ peutic regimens, efficacy in BRCA-associated breast cancer, and treatment of patients with locally advanced or metastatic breast and advanced ovarian cancer [4]. There a few other PARP inhibitors that are currently being evaluated for efficacy and tolerability, and will likely acquire more interest in the near future. Table 3 summarizes the findings of the PARP

Phase I and II trials demonstrated PARP inhibitor's favorable side effect profile; potentially including fatigue, nausea, vomiting, and mood disturbance. Also, myelosuppression has been noted in higher doses, especially when in combination with chemotherapy. These ad‐ verse effects are generally mild, especially in comparison with current chemotherapeutic

PARP inhibitors have been increasingly studied. This new group of drugs has been shown in multiple phase I and II trials to be efficacious with favorable side effect profiles. The pre‐ clinical data which suggested the effect of PARP inhibitors in BRCA deficient cells did not take into account the much larger population of tumors deficient in homologous recombina‐ tion, with physically healthy yet functionally impaired BRCA genes. This new understand‐ ing has further raised the bar for the potential of this new class. PARP inhibitors have been shown to increase progression-free-survival and overall response rate in a number of trials. While there have indeed been some setbacks in the development of these new drugs, some of the major concerns have been addressed. Though Iniparib's phase III trial failure was ini‐ tially viewed as a major disappointment, later studies proved that Iniparib does not have the same efficacy as its counterparts Olaparib/Veliparib, and ultimately may have no role in PARP inhibition of single-strand break repair. Also, the discovery that Olaparib did not show a clinically significant difference when compared to pegylated liposomal doxorubicin, led to the detection of PLD as a particularly potent agent against BRCA deficient cells. So rather than dismissing Olaparib's effect as not clinically significant, this particular study en‐ couraged further investigation of PLD in this special population. Furthermore, the efficacy of PARP inhibition is evident, but the ideal population that could benefit most from this new class has yet to be determined. Future trials involving PARP inhibitors should undergo extremely strict stratification, as it is crucial to reveal precisely which population stands to benefit. Other concerns that need to be further investigated include mechanisms of resist‐ ance, use as frontline vs. maintenance vs. recurrent disease therapy, use in mono-therapy vs.

combination with chemo or combination with other targeted therapies.

inhibitors discussed above.

316 Ovarian Cancer - A Clinical and Translational Update

regimens.

**8. Conclusion**

Jeanine Staples1 and Annekathryn Goodman2\*

\*Address all correspondence to: agoodman@partners.org

1 Boston University School of Medicine, Boston, USA

2 Harvard Medical School, Massachusetts General Hospital, Department of Obstetrics and Gynecology, Boston, USA

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[54] Penson R, Whalen C, Lasonde B, Krasner CN, Konstantinopoulos P, Stallings TE, Bradley CR, Birrer MJ, Matulonis U. A Phase II trial of Iniparib in combination with gemcitabin/carboplatin in patients with platinum-sensitive recurrent ovarian cancer. Presented at 2011 ASCO Annual Meeting. http://www.asco.org/ascov2/Meetings/ Abstracts?&vmview=abst\_detail\_view&confID=102&abstractID=82124 accessed Au‐

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322 Ovarian Cancer - A Clinical and Translational Update


**Section 5**

**Biomarkers**

**Section 5**

### **Biomarkers**

**Chapter 15**

**The Merit of Alternative Messenger RNA Splicing as a**

In this review, we discuss the merit of splicing isoforms as a source of biomarkers for ovari‐ an cancer with a special focus on features that distinguish splice variants from global gene expression based markers. Key examples demonstrating the usefulness of alternative splic‐

Ovarian cancer is a low incidence cancer with high mortality rate [1]. The asymptomatic na‐ ture of this cancer and the late stage diagnosis of most tumors are the reasons for ineffective surgery and chemotherapy [2]. In this sense, intensive research aim at increasing overall pa‐ tient survival and quality of life by providing biomarkers for 1) early detection and 2) pre‐ diction of chemotherapy response and/or suggestion of alternative strategies. CA-125 is a glycoprotein that is usually expressed in a variety of epithelial cells and its serum level rise up in advance ovarian cancer [3]. However, its use as an early detection marker or as a tool to screen the general population has not been approved so far [4,5]. CA-125 level is helpful in treatment-decision making but do not retain the capacity to improve overall survival and quality of life [6,7]. Clearly, there is still great need for biomarkers or combination of bio‐ markers that could positively identify early ovarian cancer lesions with great certainty or in‐

Genome-wide mRNA profiling presents an opportunity to rapidly identify RNA markers. Microarray platform has been applied in numerous occasions to provide gene expression signature correlating prognosis or indicative of chemotherapy response (review in [8]). However, the nature of the platform used to carry the experiments and the analysis methods and sample sets makes inter-laboratory comparison very difficult and finding reliable mark‐

and reproduction in any medium, provided the original work is properly cited.

© 2013 Brosseau and Abou-Elela; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**New Mine for the Next Generation Ovarian Cancer**

Jean-Philippe Brosseau and Sherif Abou-Elela

Additional information is available at the end of the chapter

ing (AS) as markers of ovarian cancer are described.

**Biomarkers**

**1. Introduction**

crease patients' survival.

http://dx.doi.org/10.5772/53976

## **The Merit of Alternative Messenger RNA Splicing as a New Mine for the Next Generation Ovarian Cancer Biomarkers**

Jean-Philippe Brosseau and Sherif Abou-Elela

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53976

#### **1. Introduction**

In this review, we discuss the merit of splicing isoforms as a source of biomarkers for ovari‐ an cancer with a special focus on features that distinguish splice variants from global gene expression based markers. Key examples demonstrating the usefulness of alternative splic‐ ing (AS) as markers of ovarian cancer are described.

Ovarian cancer is a low incidence cancer with high mortality rate [1]. The asymptomatic na‐ ture of this cancer and the late stage diagnosis of most tumors are the reasons for ineffective surgery and chemotherapy [2]. In this sense, intensive research aim at increasing overall pa‐ tient survival and quality of life by providing biomarkers for 1) early detection and 2) pre‐ diction of chemotherapy response and/or suggestion of alternative strategies. CA-125 is a glycoprotein that is usually expressed in a variety of epithelial cells and its serum level rise up in advance ovarian cancer [3]. However, its use as an early detection marker or as a tool to screen the general population has not been approved so far [4,5]. CA-125 level is helpful in treatment-decision making but do not retain the capacity to improve overall survival and quality of life [6,7]. Clearly, there is still great need for biomarkers or combination of bio‐ markers that could positively identify early ovarian cancer lesions with great certainty or in‐ crease patients' survival.

Genome-wide mRNA profiling presents an opportunity to rapidly identify RNA markers. Microarray platform has been applied in numerous occasions to provide gene expression signature correlating prognosis or indicative of chemotherapy response (review in [8]). However, the nature of the platform used to carry the experiments and the analysis methods and sample sets makes inter-laboratory comparison very difficult and finding reliable mark‐

© 2013 Brosseau and Abou-Elela; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ers complicated. Indeed, a meta-analysis regrouping 829 samples fails to demonstrate the predictive power of 16 individual gene expression signatures [9]. Consequently, very few microarray markers reached the clinic. In contrast, high-throughput protein signature based on mass spectrometry platform appears to have much more overlap in the peaks found by different experimental studies [10]. However, the pace by which protein biomarkers are translated into clinical setting is relatively slow [11]. Clearly, there is a need for novel meth‐ odology to discover ovarian cancer biomarkers that can yield reliable results and produce tests that could be quickly integrated in normal clinical setting. In this chapter, we discuss the potential of splice variant annotations as a tool for the discovery of ovarian cancer mark‐ ers and discuss the challenges and promises of this hidden mine.

type of ASE in human is the full skipping of an exon (cassette exon, Fig. 2C). Some exons are al‐ so skipped as a bloc (multiple cassette exon, Fig. 2D) or mutually exclusive (Fig. 2E). AS could also be coupled to others regulatory mechanisms such as polyadenylation [14] (Fig. 2F). In this case, the resulting mRNA exhibits a different 3' untranslated region (UTR), which is further subjected to different regulation by small non-coding RNA (e.g. microRNA). In about 1 out of 3 cases, AS decision introduces a sequence containing premature stop codon [20]. In these cases, the resulting mRNA is flagged to be degraded by the non-sense mediated decay machinery creating an efficient mechanism that control gene expression post-transcriptionally [21] (Fig. 2G). In some cases, ASE occurs outside the coding region and influence regulatory sequence in the UTR [22]. These different forms of splicing isoforms should not be confused with those gen‐ erated by alternative transcription start site where a single gene might transcribe from differ‐ ent promoters (Fig. 2H). In this case, and unlike alternative splicing, there is little chance that the isoform will have different protein sequence unless a new protein-coding exon is added in

The Merit of Alternative Messenger RNA Splicing as a New Mine for the Next Generation Ovarian Cancer Biomarkers

http://dx.doi.org/10.5772/53976

329

**Figure 1.** Constitutive splicing. A) Consensus splicing sequences. The 5'ss, BPS, PPT and 3'ss are represented and are binds by U1 snRNP, SF1, U2AF65 and U2AF35, respectively. B) Splicing reaction. The first step involved the attack by the 2' hydroxyl of the branch point adenine on the phosphate at the 5'ss, releasing at the same time the 3'end of the mRNA. The second step involved the attack by one of the hydroxyl of the terminal phosphate on the phosphate at the

frame for translation initiation.

3'ss, liberating the intron in the form of a lariat.

#### **2. Pre-mRNA splicing mechanism and regulation**

Transcription of messenger RNA (mRNA) is the first step of converting the DNA code into functional proteins. This process was often seen as a linear cascade of events that include mRNA capping [12], splicing [13], polyadenylation [14], export to the cytoplasm [15] and trans‐ lation [16] to produce a single protein. However, in reality a single pre-mRNA can produce many mRNAs through the process of AS and this in turn lead to the production of several pro‐ teins from a single gene. Splicing is the process by which the protein coding exons (typically hundreds of nucleotides in length) are joined together after the removal of large non-coding in‐ trons (typically thousands of nucleotides in length) to form the coding sequence. In some genes, this process leads to one outcome and thus named constitutive (Fig. 1.) but in most cases it leads to more than one outcome and thus called alternative (Fig. 2). Both processes are medi‐ ated by the spliceosome, specialized machinery that recognizes consensus RNA sequences [13,17]. The spliceosome component U1snRNP binds the 5' splice site (5'ss), the splicing Factor 1 (SF1) binds the branch point site (BPS) adenine, U2 auxiliary factor 65 kDa subunit (U2AF65) binds the poly-pyrimidine tract (PPT) and U2 auxiliary factor 35 kDa subunits (U2AF35) binds the 3' splice site (3'ss) (Fig. 1A). The last two component are further replaced by U2snRNP and following complex base-paring rearrangements and RNA-protein interactions involving hun‐ dreds of protein, the spliced mRNA, the intron by-product and the spliceosome component are release [17]. Chemically speaking, the splicing reaction proceeds in two trans-esterification steps (Fig. 1B). The first step involved the attack by the 2' hydroxyl of the branch point adenine on the phosphate at the 5'ss, releasing at the same time the 3'end of the mRNA. The second step involved the attack by one of the hydroxyl of the terminal phosphate on the phosphate at the 3'ss, liberating the intron in the form of a lariat. This cycle of spliceosome assembly/disassem‐ bly is repeated for every intron of a gene on the nascent RNA transcript [18].

When the splice site for some exons become weak or introns with suboptimal sequence exist splicing may become less accurate and may depend on the factors that influence the splicing of competing exons and consequently produce mRNA versions with different exon pairs. AS af‐ fected the majority of multi-exons genes and is believed to be the principal driver of proteome diversity [19]. As illustrated in figure 2, two 5'ss can compete for a single 3'ss or inversely, two 3'ss can compete for a single 5'ss. These type of alternative splicing events (ASEs) are referred to alternative 5' (alt5', Fig. 2A) and alternative 3' (alt3' Fig. 2B), respectively. The most frequent type of ASE in human is the full skipping of an exon (cassette exon, Fig. 2C). Some exons are al‐ so skipped as a bloc (multiple cassette exon, Fig. 2D) or mutually exclusive (Fig. 2E). AS could also be coupled to others regulatory mechanisms such as polyadenylation [14] (Fig. 2F). In this case, the resulting mRNA exhibits a different 3' untranslated region (UTR), which is further subjected to different regulation by small non-coding RNA (e.g. microRNA). In about 1 out of 3 cases, AS decision introduces a sequence containing premature stop codon [20]. In these cases, the resulting mRNA is flagged to be degraded by the non-sense mediated decay machinery creating an efficient mechanism that control gene expression post-transcriptionally [21] (Fig. 2G). In some cases, ASE occurs outside the coding region and influence regulatory sequence in the UTR [22]. These different forms of splicing isoforms should not be confused with those gen‐ erated by alternative transcription start site where a single gene might transcribe from differ‐ ent promoters (Fig. 2H). In this case, and unlike alternative splicing, there is little chance that the isoform will have different protein sequence unless a new protein-coding exon is added in frame for translation initiation.

ers complicated. Indeed, a meta-analysis regrouping 829 samples fails to demonstrate the predictive power of 16 individual gene expression signatures [9]. Consequently, very few microarray markers reached the clinic. In contrast, high-throughput protein signature based on mass spectrometry platform appears to have much more overlap in the peaks found by different experimental studies [10]. However, the pace by which protein biomarkers are translated into clinical setting is relatively slow [11]. Clearly, there is a need for novel meth‐ odology to discover ovarian cancer biomarkers that can yield reliable results and produce tests that could be quickly integrated in normal clinical setting. In this chapter, we discuss the potential of splice variant annotations as a tool for the discovery of ovarian cancer mark‐

Transcription of messenger RNA (mRNA) is the first step of converting the DNA code into functional proteins. This process was often seen as a linear cascade of events that include mRNA capping [12], splicing [13], polyadenylation [14], export to the cytoplasm [15] and trans‐ lation [16] to produce a single protein. However, in reality a single pre-mRNA can produce many mRNAs through the process of AS and this in turn lead to the production of several pro‐ teins from a single gene. Splicing is the process by which the protein coding exons (typically hundreds of nucleotides in length) are joined together after the removal of large non-coding in‐ trons (typically thousands of nucleotides in length) to form the coding sequence. In some genes, this process leads to one outcome and thus named constitutive (Fig. 1.) but in most cases it leads to more than one outcome and thus called alternative (Fig. 2). Both processes are medi‐ ated by the spliceosome, specialized machinery that recognizes consensus RNA sequences [13,17]. The spliceosome component U1snRNP binds the 5' splice site (5'ss), the splicing Factor 1 (SF1) binds the branch point site (BPS) adenine, U2 auxiliary factor 65 kDa subunit (U2AF65) binds the poly-pyrimidine tract (PPT) and U2 auxiliary factor 35 kDa subunits (U2AF35) binds the 3' splice site (3'ss) (Fig. 1A). The last two component are further replaced by U2snRNP and following complex base-paring rearrangements and RNA-protein interactions involving hun‐ dreds of protein, the spliced mRNA, the intron by-product and the spliceosome component are release [17]. Chemically speaking, the splicing reaction proceeds in two trans-esterification steps (Fig. 1B). The first step involved the attack by the 2' hydroxyl of the branch point adenine on the phosphate at the 5'ss, releasing at the same time the 3'end of the mRNA. The second step involved the attack by one of the hydroxyl of the terminal phosphate on the phosphate at the 3'ss, liberating the intron in the form of a lariat. This cycle of spliceosome assembly/disassem‐

ers and discuss the challenges and promises of this hidden mine.

328 Ovarian Cancer - A Clinical and Translational Update

**2. Pre-mRNA splicing mechanism and regulation**

bly is repeated for every intron of a gene on the nascent RNA transcript [18].

When the splice site for some exons become weak or introns with suboptimal sequence exist splicing may become less accurate and may depend on the factors that influence the splicing of competing exons and consequently produce mRNA versions with different exon pairs. AS af‐ fected the majority of multi-exons genes and is believed to be the principal driver of proteome diversity [19]. As illustrated in figure 2, two 5'ss can compete for a single 3'ss or inversely, two 3'ss can compete for a single 5'ss. These type of alternative splicing events (ASEs) are referred to alternative 5' (alt5', Fig. 2A) and alternative 3' (alt3' Fig. 2B), respectively. The most frequent

**Figure 1.** Constitutive splicing. A) Consensus splicing sequences. The 5'ss, BPS, PPT and 3'ss are represented and are binds by U1 snRNP, SF1, U2AF65 and U2AF35, respectively. B) Splicing reaction. The first step involved the attack by the 2' hydroxyl of the branch point adenine on the phosphate at the 5'ss, releasing at the same time the 3'end of the mRNA. The second step involved the attack by one of the hydroxyl of the terminal phosphate on the phosphate at the 3'ss, liberating the intron in the form of a lariat.

**Figure 3.** Schematic representation of the different mechanisms regulating alternative splicing. A) SR protein bind to exonic splicing enhancer (ESE) and favor exon inclusion by recruting U1snRNP to 5'ss or U2 snRNP to 3'ss. B) HnRNPs binding to exonic splicing silencer (ESS) and subsequent 3' to 5' oligomerization through exon favor exon exclusion by blocking SR protein access to their exonic splicing enhancer (ESE). C) Intronic splicing enhancer (ISE) binding of hnRNP interferes with intron definition and favor exon definition. D) HnRNP intron looping out. E) SR protein and hnRNPs functional antagonism. F) Tissu-specific splicing factor tend to favor exon inclusion when bound to intronic splicing silencer (ISS) in intron downstream of alternative exon and exclusion when bound upstream. Gray boxes = constitutive

The Merit of Alternative Messenger RNA Splicing as a New Mine for the Next Generation Ovarian Cancer Biomarkers

http://dx.doi.org/10.5772/53976

331

**3. The advantage of alternative splicing as a source of ovarian cancer**

Analysis of the ovarian cancer proteome using mass spectrometry is undoubtedly the most di‐ rect approach for the identification of biomarkers that could be readily implemented in the clinic. However, the difficulty generating specific antibodies for the large number of potential markers generated via this approach makes marker validation very difficult. In contrast, the validation of nucleic acid markers generated through microarray or deep-sequencing screen is fairly simple and is often achieved by polymerase chain reaction (PCR) [34,37]. Furthermore, the function of these potential markers can easily be verified through the knockdown of gene expression using RNA interference (RNAi) strategies [38]. However, scoring global changes in gene expression as markers for ovarian cancer limits the assay to ~25000 genes in the genome, while it is estimate that the human cells contains at least >100 000 proteins. This limitation is no longer an issue when we consider the expression of specific splice variants, the number of which equal or exceeds the number of cellular proteins [39]. In addition, it is much easier to pre‐ dict the function of an alternative splice variant than predicting the function of a peptide mark‐ er. For examples, while the role of the well established markers CA-125 remain unclear after 25 years of research [40], one could easily predict the function of a marker by the protein domain eliminated or included through AS as is the case of the tyrosine kinase SYK. In this case, exon skipping remove a nuclear localization domain leading to the accumulation of protein in the

exons; white boxes = alternative exons; 5' = 5' splice site; 3' = 3' splice site.

**biomarker**

**Figure 2.** Alternative splicing may take many different forms. A) Alt5'. B) Alt3'. C) Cassette exon. D) Multiple cassette exons. E) Mutually exclusive exons. F) Coupled to alternative polyadenylation. G) Coupled to NMD. H) Alternative tran‐ scription initiation. Gray boxes = constitutive exons; white boxes = alternative exons; 5' = 5' splice site; 3' = 3' splice site; A = polyadenylation site.

ASEs are normally associated with low sequence conservation near the splice site and instead are usually linked to RNA binding motifs that may enhance or repress exon inclusion [23,24]. Motifs that enhance exon inclusion often recruit splicing factors like the SR protein family, which in turn interact with the spliceosome via an arginine serine rich domain to increase weak 5'ss and 3'ss recognition [25] (Fig. 3A). On the other hand, splicing motifs that promotes exon exclusion by binding members of the hnRNP family oligomerized through exon [26], block UsnRNA recruitment [27] or loop out the alternative exon [28] (Fig. 3B to D). Similarly, se‐ quence motifs in intron may bind to SR or hnRNP proteins to influence splicing, but in this case, the SR proteins results in exon exclusion and hnRNP in exon inclusion. This is most likely be‐ cause hnRNPs define intronic region and SR protein define exons location. Usually, these dif‐ ferent enhancers and repressor protein families work together to define the final outcome of any ASEs (Fig. 3E) [29,30]. One of the most conserved intronic motif downstream of alternative exons is the UGCAUG motif [31,32], which bind the tissue-specific splicing factors family RBFOX. In general it is suggested that tissues specific splicing factors favor exon inclusion when bound to introns downstream of alternative exons and exclusion when bound upstream. This rule is beginning to be appreciated for several splicing factor such as Celf [33], epithelialspecific regulatory protein [34], Nova [35] and RBFOX [36] (Fig. 3F).

**Figure 3.** Schematic representation of the different mechanisms regulating alternative splicing. A) SR protein bind to exonic splicing enhancer (ESE) and favor exon inclusion by recruting U1snRNP to 5'ss or U2 snRNP to 3'ss. B) HnRNPs binding to exonic splicing silencer (ESS) and subsequent 3' to 5' oligomerization through exon favor exon exclusion by blocking SR protein access to their exonic splicing enhancer (ESE). C) Intronic splicing enhancer (ISE) binding of hnRNP interferes with intron definition and favor exon definition. D) HnRNP intron looping out. E) SR protein and hnRNPs functional antagonism. F) Tissu-specific splicing factor tend to favor exon inclusion when bound to intronic splicing silencer (ISS) in intron downstream of alternative exon and exclusion when bound upstream. Gray boxes = constitutive exons; white boxes = alternative exons; 5' = 5' splice site; 3' = 3' splice site.

#### **3. The advantage of alternative splicing as a source of ovarian cancer biomarker**

**Figure 2.** Alternative splicing may take many different forms. A) Alt5'. B) Alt3'. C) Cassette exon. D) Multiple cassette exons. E) Mutually exclusive exons. F) Coupled to alternative polyadenylation. G) Coupled to NMD. H) Alternative tran‐ scription initiation. Gray boxes = constitutive exons; white boxes = alternative exons; 5' = 5' splice site; 3' = 3' splice

ASEs are normally associated with low sequence conservation near the splice site and instead are usually linked to RNA binding motifs that may enhance or repress exon inclusion [23,24]. Motifs that enhance exon inclusion often recruit splicing factors like the SR protein family, which in turn interact with the spliceosome via an arginine serine rich domain to increase weak 5'ss and 3'ss recognition [25] (Fig. 3A). On the other hand, splicing motifs that promotes exon exclusion by binding members of the hnRNP family oligomerized through exon [26], block UsnRNA recruitment [27] or loop out the alternative exon [28] (Fig. 3B to D). Similarly, se‐ quence motifs in intron may bind to SR or hnRNP proteins to influence splicing, but in this case, the SR proteins results in exon exclusion and hnRNP in exon inclusion. This is most likely be‐ cause hnRNPs define intronic region and SR protein define exons location. Usually, these dif‐ ferent enhancers and repressor protein families work together to define the final outcome of any ASEs (Fig. 3E) [29,30]. One of the most conserved intronic motif downstream of alternative exons is the UGCAUG motif [31,32], which bind the tissue-specific splicing factors family RBFOX. In general it is suggested that tissues specific splicing factors favor exon inclusion when bound to introns downstream of alternative exons and exclusion when bound upstream. This rule is beginning to be appreciated for several splicing factor such as Celf [33], epithelial-

specific regulatory protein [34], Nova [35] and RBFOX [36] (Fig. 3F).

site; A = polyadenylation site.

330 Ovarian Cancer - A Clinical and Translational Update

Analysis of the ovarian cancer proteome using mass spectrometry is undoubtedly the most di‐ rect approach for the identification of biomarkers that could be readily implemented in the clinic. However, the difficulty generating specific antibodies for the large number of potential markers generated via this approach makes marker validation very difficult. In contrast, the validation of nucleic acid markers generated through microarray or deep-sequencing screen is fairly simple and is often achieved by polymerase chain reaction (PCR) [34,37]. Furthermore, the function of these potential markers can easily be verified through the knockdown of gene expression using RNA interference (RNAi) strategies [38]. However, scoring global changes in gene expression as markers for ovarian cancer limits the assay to ~25000 genes in the genome, while it is estimate that the human cells contains at least >100 000 proteins. This limitation is no longer an issue when we consider the expression of specific splice variants, the number of which equal or exceeds the number of cellular proteins [39]. In addition, it is much easier to pre‐ dict the function of an alternative splice variant than predicting the function of a peptide mark‐ er. For examples, while the role of the well established markers CA-125 remain unclear after 25 years of research [40], one could easily predict the function of a marker by the protein domain eliminated or included through AS as is the case of the tyrosine kinase SYK. In this case, exon skipping remove a nuclear localization domain leading to the accumulation of protein in the cytoplasm, elegantly explaining the lost of nuclear function associated with cancer [38]. Pre‐ dicting the impact of AS is particularly attractive for biomarkers development when the alter‐ native exon encodes a plasma transmembrane domain or an extracellular protease cleavage site [41]. In these cases, one would be able to predict whether the cancer associated marker leads to an increase or decrease in the secretion of membrane anchored protein, an information that is difficult to obtain using global gene expression profiles.

Back in the 1980's, splicing isoforms were mainly detected by Northern-Blot, which separate transcripts by size [42] and estimate relative mRNA abundance using internal controls. However, this method is difficult to adopt in a clinical setting and require a large amount of RNA (µg), which is difficult to obtain from clinical samples. Later, the discovery of reverse transcription and PCR amplification greatly facilitated the detection of splice variants [43]. Splicing isoform amplification is achived by using PCR primers that are designed to hybrid‐ ize to constitutive exons flanking the ASE of interest (Fig. 4A). The products are separated in agarose gels or capillary gel electrophoresis [44] and the ratio of the long and short isoform quantified and presented as ψ (percent of splicing index): the molarity of the long over the sum of the long and the short isoforms (Fig. 4A). Even if competitive PCR reaction are limit‐ ed to a narrow range [43,45], endpoint PCR is still the preferred technique to detect splicing

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The gold standard for the mRNA quantification is real-time PCR [46], which unlike stand‐ ard endpoint PCR, detects the amount of products accumulating after each cycle of amplifi‐ cation and permits accurate comparison of different samples. This type of PCR requires the use of fluorescent probes [47] or dyes [48] that permit detection by specialized sensors. De‐ spite the accuracy of this detection method it is rarely used for the detection of splice var‐ iants due to difficulty in achieving isoform specific amplification [43,49]. Primers required for the amplification of the short isoform need to bind to a short unique sequence created by the exon-exon junction, which severely restrict the design (Fig. 4B). However, systematic evaluation of isoform specific design parameters and the availability of new algorithms for primer selection greatly facilitated the detection of ASEs from any species [49]. Indeed, uni‐ versal PCR conditions and ease of primer design makes real-time PCR reaches the point where it can compete with high-throughput detection methods like microarray in term of

Microarray as a method for genome-wide expression profiling was discovered in 1995 [50], but the use of this method to detect splice variants was reported only in 2003 [51]. It took 8 years to develop methods that could distinguish between the hybridization patterns of two closely related transcripts and develop chips with high enough density to accommodate the thousands of splicing isoforms [51] (Fig. 4C) Early attempts to extract splicing pattern from expression microarrays generated high false positive rate [52]. Therefore, strategies where developed to probe exon-exon junction (junction array) [51]. In this case, alternative exons are defined by very low or very high signals emanating from two consecutive splice junc‐ tions [51]. Another popular strategy is to use exonic probe in addition to exon-exon junction probe (exon/junction array) [53]. In every case, the high similarity of exon-exon junction to favor non-specific hybridization and in some analysis procedures the information is restrict‐ ed to splicing isoform "detection" rather than true quantification [54]. The most successful quantification of splicing isoform by microarray was achieved by relying solely on exonic probe [54-56]. However, the success of this method was limited by its dependence on a small set of pre-selected splice variants [53,57]. To allow the discovery of new splicing iso‐ forms, a fourth strategy that consider all putative exons (tiling array) was developed [58]. However, the high number of probe required for this methods restricted coverage to only a

isoforms due to the ease of use and low cost of the experiments.

ASE coverage [49].

#### **4. The challenges of detecting splicing isoforms**

Examples of alternatively spliced genes are steadily accumulating in the literature for more than 20 years and the discovery rate was greatly accelerated by recent technological advances like transcriptome sequencing techniques. Indeed, while early estimation of alternatively spliced genes based on Northern-Blots and endpoint RT-PCR were around 5% of the human genome, transcriptome sequencing revealed ASEs in 95% of the human genes with multiple in‐ trons [39]. Different techniques have different capacity to illustrate the number of ASEs (see Ta‐ ble 1) and detecting splice variants remained difficult to detect for many years, which explains the reason they are not regularly considered as a source of biomarkers by most clinicians.


**Table 1.** Techniques used for the detection of alternative splicing

Back in the 1980's, splicing isoforms were mainly detected by Northern-Blot, which separate transcripts by size [42] and estimate relative mRNA abundance using internal controls. However, this method is difficult to adopt in a clinical setting and require a large amount of RNA (µg), which is difficult to obtain from clinical samples. Later, the discovery of reverse transcription and PCR amplification greatly facilitated the detection of splice variants [43]. Splicing isoform amplification is achived by using PCR primers that are designed to hybrid‐ ize to constitutive exons flanking the ASE of interest (Fig. 4A). The products are separated in agarose gels or capillary gel electrophoresis [44] and the ratio of the long and short isoform quantified and presented as ψ (percent of splicing index): the molarity of the long over the sum of the long and the short isoforms (Fig. 4A). Even if competitive PCR reaction are limit‐ ed to a narrow range [43,45], endpoint PCR is still the preferred technique to detect splicing isoforms due to the ease of use and low cost of the experiments.

cytoplasm, elegantly explaining the lost of nuclear function associated with cancer [38]. Pre‐ dicting the impact of AS is particularly attractive for biomarkers development when the alter‐ native exon encodes a plasma transmembrane domain or an extracellular protease cleavage site [41]. In these cases, one would be able to predict whether the cancer associated marker leads to an increase or decrease in the secretion of membrane anchored protein, an information

Examples of alternatively spliced genes are steadily accumulating in the literature for more than 20 years and the discovery rate was greatly accelerated by recent technological advances like transcriptome sequencing techniques. Indeed, while early estimation of alternatively spliced genes based on Northern-Blots and endpoint RT-PCR were around 5% of the human genome, transcriptome sequencing revealed ASEs in 95% of the human genes with multiple in‐ trons [39]. Different techniques have different capacity to illustrate the number of ASEs (see Ta‐ ble 1) and detecting splice variants remained difficult to detect for many years, which explains the reason they are not regularly considered as a source of biomarkers by most clinicians.

> Several isoforms can be detected in a single sample

standard for validation Results easy to analyze Throughput is enhanced when

coupled to capillary electrophoresis

Large quantitative range Accurate data in fixed tissues

(Discovery of novel splicing

data

available

annotation

isoforms)

Labour-intense

products

Large amount of RNA needed Restricted by gel resolution

Labour-intense if polyacrylamide gels are used to separate PCR

Low quantitative range Restricted by gel resolution

Custom primer design often needed

Complex analysis

Complex analysis

Results need PCR validation

High cost prevent the use of biological replicates Long multi-step procedure

Results need PCR validation

that is difficult to obtain using global gene expression profiles.

332 Ovarian Cancer - A Clinical and Translational Update

**4. The challenges of detecting splicing isoforms**

**Technique Throughput Advantages Limitations**

Northern-Blot Low No amplification

Endpoint RT-PCR Low to medium Considered as the gold

Real-time PCR Low to medium Provided already validated

Microarray medium Some array are commercially

Next generation Sequencing Medium to High Independent of genome

**Table 1.** Techniques used for the detection of alternative splicing

The gold standard for the mRNA quantification is real-time PCR [46], which unlike stand‐ ard endpoint PCR, detects the amount of products accumulating after each cycle of amplifi‐ cation and permits accurate comparison of different samples. This type of PCR requires the use of fluorescent probes [47] or dyes [48] that permit detection by specialized sensors. De‐ spite the accuracy of this detection method it is rarely used for the detection of splice var‐ iants due to difficulty in achieving isoform specific amplification [43,49]. Primers required for the amplification of the short isoform need to bind to a short unique sequence created by the exon-exon junction, which severely restrict the design (Fig. 4B). However, systematic evaluation of isoform specific design parameters and the availability of new algorithms for primer selection greatly facilitated the detection of ASEs from any species [49]. Indeed, uni‐ versal PCR conditions and ease of primer design makes real-time PCR reaches the point where it can compete with high-throughput detection methods like microarray in term of ASE coverage [49].

Microarray as a method for genome-wide expression profiling was discovered in 1995 [50], but the use of this method to detect splice variants was reported only in 2003 [51]. It took 8 years to develop methods that could distinguish between the hybridization patterns of two closely related transcripts and develop chips with high enough density to accommodate the thousands of splicing isoforms [51] (Fig. 4C) Early attempts to extract splicing pattern from expression microarrays generated high false positive rate [52]. Therefore, strategies where developed to probe exon-exon junction (junction array) [51]. In this case, alternative exons are defined by very low or very high signals emanating from two consecutive splice junc‐ tions [51]. Another popular strategy is to use exonic probe in addition to exon-exon junction probe (exon/junction array) [53]. In every case, the high similarity of exon-exon junction to favor non-specific hybridization and in some analysis procedures the information is restrict‐ ed to splicing isoform "detection" rather than true quantification [54]. The most successful quantification of splicing isoform by microarray was achieved by relying solely on exonic probe [54-56]. However, the success of this method was limited by its dependence on a small set of pre-selected splice variants [53,57]. To allow the discovery of new splicing iso‐ forms, a fourth strategy that consider all putative exons (tiling array) was developed [58]. However, the high number of probe required for this methods restricted coverage to only a small fraction of the genome. Not surprisingly, these difficulties hampered the application of this method for the study of ovarian cancer splicing isforms. Indeed, to date there is no report of microarray based profiling of ovarian cancer splice variants.

**Gene Technique Marker type and isoform description Reference**

The Merit of Alternative Messenger RNA Splicing as a New Mine for the Next Generation Ovarian Cancer Biomarkers

AML1bDel179-242 expression inversely correlates with overall survival

Numerous DNA mutation inactivated BRCA1/2 function (deletion of essential domain or protein truncation) through

The ratio of total to full length KLF6 expression correlates with

p53δ expression is associated to impaired response to first line

\*Osteopontin-c is undetectable in normal ovaries and present

This signature distinguishes normal ovaries from ovarian tumors regardless of grade, stage or histotype (serous,

This signature distinguishes normal ovaries from serous high

This cancer epithelial signature (CES) distinguishes normal Fallopian tube epithelium tissues from ovarian epithelial

This cancer stromal signature (CSS) distinguishes normal tissues from ovarian tumors independent of the epithelial

\*The ratio of Fibulin C/D increase in ovarian tumors

**Number of ASEs Technique Marker type and splicing signature description Reference**

muscinous, endometriod, mix type)

content of the tissue compared

[67]

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335

[69]

[72]

[75]

[77]

[85]

[44]

[36]

[84]

[84]

Prognosis

Prognosis

Grade

aberrant splicing.

ovarian tumor grade

Chemoresistance

chemotherapy

in ovarian tumors

Diagnosis

Diagnosis

Diagnosis

cancer cells

Diagnosis

**Table 3.** Ovarian cancer splicing markers derived from high-throughput expression profiling.

grade ovarian tumors.

Diagnosis

Diagnosis

**Table 2.** Ovarian cancer splicing markers discovered through single gene analysis

BRCA1/2 Endpoint

KLF6 Real-time

TP53 Real-time

FBLN1 Endpoint

SPP1 Real-time

\*Potential serum marker

48 ASEs Endpoint

288 ASEs Endpoint

8 ASEs Real-time

10 ASEs Real-time

RT-PCR

RT-PCR

RT-PCR

RT-PCR

RUNX1 (AML1) RT-PCR

Real-time RT-PCR

RT-PCR

RT-PCR

RT-PCR

RT-PCR

In theory, the most promising approach for the detection of ovarian cancer splicing iso‐ form is the transcriptome sequencing [39]. Next generation sequencing (NGS) technology provide massively parallel sequencing of nucleotidic sequences in miniaturized microsys‐ tem. Several platforms are commercially available and their unique technology are dis‐ cussed elsewhere [59,60]. The specific application of mRNA quantification through sequencing (RNA-seq) was demonstrated for different cancer types (e.g. lung [61] and prostate [62]) but not ovarian cancer thus far. Encouraging development in the refinement of the analytical pipeline to allow accurate quantification of splicing isoforms was recently made [37,63,64]. However, the complexity of the analytical pipeline of sequencing data and the cost of the sequencing read necessary to detect splice variants will reduce the speed by which this technique is applied to the discovery of splicing dependent biomarkers (Fig. 4D). In addition, secondary techniques like PCR will still be needed to confirm and vali‐ date the accuracy of the data generated and confirm it in a large number of clinical sam‐ ple. Indeed, the majority of the AS information in ovarian cancer are derived from PCRbased techniques (see Table 2 and 3).

**Figure 4.** Methodology for splicing isoforms detection. A) Endpoint PCR. B) Real-time PCR. C) Microarray. D) Next Gen‐ eration Sequencing. Red arrows and lines refer to short isoform specific detection. Gray boxes = constitutive exons; white boxes = alternative exons.

The Merit of Alternative Messenger RNA Splicing as a New Mine for the Next Generation Ovarian Cancer Biomarkers http://dx.doi.org/10.5772/53976 335


\*Potential serum marker

small fraction of the genome. Not surprisingly, these difficulties hampered the application of this method for the study of ovarian cancer splicing isforms. Indeed, to date there is no

In theory, the most promising approach for the detection of ovarian cancer splicing iso‐ form is the transcriptome sequencing [39]. Next generation sequencing (NGS) technology provide massively parallel sequencing of nucleotidic sequences in miniaturized microsys‐ tem. Several platforms are commercially available and their unique technology are dis‐ cussed elsewhere [59,60]. The specific application of mRNA quantification through sequencing (RNA-seq) was demonstrated for different cancer types (e.g. lung [61] and prostate [62]) but not ovarian cancer thus far. Encouraging development in the refinement of the analytical pipeline to allow accurate quantification of splicing isoforms was recently made [37,63,64]. However, the complexity of the analytical pipeline of sequencing data and the cost of the sequencing read necessary to detect splice variants will reduce the speed by which this technique is applied to the discovery of splicing dependent biomarkers (Fig. 4D). In addition, secondary techniques like PCR will still be needed to confirm and vali‐ date the accuracy of the data generated and confirm it in a large number of clinical sam‐ ple. Indeed, the majority of the AS information in ovarian cancer are derived from PCR-

**Figure 4.** Methodology for splicing isoforms detection. A) Endpoint PCR. B) Real-time PCR. C) Microarray. D) Next Gen‐ eration Sequencing. Red arrows and lines refer to short isoform specific detection. Gray boxes = constitutive exons;

report of microarray based profiling of ovarian cancer splice variants.

based techniques (see Table 2 and 3).

334 Ovarian Cancer - A Clinical and Translational Update

white boxes = alternative exons.

**Table 2.** Ovarian cancer splicing markers discovered through single gene analysis


**Table 3.** Ovarian cancer splicing markers derived from high-throughput expression profiling.

#### **5. Example of alternative splicing based ovarian cancer biomarkers**

#### **5.1. Gene specific discovery of splicing markers**

PCR-based techniques of specific genes associated with ovarian cancer revealed a number of ovarian cancer associated splicing events. The most promising of these potential biomarkers for diagnosis, prognosis, chemoresistance and grade biomarkers are listed in table 2 and are further described in the text below.

tal alt5' ss and produce a protein version lacking the characteristic zinc finger binding do‐ main (KLF6 SV1) and act as a dominant-negative [72]. Although the technical challenges of amplifying specific KLF6 isoforms preclude pinpointing the KLF6 SV1 as the isoform that correlates with grade tumor, a series of *in vitro* and *in vivo* evidence making it likely [72]. It remains to be established whether or not the full length KLF6 over KLF6 SV1 isoform ratio

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**TP53.** The tumor suppressor gene TP53 is mutated in several solid tumors and in almost all of the serous ovarian tumors [73]. Mutations affecting splice site of TP53 are very common [74] and leads to a complex pattern of splicing isoforms that add up to the already complex picture of this gatekeeper. Indeed, the point mutation IVS9-2A>G destroys the splice accept‐ or and redirect the splicing to include exon 9c. The resulting p53δ protein isoform is truncat‐ ed in the oligomerization domain and have a new stretch of 27 new residues. In a cohort of 245 ovarian samples, the expression of this isoform is significantly correlated with poor overall survival in multivariate analyses. Moreover, patient having tumor that express p53δ have a higher chance of early relapse after first-line chemotherapy [75]. Since isoform p53δ doesn't correlate with the debulking status, it suggests that expression of p53δ impair plati‐ num-based chemotherapy [75]. In respect to personalized medicine, it would be very inter‐ esting to sensitize these tumors by targeting p53δ. Thus, p53δ is not only a potential adverse prognosis marker but could also be a promising target for a subclass of ovarian tumors.

**FBLN1.** Fibulin (FBLN) 1 is an extracellular matrix (ECM)-associated protein produced by both stromal cells and epithelial cells [76]. Its presence near sites of epithelial cells locally invading stromal boundaries suggests its implication in cell adhesion/motility (see [77] and reference there in). Different C-terminal exons of the FBLN1 gene are alternatively spliced to generate four isoforms. In ovarian tumors, the ratio fibulin 1C / fibulin 1D is significantly increased compared to benign ovarian cystic sample [77]. Interestingly, the isoform ratio be‐ tween normal and benign cyst is slightly increased in cystic samples (although not signifi‐ cant). It raises the hypothesis that the ratio fibulin 1C / fibulin 1D could potentially serves as an early diagnostic marker. Importantly, the sensitivity and specificity of fibulin splicing iso‐ forms remains to be firmly established using a panel of normal and early lesion tissues and

**SPP1.** Osteopontin is a member of the small integrin-binding ligand, N-linked glycoprotein (SIBLING) family of proteins [78]. It is an important component of the ECM that is secreted by both cancer cells and stromal cells in the tumor microenvironment [79]. Osteopontin in‐ teracts with various integrin receptors [80,81] as well as the CD44 receptor [82] to activate the angiogenic switch or enhance cancer cell motility [79]. The level of osteopontin is elevat‐ ed in patient's plasma when compared to healthy controls by enzyme-linked immuno assay (ELISA) [83]. However, the specificity (80,4%) and sensitivity (80,4%) for the detection of early stage disease are not convincing [83]. These parameters could be increase if one takes advantages of AS. A recent report conducted by real-time PCR indicated that the isoform osteopontin c (excluding exon 4) is absent in normal or benign tissues but always present in ovarian cancer samples. This is supported by our own data from microdissected normal and cancerous ovarian cells indicating that the expression of osteopontin c comes specifically

could serve as a prognosis or even early marker.

ultimately in patient's serum.

**BRCA1/2.** The ability of DNA mutation in the hereditary gene BRCA1 and BRCA2 (BRCA1/2) to predict the risk of ovarian and breast cancer is known for decades [65,66]. However, in several instance, the clinical relevance of DNA mutation is unknown, making the clinical management difficult to establish properly. Furthermore, the role of these muta‐ tions in varying the splicing of BRCA genes was largely ignored despite the fact that any nucleotide changes in the splice site consensus sequences or in any AS regulatory sequence could produce aberrant splicing isoforms. Recently, *in vitro* splicing assay of BRCA1/2 muta‐ tion [67], revealed that many cancer associated variants including those with unsuspected synonymous mutations have dramatic effect on splicing. Strikingly, six of the most frequent DNA variants representing 58,5% of BRCA1 families induced aberrant splicing profile [67]. These results, clearly demonstrate the importance of studying cancer associated splicing since it may in many case help explain mutation that cannot be associated with changes in protein sequence. However, the fidelity of splicing signature as a diagnostic marker as com‐ pared to DNA sequencing remains to be established.

**RUNX1.** Runt-related transcription factor 1 is a transcriptional regulator harboring a DNAbinding runt homology domain (RHD). Several layers of regulation (transcription, splicing and translation) fine-tune its tissue-specific expression [68]. RUNX1 is also known as Acute myeloid leukemia (AML) 1 and is often found as oncogenic fusion in leukemia. Nanjundan M and collaborators [69] report the fortuit discovery of a novel isoform that lack exon 6 as compared to isoform AML1b during classical cloning procedure. This novel isoform, subse‐ quently named AML1bDel179-242 was found to be the dominant isoform in the majority of the 42 ovarian tumors studied. Functionally speaking, skipping of exon 6 severely abrogated the transactivation potential of the resulting protein and inhibits its tumor suppressive func‐ tions. Interestingly, AML1bDel179-242 level was either not different from normal cell line (lung cancer) or significantly decreased (breast cancer), suggesting that it may be an ovarian spe‐ cific marker [69]. Noneoftheless, AML1bDel179-242 expression is inversely proportional to the survival rate of patient, suggesting is used as a prognosis marker [69]. As attractive as it sounds, AML1bDel179-242 certainly represent an excellent potential target and marker but re‐ quired further validation in larger cohort and by independent research group.

**KLF6.** Kruppel-like factor (KLF) 6 is a transcription factor from the well conserved KLF gene family implicated in differentiation, development and cell growth [70,71]. KLF6 is a suspect‐ ed tumor suppressor gene in several epithelial cancer (see [72] and reference there in). In ovarian cancer, an increase in KLF6 isoforms was noted and correlates with the aggressivity of the tumor in tissues (grade). One of these isoform is produced by the use of the more dis‐ tal alt5' ss and produce a protein version lacking the characteristic zinc finger binding do‐ main (KLF6 SV1) and act as a dominant-negative [72]. Although the technical challenges of amplifying specific KLF6 isoforms preclude pinpointing the KLF6 SV1 as the isoform that correlates with grade tumor, a series of *in vitro* and *in vivo* evidence making it likely [72]. It remains to be established whether or not the full length KLF6 over KLF6 SV1 isoform ratio could serve as a prognosis or even early marker.

**5. Example of alternative splicing based ovarian cancer biomarkers**

PCR-based techniques of specific genes associated with ovarian cancer revealed a number of ovarian cancer associated splicing events. The most promising of these potential biomarkers for diagnosis, prognosis, chemoresistance and grade biomarkers are listed in table 2 and are

**BRCA1/2.** The ability of DNA mutation in the hereditary gene BRCA1 and BRCA2 (BRCA1/2) to predict the risk of ovarian and breast cancer is known for decades [65,66]. However, in several instance, the clinical relevance of DNA mutation is unknown, making the clinical management difficult to establish properly. Furthermore, the role of these muta‐ tions in varying the splicing of BRCA genes was largely ignored despite the fact that any nucleotide changes in the splice site consensus sequences or in any AS regulatory sequence could produce aberrant splicing isoforms. Recently, *in vitro* splicing assay of BRCA1/2 muta‐ tion [67], revealed that many cancer associated variants including those with unsuspected synonymous mutations have dramatic effect on splicing. Strikingly, six of the most frequent DNA variants representing 58,5% of BRCA1 families induced aberrant splicing profile [67]. These results, clearly demonstrate the importance of studying cancer associated splicing since it may in many case help explain mutation that cannot be associated with changes in protein sequence. However, the fidelity of splicing signature as a diagnostic marker as com‐

**RUNX1.** Runt-related transcription factor 1 is a transcriptional regulator harboring a DNAbinding runt homology domain (RHD). Several layers of regulation (transcription, splicing and translation) fine-tune its tissue-specific expression [68]. RUNX1 is also known as Acute myeloid leukemia (AML) 1 and is often found as oncogenic fusion in leukemia. Nanjundan M and collaborators [69] report the fortuit discovery of a novel isoform that lack exon 6 as compared to isoform AML1b during classical cloning procedure. This novel isoform, subse‐ quently named AML1bDel179-242 was found to be the dominant isoform in the majority of the 42 ovarian tumors studied. Functionally speaking, skipping of exon 6 severely abrogated the transactivation potential of the resulting protein and inhibits its tumor suppressive func‐ tions. Interestingly, AML1bDel179-242 level was either not different from normal cell line (lung cancer) or significantly decreased (breast cancer), suggesting that it may be an ovarian spe‐ cific marker [69]. Noneoftheless, AML1bDel179-242 expression is inversely proportional to the survival rate of patient, suggesting is used as a prognosis marker [69]. As attractive as it sounds, AML1bDel179-242 certainly represent an excellent potential target and marker but re‐

quired further validation in larger cohort and by independent research group.

**KLF6.** Kruppel-like factor (KLF) 6 is a transcription factor from the well conserved KLF gene family implicated in differentiation, development and cell growth [70,71]. KLF6 is a suspect‐ ed tumor suppressor gene in several epithelial cancer (see [72] and reference there in). In ovarian cancer, an increase in KLF6 isoforms was noted and correlates with the aggressivity of the tumor in tissues (grade). One of these isoform is produced by the use of the more dis‐

**5.1. Gene specific discovery of splicing markers**

pared to DNA sequencing remains to be established.

further described in the text below.

336 Ovarian Cancer - A Clinical and Translational Update

**TP53.** The tumor suppressor gene TP53 is mutated in several solid tumors and in almost all of the serous ovarian tumors [73]. Mutations affecting splice site of TP53 are very common [74] and leads to a complex pattern of splicing isoforms that add up to the already complex picture of this gatekeeper. Indeed, the point mutation IVS9-2A>G destroys the splice accept‐ or and redirect the splicing to include exon 9c. The resulting p53δ protein isoform is truncat‐ ed in the oligomerization domain and have a new stretch of 27 new residues. In a cohort of 245 ovarian samples, the expression of this isoform is significantly correlated with poor overall survival in multivariate analyses. Moreover, patient having tumor that express p53δ have a higher chance of early relapse after first-line chemotherapy [75]. Since isoform p53δ doesn't correlate with the debulking status, it suggests that expression of p53δ impair plati‐ num-based chemotherapy [75]. In respect to personalized medicine, it would be very inter‐ esting to sensitize these tumors by targeting p53δ. Thus, p53δ is not only a potential adverse prognosis marker but could also be a promising target for a subclass of ovarian tumors.

**FBLN1.** Fibulin (FBLN) 1 is an extracellular matrix (ECM)-associated protein produced by both stromal cells and epithelial cells [76]. Its presence near sites of epithelial cells locally invading stromal boundaries suggests its implication in cell adhesion/motility (see [77] and reference there in). Different C-terminal exons of the FBLN1 gene are alternatively spliced to generate four isoforms. In ovarian tumors, the ratio fibulin 1C / fibulin 1D is significantly increased compared to benign ovarian cystic sample [77]. Interestingly, the isoform ratio be‐ tween normal and benign cyst is slightly increased in cystic samples (although not signifi‐ cant). It raises the hypothesis that the ratio fibulin 1C / fibulin 1D could potentially serves as an early diagnostic marker. Importantly, the sensitivity and specificity of fibulin splicing iso‐ forms remains to be firmly established using a panel of normal and early lesion tissues and ultimately in patient's serum.

**SPP1.** Osteopontin is a member of the small integrin-binding ligand, N-linked glycoprotein (SIBLING) family of proteins [78]. It is an important component of the ECM that is secreted by both cancer cells and stromal cells in the tumor microenvironment [79]. Osteopontin in‐ teracts with various integrin receptors [80,81] as well as the CD44 receptor [82] to activate the angiogenic switch or enhance cancer cell motility [79]. The level of osteopontin is elevat‐ ed in patient's plasma when compared to healthy controls by enzyme-linked immuno assay (ELISA) [83]. However, the specificity (80,4%) and sensitivity (80,4%) for the detection of early stage disease are not convincing [83]. These parameters could be increase if one takes advantages of AS. A recent report conducted by real-time PCR indicated that the isoform osteopontin c (excluding exon 4) is absent in normal or benign tissues but always present in ovarian cancer samples. This is supported by our own data from microdissected normal and cancerous ovarian cells indicating that the expression of osteopontin c comes specifically from ovarian epithelial cancer cells [84]. Conditioned medium overexpressing osteopontin c stimulate proliferation of cancer cells more efficiently than either osteopontin a or b, and this effect is revert by specific antibodies against osteopontin c [85]. Based on these data, the bio‐ marker capacity of osteopontin in patient's blood need to be re-established using isoformspecific methodology. As the secretion of osteopontin might be an early event [86-88], it is tempting to speculate that osteopontin c could be an early marker.

ovaries (1% epithelial cells) are used as normal reference. As a consequence, prognosis marker derived from cancer samples comparison should yield more reliable splicing mark‐

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339

Interestingly, a number of RNA splicing isoforms markers might be amenable to detection at the protein level using isoform-specific antibodies. Ultimately, these could serve as diag‐ nostic or prognostic tool to either directly detect the presence of cancer cells or indirectly the protein in patient's fluid. Indeed, the product of the genes encoding fibronectin 1, fibulin, osteopontin, galectin 9, platelet derived growth factor A, extracellular sulfatase 2 and slit ho‐ molog 2 are all secreted in the extracellular matrix. Even some cytosolic proteins such as utrophin and serine hydroxymethyltransferase 1 were found in patient's serum [89]. Others are cell surface protein (amyloid beta A4 protein, stromal interaction molecule 1, CD97, pep‐ tidyl-glycine alpha-amidating monooxygenase and chemokine-like factor) harboring an ASE that encodes for an extracellular domain. More impressively, the exon encoding the trans‐ membrane domain of betacellulin is preferentially excluded in ovarian tumors [44], leading to a secreted version of the protein [90]. Thus in every cases, isoform-specific antibodies could be theoretically raised against the cancer associated isoform to ultimately serve as di‐ agnostic/prognostic tool to either detect cancer cells or detect the protein in patient's fluid.

Inversely, the splicing isoforms of the cell surface receptor Fas and CD44 were mostly stud‐ ied at the protein level by either immunohistochemistry (IHC) or ELISA. Fas linked extracel‐ lular apoptotic signals that converge to the programmed cell death pathway through caspase 8 and 10. Differential usage of exon 6, which encodes the single pass transmembrane domain, results in a soluble version (sFas) and a membrane anchored version (mFas). The level of sFas is increase in ovarian tumor of higher grade compared to low grade [91,92] and correlates with worst prognosis for these patient [91]. Although these studies were per‐ formed in small cohort, it elegantly demonstrated that AS can produce isoforms detectable

The glycoprotein CD44 is a cell surface receptor that binds diverse extracellular matrix li‐ gands such as hyaluronic acid, fibronectin, osteopontin, collagen and laminin [93]. The bind‐ ing of low molecular weight hyaluronan polymer promotes the motility and invasion properties of CD44 (review in [93]). It is encoded as a 20 exons gene that exhibit extensive AS of the extracellular domain of exons 6 to 15 (also called variable exons 1 to 10). The major isoform present in normal epithelial [94,95] or stromal [96] ovarian cells is the shorter iso‐ form CD44s lacking all variable exons (CD44s for standard isoform). In contrast, a complex pattern of splicing isoforms were detected in cancer tissues, including most of ovarian tu‐ mors by mean of RT-PCR [94,97,98] or by IHC using isoform specific antibodies [95,96,99,100]. One of these splicing isoforms, the inclusion of exon v10, appears to correlate with prognosis and is indicative of improved survival in a multivariate analysis of a 142 pa‐ tient cohort by IHC [96]. However, these findings contrast the initial study of Schroder who found no exon v10 expression although it relies on a smaller cohort [100]. Intriguingly, in‐ clusion of exon v10 in metastatic tumors was correlated with decrease survival [96]. This ap‐

ers than diagnosis marker normalized with normal ovaries.

**5.3. Alternative splicing associated protein markers**

in patient's serum.

#### **5.2. Splicing markers generated through genome-wide expression profiling**

The advent of splicing sensitive high-throughput technique opens the doors to monitor a large number of randomly selected ASEs rather than be limited to few candidate genes (see Table 3). The recent use of high-throughput RT-PCR by coupling PCR reaction in 384 wells plate to capillary gel electrophoresis in 96 well Caliper station dramatically increased the number of confirmed ovarian cancer associated splicing events. Initially, exon-exon junc‐ tions were systematically analyzed for a set of 600 cancer related genes in four different pools of normal and cancer ovarian samples. The resulting ASEs were subsequently validat‐ ed using an independent set of 21 normal ovaries and 25 ovarian cancer samples, yielding 48 ASE markers [44]. Later on, a focus on a collection of 2168 highly curated ASEs (RefSeq NCBI build 36) subsequently yield 288 ASEs markers using roughly the same sample set [36]. The relatively high number of ASEs markers found coupled to the fact that several were related to the epithelial-mesenchymal transition raised the possibility that a large frac‐ tion of the discovered events might result from difference in the cell type compared (normal ovaries are largely composed of stromal cells where as ovarian tumors have a typical epithe‐ lial content around 75% [36]). This question was answered when 9 ovarian tumors were mi‐ crodissected to isolated the RNA from stromal (tumor microenvironment) and epithelial cancer cells separately. A real-time PCR-based screening strategy coupled to an update ver‐ sion of RefSeq NCBI build 36 (3313 ASEs) yield a low but unambiguous set of cancer-specif‐ ic splicing isoforms, the cancer epithelial signature (CES) [84]. Surprisingly, the tumor microenvironment appears to contain promising splicing isoforms RNA markers. Indeed, this cancer stromal signature (CSS) might be able to diagnosis early ovarian tumors as it clusters low malignant potential and low-grade tumors within normal ovaries and Fallopian tube samples, although this study was performed on a low number of tissues [84].

The possibility that ovarian tumor microenvironment may be a source of splicing isoforms markers raise interesting questions regarding the studies conducted on whole tumors. First, some of the RNA transcripts detected may actually come from the microenvironment cells. For exemple, fibulin and fibronectin are two ECM components known to be produced and secreted by stromal cells. Pinpointing the cell type that produced those splicing deregulated secreted proteins will certainly help to rationalize the complex autocrine and paracrine path‐ ways implicated in the cell to cell communication that take place into and surrounding the ovarian tumor. Second, AS is a highly tissue-specific process, some of the splicing pattern changes might be the reflection of the different proportion of stromal and epithelial cells of ovarian tumors. Theoretically, those effects would be minimal when ovarian tumors of equivalent epithelial content (typically 50-75%) are compared but maximal when normal ovaries (1% epithelial cells) are used as normal reference. As a consequence, prognosis marker derived from cancer samples comparison should yield more reliable splicing mark‐ ers than diagnosis marker normalized with normal ovaries.

#### **5.3. Alternative splicing associated protein markers**

from ovarian epithelial cancer cells [84]. Conditioned medium overexpressing osteopontin c stimulate proliferation of cancer cells more efficiently than either osteopontin a or b, and this effect is revert by specific antibodies against osteopontin c [85]. Based on these data, the bio‐ marker capacity of osteopontin in patient's blood need to be re-established using isoformspecific methodology. As the secretion of osteopontin might be an early event [86-88], it is

The advent of splicing sensitive high-throughput technique opens the doors to monitor a large number of randomly selected ASEs rather than be limited to few candidate genes (see Table 3). The recent use of high-throughput RT-PCR by coupling PCR reaction in 384 wells plate to capillary gel electrophoresis in 96 well Caliper station dramatically increased the number of confirmed ovarian cancer associated splicing events. Initially, exon-exon junc‐ tions were systematically analyzed for a set of 600 cancer related genes in four different pools of normal and cancer ovarian samples. The resulting ASEs were subsequently validat‐ ed using an independent set of 21 normal ovaries and 25 ovarian cancer samples, yielding 48 ASE markers [44]. Later on, a focus on a collection of 2168 highly curated ASEs (RefSeq NCBI build 36) subsequently yield 288 ASEs markers using roughly the same sample set [36]. The relatively high number of ASEs markers found coupled to the fact that several were related to the epithelial-mesenchymal transition raised the possibility that a large frac‐ tion of the discovered events might result from difference in the cell type compared (normal ovaries are largely composed of stromal cells where as ovarian tumors have a typical epithe‐ lial content around 75% [36]). This question was answered when 9 ovarian tumors were mi‐ crodissected to isolated the RNA from stromal (tumor microenvironment) and epithelial cancer cells separately. A real-time PCR-based screening strategy coupled to an update ver‐ sion of RefSeq NCBI build 36 (3313 ASEs) yield a low but unambiguous set of cancer-specif‐ ic splicing isoforms, the cancer epithelial signature (CES) [84]. Surprisingly, the tumor microenvironment appears to contain promising splicing isoforms RNA markers. Indeed, this cancer stromal signature (CSS) might be able to diagnosis early ovarian tumors as it clusters low malignant potential and low-grade tumors within normal ovaries and Fallopian

tempting to speculate that osteopontin c could be an early marker.

338 Ovarian Cancer - A Clinical and Translational Update

**5.2. Splicing markers generated through genome-wide expression profiling**

tube samples, although this study was performed on a low number of tissues [84].

The possibility that ovarian tumor microenvironment may be a source of splicing isoforms markers raise interesting questions regarding the studies conducted on whole tumors. First, some of the RNA transcripts detected may actually come from the microenvironment cells. For exemple, fibulin and fibronectin are two ECM components known to be produced and secreted by stromal cells. Pinpointing the cell type that produced those splicing deregulated secreted proteins will certainly help to rationalize the complex autocrine and paracrine path‐ ways implicated in the cell to cell communication that take place into and surrounding the ovarian tumor. Second, AS is a highly tissue-specific process, some of the splicing pattern changes might be the reflection of the different proportion of stromal and epithelial cells of ovarian tumors. Theoretically, those effects would be minimal when ovarian tumors of equivalent epithelial content (typically 50-75%) are compared but maximal when normal

Interestingly, a number of RNA splicing isoforms markers might be amenable to detection at the protein level using isoform-specific antibodies. Ultimately, these could serve as diag‐ nostic or prognostic tool to either directly detect the presence of cancer cells or indirectly the protein in patient's fluid. Indeed, the product of the genes encoding fibronectin 1, fibulin, osteopontin, galectin 9, platelet derived growth factor A, extracellular sulfatase 2 and slit ho‐ molog 2 are all secreted in the extracellular matrix. Even some cytosolic proteins such as utrophin and serine hydroxymethyltransferase 1 were found in patient's serum [89]. Others are cell surface protein (amyloid beta A4 protein, stromal interaction molecule 1, CD97, pep‐ tidyl-glycine alpha-amidating monooxygenase and chemokine-like factor) harboring an ASE that encodes for an extracellular domain. More impressively, the exon encoding the trans‐ membrane domain of betacellulin is preferentially excluded in ovarian tumors [44], leading to a secreted version of the protein [90]. Thus in every cases, isoform-specific antibodies could be theoretically raised against the cancer associated isoform to ultimately serve as di‐ agnostic/prognostic tool to either detect cancer cells or detect the protein in patient's fluid.

Inversely, the splicing isoforms of the cell surface receptor Fas and CD44 were mostly stud‐ ied at the protein level by either immunohistochemistry (IHC) or ELISA. Fas linked extracel‐ lular apoptotic signals that converge to the programmed cell death pathway through caspase 8 and 10. Differential usage of exon 6, which encodes the single pass transmembrane domain, results in a soluble version (sFas) and a membrane anchored version (mFas). The level of sFas is increase in ovarian tumor of higher grade compared to low grade [91,92] and correlates with worst prognosis for these patient [91]. Although these studies were per‐ formed in small cohort, it elegantly demonstrated that AS can produce isoforms detectable in patient's serum.

The glycoprotein CD44 is a cell surface receptor that binds diverse extracellular matrix li‐ gands such as hyaluronic acid, fibronectin, osteopontin, collagen and laminin [93]. The bind‐ ing of low molecular weight hyaluronan polymer promotes the motility and invasion properties of CD44 (review in [93]). It is encoded as a 20 exons gene that exhibit extensive AS of the extracellular domain of exons 6 to 15 (also called variable exons 1 to 10). The major isoform present in normal epithelial [94,95] or stromal [96] ovarian cells is the shorter iso‐ form CD44s lacking all variable exons (CD44s for standard isoform). In contrast, a complex pattern of splicing isoforms were detected in cancer tissues, including most of ovarian tu‐ mors by mean of RT-PCR [94,97,98] or by IHC using isoform specific antibodies [95,96,99,100]. One of these splicing isoforms, the inclusion of exon v10, appears to correlate with prognosis and is indicative of improved survival in a multivariate analysis of a 142 pa‐ tient cohort by IHC [96]. However, these findings contrast the initial study of Schroder who found no exon v10 expression although it relies on a smaller cohort [100]. Intriguingly, in‐ clusion of exon v10 in metastatic tumors was correlated with decrease survival [96]. This ap‐

parent discrepancy could be rationalized if the exon v10 inclusion is seen as crucial to maintain proper cell adhesion and avoid cell detachment [101]. It remains to be determined if any of the variable exons of CD44 could serve as biomarker at the RNA level.

[3] Bast RC,Jr, Klug TL, St John E, Jenison E, Niloff JM, Lazarus H, et al. A radioimmu‐ noassay using a monoclonal antibody to monitor the course of epithelial ovarian can‐

The Merit of Alternative Messenger RNA Splicing as a New Mine for the Next Generation Ovarian Cancer Biomarkers

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341

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#### **6. Concluding remarks**

AS dramatically increase the diversity of protein expression in human cells and therefore ex‐ ponentially increase the number of potential disease markers. However, the complexity in detecting AS and the unclear function of the majority of splice variants greatly reduced the rate of AS based ovarian cancer biomarkers. This trend is likely to change in the next few years with the explosion of whole transcriptome sequencing efforts and the inevitable iden‐ tification of splice variants as byproducts of next generations' expression profiles. The real challenge now is to develop techniques allowing the use of splicing markers in the clinic and prepare pathologists to this new wave. Clearly, a compelling argument is needed to drive this drastic change in clinical practice and it will most likely be driven by the success of AS based screens in rationally predicting secreted protein that may serve as non-invasive ovari‐ an cancer markers.

#### **Author details**

Jean-Philippe Brosseau1,2 and Sherif Abou-Elela1,3

\*Address all correspondence to: sherif.abou.elela@usherbrooke.ca

1 Laboratoire de Génomique Fonctionnelle de l'Université de Sherbrooke, Canada

2 Département de Biochimie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada

3 Département de Microbiologie et d'Infectiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada

#### **References**


[3] Bast RC,Jr, Klug TL, St John E, Jenison E, Niloff JM, Lazarus H, et al. A radioimmu‐ noassay using a monoclonal antibody to monitor the course of epithelial ovarian can‐ cer. N Engl J Med 1983 Oct 13;309[15]:883-887.

parent discrepancy could be rationalized if the exon v10 inclusion is seen as crucial to maintain proper cell adhesion and avoid cell detachment [101]. It remains to be determined

AS dramatically increase the diversity of protein expression in human cells and therefore ex‐ ponentially increase the number of potential disease markers. However, the complexity in detecting AS and the unclear function of the majority of splice variants greatly reduced the rate of AS based ovarian cancer biomarkers. This trend is likely to change in the next few years with the explosion of whole transcriptome sequencing efforts and the inevitable iden‐ tification of splice variants as byproducts of next generations' expression profiles. The real challenge now is to develop techniques allowing the use of splicing markers in the clinic and prepare pathologists to this new wave. Clearly, a compelling argument is needed to drive this drastic change in clinical practice and it will most likely be driven by the success of AS based screens in rationally predicting secreted protein that may serve as non-invasive ovari‐

if any of the variable exons of CD44 could serve as biomarker at the RNA level.

**6. Concluding remarks**

340 Ovarian Cancer - A Clinical and Translational Update

an cancer markers.

**Author details**

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1105-7.

Jean-Philippe Brosseau1,2 and Sherif Abou-Elela1,3

Sherbrooke, Sherbrooke, Québec, Canada

\*Address all correspondence to: sherif.abou.elela@usherbrooke.ca

Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada

1 Laboratoire de Génomique Fonctionnelle de l'Université de Sherbrooke, Canada

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[59] Metzker ML. Sequencing technologies - the next generation. Nat Rev Genet 2010 Jan;

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[86] Mi Z, Bhattacharya SD, Kim VM, Guo H, Talbot LJ, Kuo PC. Osteopontin promotes CCL5-mesenchymal stromal cell-mediated breast cancer metastasis. Carcinogenesis

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[87] McAllister SS, Gifford AM, Greiner AL, Kelleher SP, Saelzler MP, Ince TA, et al. Sys‐ temic endocrine instigation of indolent tumor growth requires osteopontin. Cell 2008

[88] Pazolli E, Luo X, Brehm S, Carbery K, Chung JJ, Prior JL, et al. Senescent stromal-de‐ rived osteopontin promotes preneoplastic cell growth. Cancer Res 2009 Feb 1;69(3):

[89] Lomnytska M, Dubrovska A, Hellman U, Volodko N, Souchelnytskyi S. Increased expression of cSHMT, Tbx3 and utrophin in plasma of ovarian and breast cancer pa‐

[90] Dunbar AJ, Goddard C. Identification of an alternatively spliced mRNA transcript of human betacellulin lacking the C-loop of the EGF motif and the transmembrane do‐

[91] Konno R, Takano T, Sato S, Yajima A. Serum soluble fas level as a prognostic factor in patients with gynecological malignancies. Clin Cancer Res 2000 Sep;6(9):

[92] Midis GP, Shen Y, Owen-Schaub LB. Elevated soluble Fas (sFas) levels in nonhema‐

[93] Louderbough JM, Schroeder JA. Understanding the dual nature of CD44 in breast

[94] Cannistra SA, Abu-Jawdeh G, Niloff J, Strobel T, Swanson L, Andersen J, et al. CD44 variant expression is a common feature of epithelial ovarian cancer: lack of associa‐

[95] Darai E, Walker-Combrouze F, Fauconnier A, Madelenat P, Potet F, Scoazec JY. Anal‐ ysis of CD44 expression in serous and mucinous borderline tumours of the ovary: comparison with cystadenomas and overt carcinomas. Histopathology 1998 Feb;

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**Chapter 16**

**Ovarian Cancer** *in vitro* **Diagnostics:**

Jorge Tapia, Mercedes de Alvarado, Benjamín Bustos, Vania Lukoviek and

Additional information is available at the end of the chapter

The overall mortality of ovarian cancer has remained unchanged despite new chemothera‐ peutic agents that have improved 5-year survival rates. In the United States, ovarian cancer is among the most lethal malignant gynaecological pathology. Each year, more than 230,000 new cases of ovarian cancer are diagnosed. More than 90% of these cases occur in women without clearly identifiable risk factors. In the majority of cases, ovarian cancer is first diag‐ nosed as disseminated disease that has a five-year survival rate of less than 30%. Ovarian cancer, thus, remains a significant health care challenge and the most lethal of women's re‐

Although ovarian cancer is often considered to be a single disease, it is composed of several related but distinct tumour categories, including: surface epithelial tumours, sex-cord stro‐ mal tumours, germ cell tumours, and metastatic tumours. The most frequent are the epithe‐ lial tumours that are also divided according to their histologic types: serous, mucinous, endometrioid, clear cell, and transitional. Epithelial tumours may be classified into two fur‐ ther groups, according to their clinical behaviour: either low malignant potential (LMP) or high malignant potential (HMP). In addition, HMP epithelial tumours are also divided into type 1 and 2 depending upon whether or not there is a pre-malignant lesion. Considering this new classification, specific mutations have been isolated depending on the type of tu‐ mour. Furthermore, the primary origin of serous epithelial ovarian cancer has been ques‐ tioned. Crum *et al*. (2007), proposed that the majority of ovarian carcinomas originate outside the ovary and are derived from fallopian tube epithelial cells. The identification of

> © 2013 Tapia et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Gregory E. Rice

**1. Introduction**

productive tract cancers.

http://dx.doi.org/10.5772/53953

**New Approaches to Earlier Detection**


**Chapter 16**

### **Ovarian Cancer** *in vitro* **Diagnostics: New Approaches to Earlier Detection**

Jorge Tapia, Mercedes de Alvarado, Benjamín Bustos, Vania Lukoviek and Gregory E. Rice

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53953

#### **1. Introduction**

[99] Uhl-Steidl M, Muller-Holzner E, Zeimet AG, Adolf GR, Daxenbichler G, Marth C, et al. Prognostic value of CD44 splice variant expression in ovarian cancer. Oncology

[100] Schroder W, Rudlowski C, Biesterfeld S, Knobloch C, Hauptmann S, Rath W. Expres‐ sion of CD44(v5-10) splicing variants in primary ovarian cancer and lymph node

[101] Chiu RK, Droll A, Dougherty ST, Carpenito C, Cooper DL, Dougherty GJ. Alterna‐ tively spliced CD44 isoforms containing exon v10 promote cellular adhesion through the recognition of chondroitin sulfate-modified CD44. Exp Cell Res 1999 Apr

metastases. Anticancer Res 1999 Sep-Oct;19(5B):3901-3906.

1995 Sep-Oct;52(5):400-406.

348 Ovarian Cancer - A Clinical and Translational Update

10;248(1):314-321.

The overall mortality of ovarian cancer has remained unchanged despite new chemothera‐ peutic agents that have improved 5-year survival rates. In the United States, ovarian cancer is among the most lethal malignant gynaecological pathology. Each year, more than 230,000 new cases of ovarian cancer are diagnosed. More than 90% of these cases occur in women without clearly identifiable risk factors. In the majority of cases, ovarian cancer is first diag‐ nosed as disseminated disease that has a five-year survival rate of less than 30%. Ovarian cancer, thus, remains a significant health care challenge and the most lethal of women's re‐ productive tract cancers.

Although ovarian cancer is often considered to be a single disease, it is composed of several related but distinct tumour categories, including: surface epithelial tumours, sex-cord stro‐ mal tumours, germ cell tumours, and metastatic tumours. The most frequent are the epithe‐ lial tumours that are also divided according to their histologic types: serous, mucinous, endometrioid, clear cell, and transitional. Epithelial tumours may be classified into two fur‐ ther groups, according to their clinical behaviour: either low malignant potential (LMP) or high malignant potential (HMP). In addition, HMP epithelial tumours are also divided into type 1 and 2 depending upon whether or not there is a pre-malignant lesion. Considering this new classification, specific mutations have been isolated depending on the type of tu‐ mour. Furthermore, the primary origin of serous epithelial ovarian cancer has been ques‐ tioned. Crum *et al*. (2007), proposed that the majority of ovarian carcinomas originate outside the ovary and are derived from fallopian tube epithelial cells. The identification of

© 2013 Tapia et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

cells with a molecular phenotype similar to Type 2 ovarian cancer within the fimbria is con‐ sistent with the hypothesis that ovarian cancer may indeed originate from intraepithelial carcinomas of the fallopian tubule.

TP53 pathway, although there is little information relating to other molecular mutations. [6,7]. When stratified by type (*i.e.* high-grade and low-grade serous carcinoma), it be‐ came evident that the mutation TP53 is present in almost 100% of type II high-grade se‐ rous carcinomas [8]. Taking into account that TP53 mutation is precocious and ubiquitous (at least in advance stages) it remains to be proven whether or not this muta‐

Women with mutations in BRCA 1-2 genes have around 30-70% probability of developing ovarian cancer before reaching old age, in most cases, type II HMP tumours.[9] The BRCA 1-2 genes are crucial components in the DNA repair pathway of homologous recombinant required to resolve errors in the double-stranded DNA [10]. It is likely that inherited muta‐ tions in BRCA 1-2 genes predispose the epithelial ovarian surface to neoplastic transforma‐

lethal to the cell because of the associated apoptotic response with p53.[11] Since the loss of BRCA gene function is very common in high-grade serous carcinomas, secondary mutations

There are undoubtedly many mutations involved in the survival and adaptation of epithe‐ lial ovarian carcinomas that have yet to be studied. Currently, the processes that occur be‐ tween an initial carcinoma and its progression to widely disseminated disease remain unknown. It is presumed that there are multiple mutations in the tumourigenesis pathways that allow the tumour to overcome hypoxia, cytokines, the detachment from the basal mem‐

Within the advances in histopathological and genetic investigations, recent dogma regard‐ ing the origin of serous ovarian cancer involving pre-cancerous lesions from the ovarian sur‐ face epithelium or intra–ovarian inclusion cysts has been questioned. In women with BRCA-1 and BRCA-2 germline mutations, tubal intra-epithelial carcinoma in the fimbria has been identified as a very probable precursor of advanced high-grade serous ovarian cancer (particularly in Type 2 ovarian cancer) [12]-[14]. This is also validated by the coexistence of identical TP53 mutations in tubal intra-epithelial carcinoma and in those tumours classified

This evidence is consistent with the idea that the fallopian tube (especially its distal portion: the fimbria) is an important site for the initiation of high-grade serous ovarian cancer [16]. Crum et al. (2007), further, proposed that most ovarian carcinomas originate outside the ovary and are derived from fallopian tube epithelial cells. They suggest that fimbrial epithe‐ lial cells detach and implant on the deluded, damaged surface of the ovary resulting in the formation of inclusion cysts that subsequently give rise to what until now was known as "ovarian" cancer. The identification of cells with a molecular phenotype similar to Type 2 ovarian cancer within the fimbria is consistent with the hypothesis that ovarian cancer may

indeed originate from intraepithelial carcinomas of the fallopian tubule [17].

. The loss of function in the BRCA 1-2 genes is often

Ovarian Cancer *in vitro* Diagnostics: New Approaches to Earlier Detection

http://dx.doi.org/10.5772/53953

351

.

tion plays an aetiological role in the development of this phenotype.

are expected to be present to ensure the survival of the cells involved 3

brane and the metabolic demands of many rapidly dividing cells. 7

**3. Fallopian tubule involvement in ovarian cancer**

tion secondary to genetic instability 5

as ovarian in origin [15].

Despite significant advances in the development of mathematical modelling and validation of *in vitro* diagnostics, to date none have achieved the level of diagnostic performance re‐ quired for implementation as a screening test for asymptomatic women in the general popu‐ lation. In the absence of a screening test, however, it is important for women presenting to primary care to be diagnosed in the most effective and timely way to ensure that they are directed to the most appropriate clinical treatment available.

Ongoing studies continue to search for the presence of other biomarkers (in addition to CA125 and ultrasound imaging) to detect ovarian cancer in its initial stages. Of recent note has been the identification of tumour-specific exosomes in the blood of women with ovarian cancer. Other novel diagnosis techniques have been described: intra-fallopian tubule sam‐ pling, uterine washing sampling and the sampling of cervicovaginal swabs. While these ap‐ proaches afford some promise of increasing diagnostic performance for asymptomatic populations, they await clinical validation.

#### **2. Reclassification of disease type**

According to the classification of the World Health Organisation in 2003, from an histopa‐ thological point of view epithelial ovarian tumour are classified in serous (60%), endome‐ trioid (10-20%), clear cell (<10%), transitional (6%), mucinous (5%), and undifferentiated (<1%) [1,2]. Furthermore, ovarian tumours are also classified, according to behaviour, into low malignant potential (LMP) and high malignant potential (HMP) depending on the grade of invasion.[3] High serous malignant tumours are divided into type I and type II [4].

Type I tumours originate from the progressive transformation of low malignant potential ovarian tumours, whose behaviour is considered to be relatively benign. This group in‐ clude: mucinous carcinoma; endometrioid carcinoma; Brenner tumours; and clear cell carcinoma. Type II tumours, however, do not display a defined pre-malignant lesion, and their behaviour is aggressive, and rapidly progressive, metastising in early stages of diagnosis. Serous carcinomas, sarcocarcinomas and undifferentiated carcinomas belong in this group. Preliminary studies report that epithelial LMP tumours (mucinous, endome‐ trioid, and clear cell carcinoma) could evolve from low to mildle undifferentiated tu‐ mours becoming HMP ovarian tumours [5].

The development of a new classification of epithelial ovarian tumours (*i.e.* type 1 and type 2) has led to the identification of specific molecular phenotypes previously unidenti‐ fied because of the confounding effects of multiple histopathological types of tumours. Type I and type II tumours display different characteristics and activation of molecular pathways. Type I tumours are associated with mutations in the Ras pathway (BRAF, KRAS, ErbB2) while, type II tumours are frequently associated with mutations in the TP53 pathway, although there is little information relating to other molecular mutations. [6,7]. When stratified by type (*i.e.* high-grade and low-grade serous carcinoma), it be‐ came evident that the mutation TP53 is present in almost 100% of type II high-grade se‐ rous carcinomas [8]. Taking into account that TP53 mutation is precocious and ubiquitous (at least in advance stages) it remains to be proven whether or not this muta‐ tion plays an aetiological role in the development of this phenotype.

Women with mutations in BRCA 1-2 genes have around 30-70% probability of developing ovarian cancer before reaching old age, in most cases, type II HMP tumours.[9] The BRCA 1-2 genes are crucial components in the DNA repair pathway of homologous recombinant required to resolve errors in the double-stranded DNA [10]. It is likely that inherited muta‐ tions in BRCA 1-2 genes predispose the epithelial ovarian surface to neoplastic transforma‐ tion secondary to genetic instability 5 . The loss of function in the BRCA 1-2 genes is often lethal to the cell because of the associated apoptotic response with p53.[11] Since the loss of BRCA gene function is very common in high-grade serous carcinomas, secondary mutations are expected to be present to ensure the survival of the cells involved 3 .

There are undoubtedly many mutations involved in the survival and adaptation of epithe‐ lial ovarian carcinomas that have yet to be studied. Currently, the processes that occur be‐ tween an initial carcinoma and its progression to widely disseminated disease remain unknown. It is presumed that there are multiple mutations in the tumourigenesis pathways that allow the tumour to overcome hypoxia, cytokines, the detachment from the basal mem‐ brane and the metabolic demands of many rapidly dividing cells. 7

#### **3. Fallopian tubule involvement in ovarian cancer**

cells with a molecular phenotype similar to Type 2 ovarian cancer within the fimbria is con‐ sistent with the hypothesis that ovarian cancer may indeed originate from intraepithelial

Despite significant advances in the development of mathematical modelling and validation of *in vitro* diagnostics, to date none have achieved the level of diagnostic performance re‐ quired for implementation as a screening test for asymptomatic women in the general popu‐ lation. In the absence of a screening test, however, it is important for women presenting to primary care to be diagnosed in the most effective and timely way to ensure that they are

Ongoing studies continue to search for the presence of other biomarkers (in addition to CA125 and ultrasound imaging) to detect ovarian cancer in its initial stages. Of recent note has been the identification of tumour-specific exosomes in the blood of women with ovarian cancer. Other novel diagnosis techniques have been described: intra-fallopian tubule sam‐ pling, uterine washing sampling and the sampling of cervicovaginal swabs. While these ap‐ proaches afford some promise of increasing diagnostic performance for asymptomatic

According to the classification of the World Health Organisation in 2003, from an histopa‐ thological point of view epithelial ovarian tumour are classified in serous (60%), endome‐ trioid (10-20%), clear cell (<10%), transitional (6%), mucinous (5%), and undifferentiated (<1%) [1,2]. Furthermore, ovarian tumours are also classified, according to behaviour, into low malignant potential (LMP) and high malignant potential (HMP) depending on the grade of invasion.[3] High serous malignant tumours are divided into type I and type II [4].

Type I tumours originate from the progressive transformation of low malignant potential ovarian tumours, whose behaviour is considered to be relatively benign. This group in‐ clude: mucinous carcinoma; endometrioid carcinoma; Brenner tumours; and clear cell carcinoma. Type II tumours, however, do not display a defined pre-malignant lesion, and their behaviour is aggressive, and rapidly progressive, metastising in early stages of diagnosis. Serous carcinomas, sarcocarcinomas and undifferentiated carcinomas belong in this group. Preliminary studies report that epithelial LMP tumours (mucinous, endome‐ trioid, and clear cell carcinoma) could evolve from low to mildle undifferentiated tu‐

The development of a new classification of epithelial ovarian tumours (*i.e.* type 1 and type 2) has led to the identification of specific molecular phenotypes previously unidenti‐ fied because of the confounding effects of multiple histopathological types of tumours. Type I and type II tumours display different characteristics and activation of molecular pathways. Type I tumours are associated with mutations in the Ras pathway (BRAF, KRAS, ErbB2) while, type II tumours are frequently associated with mutations in the

carcinomas of the fallopian tubule.

350 Ovarian Cancer - A Clinical and Translational Update

populations, they await clinical validation.

**2. Reclassification of disease type**

mours becoming HMP ovarian tumours [5].

directed to the most appropriate clinical treatment available.

Within the advances in histopathological and genetic investigations, recent dogma regard‐ ing the origin of serous ovarian cancer involving pre-cancerous lesions from the ovarian sur‐ face epithelium or intra–ovarian inclusion cysts has been questioned. In women with BRCA-1 and BRCA-2 germline mutations, tubal intra-epithelial carcinoma in the fimbria has been identified as a very probable precursor of advanced high-grade serous ovarian cancer (particularly in Type 2 ovarian cancer) [12]-[14]. This is also validated by the coexistence of identical TP53 mutations in tubal intra-epithelial carcinoma and in those tumours classified as ovarian in origin [15].

This evidence is consistent with the idea that the fallopian tube (especially its distal portion: the fimbria) is an important site for the initiation of high-grade serous ovarian cancer [16]. Crum et al. (2007), further, proposed that most ovarian carcinomas originate outside the ovary and are derived from fallopian tube epithelial cells. They suggest that fimbrial epithe‐ lial cells detach and implant on the deluded, damaged surface of the ovary resulting in the formation of inclusion cysts that subsequently give rise to what until now was known as "ovarian" cancer. The identification of cells with a molecular phenotype similar to Type 2 ovarian cancer within the fimbria is consistent with the hypothesis that ovarian cancer may indeed originate from intraepithelial carcinomas of the fallopian tubule [17].

Even though the genesis of this pathology remains unclear, there are some groups that sup‐ port the idea of "endosalpingiosis" as the preliminary event. This means that even when the primary tumour seems to originate in the ovary, it is possible that the fallopian tube epithe‐ lium provides the originating cell through earlier entrapment in the ovary [16].

cut-off values to define high probability of malignancy in premenopausal women are 5.0 and 4.4 in postmenopausal women. These tests optimise sensitivity compared to physical examination in both nongynecologic oncologists (72% to 92%) as in gynecologic oncologists (78% to 99%), even at 100% stage II in both pre- and postmenopausal women [22]. In addi‐ tion to its association with physical examination, it has a sensitivity of 96%, while physical

Ovarian Cancer *in vitro* Diagnostics: New Approaches to Earlier Detection

http://dx.doi.org/10.5772/53953

353

Even though OVA1 has a high sensitivity, its specificity is low in both nongynecologic on‐ cologists and gynecologic oncologists (being 42% and 26% respectively). Other IVDMIAs have shown greater specificity, for example OvaSure, a 6 IVDMIA analysing protein bio‐ marker, has a sensitivity of 95.3% and a specificity of 99.4% [23], however, OvaSure has yet

In a recent study, Autelitano et al. analysed a unique multianalyte test that integrates CA125, C-reactive protein, amyloid-A, plasma interleukin-6 and interleukin-8. This test has a high specificity (92.3%) and a moderate sensitivity (76.4%) for the diagnosis of ovarian cancer in symptomatic women. The panel performs significantly better than CA125 alone, as measured by the area under the receiver operator characteristic curve (88.4% and 84.3%, re‐

The development of IVDMIAs for ovarian cancer based on known candidate biomarkers of‐ fers promise for improving diagnostic efficiency of not only adrenal masses but also the ear‐

Optimising preoperative diagnosis and opportune referral to specialists, would not only as‐ sist in the development of a specific management strategy for individual patients, but would also allow for more accurate determination of perioperative morbidity and chance of surviv‐ al. Further studies, however, are needed to validate not only a comparison with classical clinical or serological parameters, but also between different IVDMIAs, to determine which

Ovarian cancer is generally diagnosed in its advanced stages due to the lack of overt symp‐ toms of disease (70% of the cases approximately), resulting in a poor prognosis (rate surviv‐ al around 30%) [25]. Only a small number of ovarian cancers are detected early and these

The reason ovarian cancer is difficult to diagnose in its initial stage is due to the lack of spe‐ cific and appropriately sensitive serum biomarkers associated with the unspecific symp‐ toms. The most utilised serum biomarker in the diagnosis of ovarian cancer is CA125, but unfortunately its ability to detect ovarian cancer in a general population is quite low [26, 27].

As a response to this difficult scenario, current investigations include the search for other serum biomarkers that would improve our ability to detect ovarian cancer in its initial

examination and CA125 alone have a sensitivity of 75% and 77%, respectively [21].

to be approved by the FDA.

spectively, p <0.001) [24].

one is the better diagnostic tool.

are the ones that can generally be treated.

lier detection of ovarian cancer and prognosis.

**5. Novel approaches to the diagnosis of ovarian cancer**

These studies potentially have significant impact on clinical practice and raise important questions, including:


Future research should be oriented towards answering these and many other questions re‐ lated to the development of new surgical and medical techniques in the treatment and pre‐ vention of ovarian cancer.

#### **4. Recent advances in the development of IVDs**

Early detection and accurate diagnosis of ovarian cancer is a pending issue in gynaeco‐ logic oncology. Tools such as physical examination, transvaginal ultrasound and serum markers (*e.g.* Ca125) have limited sensitivity. Moreover, genetic counselling is warranted only in high-risk patients, such as those with a family history of BRCA-1, BRCA-2 or Lynch syndrome [19].

Considering the high mortality of this type of cancer, it is necessary to develop new and more efficient diagnostic strategies. One recent approach to improve diagnostic efficiency has been the development of multivariate index assays (IVDMIA). IVDMIAs were de‐ fined by FDA guidelines in 2007 as a tool that: 1. Combines multiple variables using a performance function to obtain a specific result for a specific patient; and 2. Provides a result whose derivation is non-transparent and cannot be independently derived or veri‐ fied by the end user.[20] The purpose of the multivariate analysis is to integrate different biomarkers into a single test, to optimise the sensitivity and specificity of the diagnostic through non-lineal functions.[21]

To date, such tests are not methods of screening, but diagnostic tools in the evaluation of women with pelvic tumours. They help to determine the likelihood of malignancy and thus the categorisation of urgency at the time of referral to a gynaecological oncologist. [19]

OVA1 (Vermillion, Inc., Austin, TX) is the first ovarian cancer IVDMIA approved by the FDA, and combines five tests: CA125 II, prealbumin, apolipoprotein A-1, β2-microglobulin, and transferrin, obtaining a score of 0-10, in which 10 is the highest risk of malignancy. The cut-off values to define high probability of malignancy in premenopausal women are 5.0 and 4.4 in postmenopausal women. These tests optimise sensitivity compared to physical examination in both nongynecologic oncologists (72% to 92%) as in gynecologic oncologists (78% to 99%), even at 100% stage II in both pre- and postmenopausal women [22]. In addi‐ tion to its association with physical examination, it has a sensitivity of 96%, while physical examination and CA125 alone have a sensitivity of 75% and 77%, respectively [21].

Even though the genesis of this pathology remains unclear, there are some groups that sup‐ port the idea of "endosalpingiosis" as the preliminary event. This means that even when the primary tumour seems to originate in the ovary, it is possible that the fallopian tube epithe‐

These studies potentially have significant impact on clinical practice and raise important

**•** Should the complete removal of the fallopian tube during hysterectomy and/or oophorec‐ tomy be a general practice? Bowtell et al. and Dietl et al. consider this approach essential

**•** Is the removal of fallopian tubes a good idea when practicing a prophylactic hysterecto‐ my in women with BRCA mutations? According to Dietl and Wishhusen, a salpingecto‐ my-only for women at increased risk of ovarian cancer would be a proper prophylactic

Future research should be oriented towards answering these and many other questions re‐ lated to the development of new surgical and medical techniques in the treatment and pre‐

Early detection and accurate diagnosis of ovarian cancer is a pending issue in gynaeco‐ logic oncology. Tools such as physical examination, transvaginal ultrasound and serum markers (*e.g.* Ca125) have limited sensitivity. Moreover, genetic counselling is warranted only in high-risk patients, such as those with a family history of BRCA-1, BRCA-2 or

Considering the high mortality of this type of cancer, it is necessary to develop new and more efficient diagnostic strategies. One recent approach to improve diagnostic efficiency has been the development of multivariate index assays (IVDMIA). IVDMIAs were de‐ fined by FDA guidelines in 2007 as a tool that: 1. Combines multiple variables using a performance function to obtain a specific result for a specific patient; and 2. Provides a result whose derivation is non-transparent and cannot be independently derived or veri‐ fied by the end user.[20] The purpose of the multivariate analysis is to integrate different biomarkers into a single test, to optimise the sensitivity and specificity of the diagnostic

To date, such tests are not methods of screening, but diagnostic tools in the evaluation of women with pelvic tumours. They help to determine the likelihood of malignancy and thus the categorisation of urgency at the time of referral to a gynaecological oncologist. [19]

OVA1 (Vermillion, Inc., Austin, TX) is the first ovarian cancer IVDMIA approved by the FDA, and combines five tests: CA125 II, prealbumin, apolipoprotein A-1, β2-microglobulin, and transferrin, obtaining a score of 0-10, in which 10 is the highest risk of malignancy. The

lium provides the originating cell through earlier entrapment in the ovary [16].

in reducing the risk of high-grade serous cancer [16, 18].

**4. Recent advances in the development of IVDs**

questions, including:

352 Ovarian Cancer - A Clinical and Translational Update

option [18].

vention of ovarian cancer.

Lynch syndrome [19].

through non-lineal functions.[21]

Even though OVA1 has a high sensitivity, its specificity is low in both nongynecologic on‐ cologists and gynecologic oncologists (being 42% and 26% respectively). Other IVDMIAs have shown greater specificity, for example OvaSure, a 6 IVDMIA analysing protein bio‐ marker, has a sensitivity of 95.3% and a specificity of 99.4% [23], however, OvaSure has yet to be approved by the FDA.

In a recent study, Autelitano et al. analysed a unique multianalyte test that integrates CA125, C-reactive protein, amyloid-A, plasma interleukin-6 and interleukin-8. This test has a high specificity (92.3%) and a moderate sensitivity (76.4%) for the diagnosis of ovarian cancer in symptomatic women. The panel performs significantly better than CA125 alone, as measured by the area under the receiver operator characteristic curve (88.4% and 84.3%, re‐ spectively, p <0.001) [24].

The development of IVDMIAs for ovarian cancer based on known candidate biomarkers of‐ fers promise for improving diagnostic efficiency of not only adrenal masses but also the ear‐ lier detection of ovarian cancer and prognosis.

Optimising preoperative diagnosis and opportune referral to specialists, would not only as‐ sist in the development of a specific management strategy for individual patients, but would also allow for more accurate determination of perioperative morbidity and chance of surviv‐ al. Further studies, however, are needed to validate not only a comparison with classical clinical or serological parameters, but also between different IVDMIAs, to determine which one is the better diagnostic tool.

#### **5. Novel approaches to the diagnosis of ovarian cancer**

Ovarian cancer is generally diagnosed in its advanced stages due to the lack of overt symp‐ toms of disease (70% of the cases approximately), resulting in a poor prognosis (rate surviv‐ al around 30%) [25]. Only a small number of ovarian cancers are detected early and these are the ones that can generally be treated.

The reason ovarian cancer is difficult to diagnose in its initial stage is due to the lack of spe‐ cific and appropriately sensitive serum biomarkers associated with the unspecific symp‐ toms. The most utilised serum biomarker in the diagnosis of ovarian cancer is CA125, but unfortunately its ability to detect ovarian cancer in a general population is quite low [26, 27].

As a response to this difficult scenario, current investigations include the search for other serum biomarkers that would improve our ability to detect ovarian cancer in its initial stages, possibly in combination with CA125 and ultrasound imaging. The recent identifica‐ tion of tumour-specific nanoparticle (exosomes) in the blood of patients with various diseas‐ es/complications, including ovarian cancer, affords an alternative approach to the identification of more effective biomarkers.

Within the field of gynaecologic oncology, an aspect that has been particularly disappoint‐ ing is the development of early detection tests for ovarian cancer. Classical methods based on physical examination, images and some serum markers such as CA125, have not resulted in significant advances in early detection rates. Tests, such as OvaSure and OVA 1, have in‐ tegrated various clinical and serum markers for the diagnosis of cancer with different sensi‐ tivities and specificities, but are aimed at defining malignancy in patients with ovarian

A possible answer to the problem is seen with the recognition of specific membrane particles in ovarian tumors (exosomes), as well as other tissue surfaces. These particles are tissue-spe‐ cific and may allow the identification of specific cell types in preclinical stages of the disease. The potential detection of these specific exosomes in biofluids also offers new perspectives in research on the early detection of ovarian cancer. Such ovarian cancer–specific non-parti‐ cles may be present in fallopian tubule fluid, uterine washings or even cervicovaginal fluids. Further research is needed in this area to assess the utility of such approaches in order to

GER wishes to acknowledge and thank the Rotary Club of Williamstown and The RoCan Program for their support of the University of Queensland Centre for Clinical Diagnostic

, Benjamín Bustos2

1 Gynaecologic Oncologist, Unniversity of The Andes and Clinica Dávila, Santiago, Chile

3 University of Queensland Centre for Clinical Research, Herston, Queensland, Australia

[1] Tavassoli FA, Devilee P. Pathology and genetics of tumors of the breast and female genital organs. In: *World Health Organization Classification of Tumors*. Lyon, France:

2 Department of Obstetrics and Gynaecology, University of the Andes, Santiago, Chile

, Vania Lukoviek2

Ovarian Cancer *in vitro* Diagnostics: New Approaches to Earlier Detection

http://dx.doi.org/10.5772/53953

355

and

tumours, rather than providing either an earlier diagnosis or a screening test.

develop simple and safe methods of detecting ovarian cancer in its early stage.

**Acknowledgements**

**Author details**

Jorge Tapia1

Gregory E. Rice3

**References**

IARC, 2003; 113–145

ovarian cancer biomarker discovery program.

, Mercedes de Alvarado2

Exosomes are small (40-100 nm) membrane vesicles that are released following the exocytot‐ ic fusion of multi-vesicular bodies with the cell membrane. They are characterised by: a cupshaped form; a buoyant density of 1,13-1,19 g ⁄ ml [28,29] endosomal origin; and the enrichment of late endosomal membrane markers, including Tsg101, CD63, CD9 and CD81 [30-32]. Exosomes have been identified in plasma under both normal and pathological con‐ ditions, and their concentration has been reported to increase in association with disease se‐ verity and/or progression. While, the process(es) of exosome formation remains to be fully elucidated, available data support an endosomal origin and formation by the inward bud‐ ding of multi-vesicular bodies [33].

Tumor cells release exosomes into peripheral circulation [34], indeed the first vesicular structures described in plasma were observed in women with ovarian cancer [35]. In ovarian cancer, the concentration of exosomes (measured as exosomal protein in peripheral blood) increases with disease stage and are associated with tumour-specific microRNA [36]. These results suggest that microRNA profiling of circulating tumor exosomes could potentially be used as surrogate diagnostic markers and may be of utility for screening asymptomatic pop‐ ulations. Recent data further suggests that the release of exosomes from cells may represent a normal mechanism for cell-to-cell communication [37] their role in the pathogenesis of ovarian cancer, however, remain to be established.

Other novel diagnosis techniques have been described: intra-fallopian tubule sampling, the sampling of uterine washings and the sampling of cervicovaginal swabs. While these ap‐ proaches represent a very promising alternatives for the diagnosis of ovarian cancer, there is a paucity of data and clinical validation to support their implementation as viable alterna‐ tives to CA125 and ultrasound imaging.

#### **6. Concluding comments**

The alignment of metastatic and molecular phenotypes of ovarian cancer is affording new insights into the aetiology and treatment of this disease cluster.

Recent evidence supports a tubal origin of epithelial ovarian cancer, including the coexi‐ stance of similar gene mutations in the tubal intraepithelial carcinoma and those classi‐ fied as ovarian origin (*e.g*. TP53 gene mutation). On the basis of these data, some have proposed "endosalpingiosis" as the initial event in ovarian cancer, suggesting that the ep‐ ithelial cells of the tube migrate to the surface of the ovary constituting ovarian cancer genesis. If proven to be correct, new opportunities for the management of ovarian cancer may be realised, particularly for those patients carrying BRCA-1 and -2 mutations that require prophylactic surgery.

Within the field of gynaecologic oncology, an aspect that has been particularly disappoint‐ ing is the development of early detection tests for ovarian cancer. Classical methods based on physical examination, images and some serum markers such as CA125, have not resulted in significant advances in early detection rates. Tests, such as OvaSure and OVA 1, have in‐ tegrated various clinical and serum markers for the diagnosis of cancer with different sensi‐ tivities and specificities, but are aimed at defining malignancy in patients with ovarian tumours, rather than providing either an earlier diagnosis or a screening test.

A possible answer to the problem is seen with the recognition of specific membrane particles in ovarian tumors (exosomes), as well as other tissue surfaces. These particles are tissue-spe‐ cific and may allow the identification of specific cell types in preclinical stages of the disease. The potential detection of these specific exosomes in biofluids also offers new perspectives in research on the early detection of ovarian cancer. Such ovarian cancer–specific non-parti‐ cles may be present in fallopian tubule fluid, uterine washings or even cervicovaginal fluids. Further research is needed in this area to assess the utility of such approaches in order to develop simple and safe methods of detecting ovarian cancer in its early stage.

#### **Acknowledgements**

stages, possibly in combination with CA125 and ultrasound imaging. The recent identifica‐ tion of tumour-specific nanoparticle (exosomes) in the blood of patients with various diseas‐ es/complications, including ovarian cancer, affords an alternative approach to the

Exosomes are small (40-100 nm) membrane vesicles that are released following the exocytot‐ ic fusion of multi-vesicular bodies with the cell membrane. They are characterised by: a cupshaped form; a buoyant density of 1,13-1,19 g ⁄ ml [28,29] endosomal origin; and the enrichment of late endosomal membrane markers, including Tsg101, CD63, CD9 and CD81 [30-32]. Exosomes have been identified in plasma under both normal and pathological con‐ ditions, and their concentration has been reported to increase in association with disease se‐ verity and/or progression. While, the process(es) of exosome formation remains to be fully elucidated, available data support an endosomal origin and formation by the inward bud‐

Tumor cells release exosomes into peripheral circulation [34], indeed the first vesicular structures described in plasma were observed in women with ovarian cancer [35]. In ovarian cancer, the concentration of exosomes (measured as exosomal protein in peripheral blood) increases with disease stage and are associated with tumour-specific microRNA [36]. These results suggest that microRNA profiling of circulating tumor exosomes could potentially be used as surrogate diagnostic markers and may be of utility for screening asymptomatic pop‐ ulations. Recent data further suggests that the release of exosomes from cells may represent a normal mechanism for cell-to-cell communication [37] their role in the pathogenesis of

Other novel diagnosis techniques have been described: intra-fallopian tubule sampling, the sampling of uterine washings and the sampling of cervicovaginal swabs. While these ap‐ proaches represent a very promising alternatives for the diagnosis of ovarian cancer, there is a paucity of data and clinical validation to support their implementation as viable alterna‐

The alignment of metastatic and molecular phenotypes of ovarian cancer is affording new

Recent evidence supports a tubal origin of epithelial ovarian cancer, including the coexi‐ stance of similar gene mutations in the tubal intraepithelial carcinoma and those classi‐ fied as ovarian origin (*e.g*. TP53 gene mutation). On the basis of these data, some have proposed "endosalpingiosis" as the initial event in ovarian cancer, suggesting that the ep‐ ithelial cells of the tube migrate to the surface of the ovary constituting ovarian cancer genesis. If proven to be correct, new opportunities for the management of ovarian cancer may be realised, particularly for those patients carrying BRCA-1 and -2 mutations that

identification of more effective biomarkers.

354 Ovarian Cancer - A Clinical and Translational Update

ding of multi-vesicular bodies [33].

ovarian cancer, however, remain to be established.

insights into the aetiology and treatment of this disease cluster.

tives to CA125 and ultrasound imaging.

**6. Concluding comments**

require prophylactic surgery.

GER wishes to acknowledge and thank the Rotary Club of Williamstown and The RoCan Program for their support of the University of Queensland Centre for Clinical Diagnostic ovarian cancer biomarker discovery program.

#### **Author details**

Jorge Tapia1 , Mercedes de Alvarado2 , Benjamín Bustos2 , Vania Lukoviek2 and Gregory E. Rice3

1 Gynaecologic Oncologist, Unniversity of The Andes and Clinica Dávila, Santiago, Chile

2 Department of Obstetrics and Gynaecology, University of the Andes, Santiago, Chile

3 University of Queensland Centre for Clinical Research, Herston, Queensland, Australia

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[18] Dietl, J. & Wischhusen, J. The forgotten fallopian tube. 10 Feb 2011 (doi:10.1038/

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[21] Zhang Z. An In Vitro Diagnostic Multivariate Index Assay (IVDMIA) for Ovarian Cancer: Harvesting the Power of Multiple Biomarkers. Rev Obstet Gynecol.

[22] Ueland FR, Desimone CP, Seamon LG, et al. Effectiveness of a multivariate index as‐ say in the preoperative assessment of ovarian tumors. Obstet Gynecol.

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[26] Bast RC Jr, Badgwell D, Lu Z, Marquez R, Rosen D, Liu J, Baggerly KA, Atkinson EN, Skates S, Zhang Z, *et al.*: New tumor markers: CA125 and beyond. *Int J Gynecol Can‐*

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[17] Crum, C. P. *et al.* Lessons from *BRCA*: the tubal fimbria emerges as an origin for pel‐

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**Chapter 17**

**Disseminated Tumor Cells and Cancer Stem Cells in**

Ovarian cancer is currently the fifth most lethal malignancy of women in Europe and the United States [1, 2]. The prognosis of ovarian cancer patients is limited due to lack of specific early symptoms and a high rate of relapse; more than half of all patients will suffer from disease recurrence, resulting in a poor overall survival [3]. Most cases are di‐ agnosed in advanced stages, and although the initial response to chemotherapy is gener‐ ally good, a significant proportion of patients will suffer from a relapse despite optimal cytoreductive surgery [4]. Since treatment strategies are mainly developed to control lo‐ coregional cancer growth, it may be anticipated that more women will die of distant metastatic disease. The identification of novel molecular markers, reflecting current tu‐ mor activity, may improve prediction and therapy monitoring and provide valuable in‐ sights into process of carcinogenesis. In this regard, oncologic research have increasingly

The presence of disseminated tumor cell (DTC) in bone marrow (BM) is a phenomenon ob‐ served in almost all solid tumors of epithelial origin. For breast cancer, DTC presence has been demonstrated as a strong independent prognostic factor (level I evidence) [5]. Availa‐ ble data support the notion that hematogenous tumor cell dissemination may be clinically relevant in ovarian cancer as well [6-10]. Detection rates of DTC, as a surrogate parameter for occult hematogenous spread, vary between 30-50% primary ovarian cancer patients.

A provocative hypothesis has been introduced recently with respect to natural history and progression of ovarian cancer. While the 'classical' stochastic model of cancer development holds that any cell may become source of malignant transformation, emerging evidence sup‐

and reproduction in any medium, provided the original work is properly cited.

© 2013 Banys et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Ovarian Cancer**

http://dx.doi.org/10.5772/54391

**1. Introduction**

Malgorzata Banys, Natalia Krawczyk, Andreas D. Hartkopf and Tanja Fehm

Additional information is available at the end of the chapter

focused on disseminated and circulating tumor cells.


### **Disseminated Tumor Cells and Cancer Stem Cells in Ovarian Cancer**

Malgorzata Banys, Natalia Krawczyk, Andreas D. Hartkopf and Tanja Fehm

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54391

#### **1. Introduction**

[32] Taylor DD, Homesley HD, Doellgast GJ: Binding of specific peroxidase- labeled anti‐ body to placental-type phosphatase on tumor-derived membrane fragments. *Cancer*

[33] Mincheva-Nilsson L, Baranov V. The role of placental exosomes in reproduction. Am

[34] Keller S, Ridinger J, Rupp AK, Janssen JW, Altevogt P. Body fluid derived exosomes as a novel template for clinical diagnostics. Journal of translational medicine.

[35] Simons M, Raposo G. Exosomes--vesicular carriers for intercellular communication.

[36] Taylor DD, Gercel-Taylor C. MicroRNA signatures of tumor-derived exosomes as di‐ agnostic biomarkers of ovarian cancer. Gynecologic oncology. 2008;110(1):13-21.

[37] Ludwig AK, Giebel B. Exosomes: small vesicles participating in intercellular commu‐ nication. The international journal of biochemistry & cell biology. 2012;44(1):11-5.

*Res* 1980, 40:4064-4069.

358 Ovarian Cancer - A Clinical and Translational Update

2011;9:86.

J Reprod Immunol. 2010;63(6):520-33.

Current opinion in cell biology. 2009;21(4):575-81.

Ovarian cancer is currently the fifth most lethal malignancy of women in Europe and the United States [1, 2]. The prognosis of ovarian cancer patients is limited due to lack of specific early symptoms and a high rate of relapse; more than half of all patients will suffer from disease recurrence, resulting in a poor overall survival [3]. Most cases are di‐ agnosed in advanced stages, and although the initial response to chemotherapy is gener‐ ally good, a significant proportion of patients will suffer from a relapse despite optimal cytoreductive surgery [4]. Since treatment strategies are mainly developed to control lo‐ coregional cancer growth, it may be anticipated that more women will die of distant metastatic disease. The identification of novel molecular markers, reflecting current tu‐ mor activity, may improve prediction and therapy monitoring and provide valuable in‐ sights into process of carcinogenesis. In this regard, oncologic research have increasingly focused on disseminated and circulating tumor cells.

The presence of disseminated tumor cell (DTC) in bone marrow (BM) is a phenomenon ob‐ served in almost all solid tumors of epithelial origin. For breast cancer, DTC presence has been demonstrated as a strong independent prognostic factor (level I evidence) [5]. Availa‐ ble data support the notion that hematogenous tumor cell dissemination may be clinically relevant in ovarian cancer as well [6-10]. Detection rates of DTC, as a surrogate parameter for occult hematogenous spread, vary between 30-50% primary ovarian cancer patients.

A provocative hypothesis has been introduced recently with respect to natural history and progression of ovarian cancer. While the 'classical' stochastic model of cancer development holds that any cell may become source of malignant transformation, emerging evidence sup‐

© 2013 Banys et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ports the view that only a minor subpopulation of cancer cells has the potential to initiate cancer growth. These cells, called cancer stem cells (CSC), have the ability to self-renew, propagate tumorigenesis and are usually drug-resistant [11]. Experimental studies on stem cell biology have given new impetus to the cancer stem cell theory. CSC are assumed to play important role in development of various tumor entities, such as breast and gastrointestinal cancer, retinoblastoma and ovarian cancer [12], [13]. Interestingly, ovarian cancer cell lines feature "side population" cells with potential to differentiate into cancers with different his‐ tologies, suggesting the pluripotent character of stem cells [14]. Whether DTC in extraperito‐ neal sites, such as bone marrow, reflect a stem cell-like sub population of tumor cells, remains yet to be cleared.

Of all prognostic factors, monitoring of minimal residual disease is the only one available after the tumor has been removed. Beside monitoring of tumor markers, there is currently a major effort to identify other biological markers which can be assessed with minimally inva‐ sive methods and persist beyond surgery. We previously reported on a significant correla‐ tion of positive BM status with shortened relapse-free survival in ovarian cancer patients [6]. DTC persistence after completion of platinum-based chemotherapy was also found to be prognostically relevant [15]. Recently, attempts have been made to target DTC by using anti‐ body-based therapy with the trifunctional antibody catumaxomab. Wimberger et al. report‐ ed a marked decrease in tumor cells in peripheral blood following intraperitoneal catumaxomab treatment for malignant ascites, indicating a systemic effect of the therapy [16]. However, in comparison with breast cancer, data on DTC detection in gynecological malignancies are so far limited [7], [17], [18], [19], [9].

In this chapter we discuss recent advances in ovarian cancer research with respect to disse‐ minated tumor cells and cancer stem cell hypothesis. Data on prognostic and clinical rele‐ vance are presented.

**Figure 1.** Disseminated tumor cell from ovarian cancer patient with typical cytomorphology and immunophenotype (positive cytokeratin-staining, large nucleus, high nuclear to cytoplasmic ratio, nucleus partially covered by CK-stain‐

Disseminated Tumor Cells and Cancer Stem Cells in Ovarian Cancer

http://dx.doi.org/10.5772/54391

361

Detection rates of disseminated tumor cells in ovarian cancer patients stage FIGO I-III reach 20-60% [7, 9, 15, 22]. These results suggest hematogenous spread to be a comparatively fre‐ quent phenomenon in ovarian malignancies and indicate the ability of single tumor cells to disseminate to bone marrow in a very early stage of disease. DTC are routinely detected in 13-18% FIGO I ovarian cancer patients [6, 19, 22]. Since bone metastases are relatively rare in ovarian cancer patients, BM seems to serve as a temporary `homing site´ for single tumor cells, from where they are able to migrate and subsequently cause distant metastasis or local recurrence [24]. Assuming that DTC may spread by means of blood stream we cannot ex‐ clude that those may also be able to repopulate the peritoneal cavity, an environment which

Based on numerous studies, no significant relationship has been reported between clini‐ copathological characteristics of primary tumor and DTC detection. In our latest trial with 414 ovarian cancer patients, DTC status did not correlate with FIGO stage, tumor size, lymph node status, histopathologic grading or resection status [26, 27]. Braun et al., in a cohort of 108 primary ovarian cancer patients, showed no concordance between classical prognostic factors and DTC positivity. The only factor associated with positive DTC status was tumor grading (p = 0.02) [7]. These results could be also confirmed in our earlier study with 112 ovarian cancer patients [6]. Similar findings were obtained by

ing, nucleus granular [23].

easily supports ovarian cancer growth [11, 25].

other investigators [8, 9, 15, 19].

#### **2. Disseminated and circulating tumor cells in ovarian cancer**

Detection and characterization of disseminated tumor cells in bone marrow and blood of pa‐ tients with epithelial carcinomas can be accomplished by various techniques. For the detec‐ tion of isolated tumor cells, both antibody-based assays and molecular assays have been established [20, 21]. Despite advances in this field, no specific antigen or marker gene has been described for ovarian cancer so far. Therefore, immunocytochemical identification of these cells based on expression of epithelial markers remains the gold standard (Figure 1). Commonly targeted antigens are cytokeratin and EpCAM due to relatively constant and universal expression pattern in cells of epithelial origin [9, 15, 22]. A major difficulty in de‐ tecting and characterizing tumor cells is their relatively low frequency. Most protocols in‐ clude therefore a cell enrichment step (e.g. density gradient centrifugation, immunomagnetic enrichment). These obstacles highlight the need for optimization of the as‐ say (e.g. by minimizing cell loss, preserving cell morphology and producing reliable immu‐ nophenotypic and genotypic data) as it is essential for detecting, enumerating and characterizing single tumor cells [21].

ports the view that only a minor subpopulation of cancer cells has the potential to initiate cancer growth. These cells, called cancer stem cells (CSC), have the ability to self-renew, propagate tumorigenesis and are usually drug-resistant [11]. Experimental studies on stem cell biology have given new impetus to the cancer stem cell theory. CSC are assumed to play important role in development of various tumor entities, such as breast and gastrointestinal cancer, retinoblastoma and ovarian cancer [12], [13]. Interestingly, ovarian cancer cell lines feature "side population" cells with potential to differentiate into cancers with different his‐ tologies, suggesting the pluripotent character of stem cells [14]. Whether DTC in extraperito‐ neal sites, such as bone marrow, reflect a stem cell-like sub population of tumor cells,

Of all prognostic factors, monitoring of minimal residual disease is the only one available after the tumor has been removed. Beside monitoring of tumor markers, there is currently a major effort to identify other biological markers which can be assessed with minimally inva‐ sive methods and persist beyond surgery. We previously reported on a significant correla‐ tion of positive BM status with shortened relapse-free survival in ovarian cancer patients [6]. DTC persistence after completion of platinum-based chemotherapy was also found to be prognostically relevant [15]. Recently, attempts have been made to target DTC by using anti‐ body-based therapy with the trifunctional antibody catumaxomab. Wimberger et al. report‐ ed a marked decrease in tumor cells in peripheral blood following intraperitoneal catumaxomab treatment for malignant ascites, indicating a systemic effect of the therapy [16]. However, in comparison with breast cancer, data on DTC detection in gynecological

In this chapter we discuss recent advances in ovarian cancer research with respect to disse‐ minated tumor cells and cancer stem cell hypothesis. Data on prognostic and clinical rele‐

Detection and characterization of disseminated tumor cells in bone marrow and blood of pa‐ tients with epithelial carcinomas can be accomplished by various techniques. For the detec‐ tion of isolated tumor cells, both antibody-based assays and molecular assays have been established [20, 21]. Despite advances in this field, no specific antigen or marker gene has been described for ovarian cancer so far. Therefore, immunocytochemical identification of these cells based on expression of epithelial markers remains the gold standard (Figure 1). Commonly targeted antigens are cytokeratin and EpCAM due to relatively constant and universal expression pattern in cells of epithelial origin [9, 15, 22]. A major difficulty in de‐ tecting and characterizing tumor cells is their relatively low frequency. Most protocols in‐ clude therefore a cell enrichment step (e.g. density gradient centrifugation, immunomagnetic enrichment). These obstacles highlight the need for optimization of the as‐ say (e.g. by minimizing cell loss, preserving cell morphology and producing reliable immu‐ nophenotypic and genotypic data) as it is essential for detecting, enumerating and

**2. Disseminated and circulating tumor cells in ovarian cancer**

remains yet to be cleared.

360 Ovarian Cancer - A Clinical and Translational Update

vance are presented.

characterizing single tumor cells [21].

malignancies are so far limited [7], [17], [18], [19], [9].

**Figure 1.** Disseminated tumor cell from ovarian cancer patient with typical cytomorphology and immunophenotype (positive cytokeratin-staining, large nucleus, high nuclear to cytoplasmic ratio, nucleus partially covered by CK-stain‐ ing, nucleus granular [23].

Detection rates of disseminated tumor cells in ovarian cancer patients stage FIGO I-III reach 20-60% [7, 9, 15, 22]. These results suggest hematogenous spread to be a comparatively fre‐ quent phenomenon in ovarian malignancies and indicate the ability of single tumor cells to disseminate to bone marrow in a very early stage of disease. DTC are routinely detected in 13-18% FIGO I ovarian cancer patients [6, 19, 22]. Since bone metastases are relatively rare in ovarian cancer patients, BM seems to serve as a temporary `homing site´ for single tumor cells, from where they are able to migrate and subsequently cause distant metastasis or local recurrence [24]. Assuming that DTC may spread by means of blood stream we cannot ex‐ clude that those may also be able to repopulate the peritoneal cavity, an environment which easily supports ovarian cancer growth [11, 25].

Based on numerous studies, no significant relationship has been reported between clini‐ copathological characteristics of primary tumor and DTC detection. In our latest trial with 414 ovarian cancer patients, DTC status did not correlate with FIGO stage, tumor size, lymph node status, histopathologic grading or resection status [26, 27]. Braun et al., in a cohort of 108 primary ovarian cancer patients, showed no concordance between classical prognostic factors and DTC positivity. The only factor associated with positive DTC status was tumor grading (p = 0.02) [7]. These results could be also confirmed in our earlier study with 112 ovarian cancer patients [6]. Similar findings were obtained by other investigators [8, 9, 15, 19].

#### **2.1. Prognostic relevance of DTC/CTC in ovarian cancer**

Detection of disseminated tumor cells in patients with primary ovarian cancer was shown to be of prognostic value (Table 1). However, the currently available data are sparse. In our latest trial bone marrow status of 414 ovarian cancer patients was corre‐ lated with clinical follow-up [26]. The presence of DTC predicted a shorter OS (p < 0.001) and DFS (p = 0.035) compared with BM negative patients [27]; this association was highly significant and confirmed in a multivariable Cox regression analysis. Similar results were found in several smaller studies. Braun et al. demonstrated unfavorable prognosis with regard to distant DFS in BM positive patients at the time of diagnosis [7]. DTC presence remained a strong prognostic factor also in a subset of 64 optimally debulked patients (p = 0.002), which highlights the role of DTC detection especially in patients who received successful surgical cytoreduction. We previously reported a signif‐ icant correlation of positive BM status with reduced DFS in a group of 112 stage FIGO I-III ovarian cancer patients [6]. Interestingly, in some studies, the presence of isolated tumor cells in secondary sites, such as BM and blood, was also associated with higher risk for recurrence [10], [6]. Therefore, it might be speculated that hematogenous tumor cell dissemination may serve as an indicator of a more aggressive phenotype of the pri‐ mary disease that is likely to cause local relapse. In contrast, other authors reported no significant correlation between DTC detection and clinical outcome in ovarian cancer [19, 28]. This discrepancy might be due to differences in study protocols, e.g. time point of BM sample collection (pre- vs. postoperative aspiration). Hypothetically, a transient in‐ crease in cancer cell dissemination from the primary tumor due to intraoperative manip‐ ulation could contribute to false-positive results and therefore affect further analysis [29]. irrespective of tumor stage but observed no correlation with clinical outcome [19]. Interest‐ ingly, positive finding in the blood was highly associated with DTC detection in bone mar‐ row. Smaller studies showed varying CTC incidence, depending on methodology [35, 36].

Fehm [26] 414 DTC (ICC) 34 27% OS, DFS 1 Banys [6] 112 DTC (ICC) 12 25% DFS Braun [7] 108 DTC (ICC) 45 30% DFS Aktas [8] 95 DTC (ICC) 28 35% n.s. Schindlbeck [9] 90 DTC (ICC) 28 23% DDFS Marth [19] 73 DTC (immunobeads) 25 21% n.s. Wimberger [30] 62 DTC (ICC) 18 54% DFS 2 Poveda [10] 216 CTC (ICC: CellSearch) 3 14% 4 PFS, OS Sehouli [17] 167 CTC (ICC) 46 n.s. Marth [19] 90 CTC (immunomagnetic beads) 25 12% n.s. Aktas [8] 86 CTC (Multiplex-RT-PCR: AdnaTest) 28 19% OS 5 Heubner [31] 68 Circulating 20S-proteasomes 19 - OS

> CTC (immunofluorescence, cell invasion assay)

protease and caspase activity

marrow, ICC – immunocytochemistry, n.s. – not significant, PFS – progression-free survival

**Table 1.** Prognostic relevance of disseminated and circulating tumor cells in ovarian cancer.

*Abbreviations:* DFS – disease-free survival, DDFS – distant disease-free survival, DTC – disseminated tumor cells in bone

Beyond the prognostic value of DTC/CTC detection, monitoring of minimal residual disease (MRD) during and after treatment offers the opportunity to assess response to therapy and evaluate the residual risk of recurrence. Changes in MRD represent the only clinical parame‐

**Median follow-up [months]**

Disseminated Tumor Cells and Cancer Stem Cells in Ovarian Cancer

**Positivity rate Prognostic**

http://dx.doi.org/10.5772/54391

18 61% DFS

18 - DFS, OS

**significance**

363

**Author N Method**

Wimberger [30] <sup>62</sup> Circulating nucleosomes, DNA,

1 Determined by multivariate Cox regression analysis

2 DTC detected after chemotherapy

5 Both before and after chemotherapy

**2.3. Therapy monitoring**

3 Relapsed ovarian cancer

4 Two or more CTC

Fan [32] 66

#### **2.2. Circulating tumor cells**

Bone marrow biopsy represents an invasive procedure not well tolerated by many patients. Therefore, detection of circulating tumor cells (CTC) by simple blood drawing has increas‐ ingly become a focus of translational research. Prognostic significance of CTC in peripheral blood has been evaluated in breast cancer both in primary and metastatic disease [33, 34]. Two commercially available kits are currently in use for CTC detection in blood of breast cancer patients: antibody-based CellSearch and RT-PCR-based AdnaTest. Both assays were modified and validated in ovarian cancer patients (Table 1). Recently published trial by Po‐ veda et al. based on a cohort of 216 patients with recurrent ovarian cancer, represents the largest study so far on the impact of CTC presence on survival [10]. Using CellSearch test increased CTC numbers (> 1 cell / 7.5 ml blood) were found in 14% of these patients before the beginning of treatment. Detection of CTC in peripheral blood was associated with signif‐ icantly impaired prognosis. In the study by Aktas et al., including 86 ovarian cancer pa‐ tients, a modified AdnaTest kit was used to detect cells expressing EpCAM, MUC-1, HER-2 or CA 125-transcripts [8]. CTC positivity rate of 19% observed in this cohort was associated with significantly shorter survival independent of the time of blood sampling (before sur‐ gery or after chemotherapy). Similar results were obtained by Fan et al. in a trial of 66 pri‐ mary ovarian cancer patients [32]. In contrast, Marth et al. reported a 12% positivity rate irrespective of tumor stage but observed no correlation with clinical outcome [19]. Interest‐ ingly, positive finding in the blood was highly associated with DTC detection in bone mar‐ row. Smaller studies showed varying CTC incidence, depending on methodology [35, 36].


*Abbreviations:* DFS – disease-free survival, DDFS – distant disease-free survival, DTC – disseminated tumor cells in bone marrow, ICC – immunocytochemistry, n.s. – not significant, PFS – progression-free survival

1 Determined by multivariate Cox regression analysis


**2.1. Prognostic relevance of DTC/CTC in ovarian cancer**

362 Ovarian Cancer - A Clinical and Translational Update

**2.2. Circulating tumor cells**

Detection of disseminated tumor cells in patients with primary ovarian cancer was shown to be of prognostic value (Table 1). However, the currently available data are sparse. In our latest trial bone marrow status of 414 ovarian cancer patients was corre‐ lated with clinical follow-up [26]. The presence of DTC predicted a shorter OS (p < 0.001) and DFS (p = 0.035) compared with BM negative patients [27]; this association was highly significant and confirmed in a multivariable Cox regression analysis. Similar results were found in several smaller studies. Braun et al. demonstrated unfavorable prognosis with regard to distant DFS in BM positive patients at the time of diagnosis [7]. DTC presence remained a strong prognostic factor also in a subset of 64 optimally debulked patients (p = 0.002), which highlights the role of DTC detection especially in patients who received successful surgical cytoreduction. We previously reported a signif‐ icant correlation of positive BM status with reduced DFS in a group of 112 stage FIGO I-III ovarian cancer patients [6]. Interestingly, in some studies, the presence of isolated tumor cells in secondary sites, such as BM and blood, was also associated with higher risk for recurrence [10], [6]. Therefore, it might be speculated that hematogenous tumor cell dissemination may serve as an indicator of a more aggressive phenotype of the pri‐ mary disease that is likely to cause local relapse. In contrast, other authors reported no significant correlation between DTC detection and clinical outcome in ovarian cancer [19, 28]. This discrepancy might be due to differences in study protocols, e.g. time point of BM sample collection (pre- vs. postoperative aspiration). Hypothetically, a transient in‐ crease in cancer cell dissemination from the primary tumor due to intraoperative manip‐ ulation could contribute to false-positive results and therefore affect further analysis [29].

Bone marrow biopsy represents an invasive procedure not well tolerated by many patients. Therefore, detection of circulating tumor cells (CTC) by simple blood drawing has increas‐ ingly become a focus of translational research. Prognostic significance of CTC in peripheral blood has been evaluated in breast cancer both in primary and metastatic disease [33, 34]. Two commercially available kits are currently in use for CTC detection in blood of breast cancer patients: antibody-based CellSearch and RT-PCR-based AdnaTest. Both assays were modified and validated in ovarian cancer patients (Table 1). Recently published trial by Po‐ veda et al. based on a cohort of 216 patients with recurrent ovarian cancer, represents the largest study so far on the impact of CTC presence on survival [10]. Using CellSearch test increased CTC numbers (> 1 cell / 7.5 ml blood) were found in 14% of these patients before the beginning of treatment. Detection of CTC in peripheral blood was associated with signif‐ icantly impaired prognosis. In the study by Aktas et al., including 86 ovarian cancer pa‐ tients, a modified AdnaTest kit was used to detect cells expressing EpCAM, MUC-1, HER-2 or CA 125-transcripts [8]. CTC positivity rate of 19% observed in this cohort was associated with significantly shorter survival independent of the time of blood sampling (before sur‐ gery or after chemotherapy). Similar results were obtained by Fan et al. in a trial of 66 pri‐ mary ovarian cancer patients [32]. In contrast, Marth et al. reported a 12% positivity rate

5 Both before and after chemotherapy

**Table 1.** Prognostic relevance of disseminated and circulating tumor cells in ovarian cancer.

#### **2.3. Therapy monitoring**

Beyond the prognostic value of DTC/CTC detection, monitoring of minimal residual disease (MRD) during and after treatment offers the opportunity to assess response to therapy and evaluate the residual risk of recurrence. Changes in MRD represent the only clinical parame‐ ter available after surgical removal of the primary tumor. While tumor markers are estab‐ lished tools for the evaluation of treatment efficacy in patients with advanced ovarian cancer, CA 125 levels fall during adjuvant chemotherapy and remain often below cut-off values after completion of first line systemic treatment even though significant number of patients will suffer from relapse within five years. Furthermore, the clinical relevance of se‐ rial CA125 measurements for early detection and treatment of disease recurrence is current‐ ly being controversially discussed [37]. In this regard, the detection of isolated tumor cells in bone marrow or peripheral blood might serve as a parameter for occult tumor load after completion of first line therapy. DTC persistence despite adjuvant treatment is so far an in‐ dependent prognostic factor in patients with primary breast cancer [38]. Whether persistent DTC influence prognosis in ovarian cancer patients, is currently being investigated. In the study by Wimberger et al. DTC counts before and after the first line systemic treatment were correlated to clinical course of disease in 30 ovarian cancer patients; 54% of these patients presented with DTC after first-line chemotherapy. Marked increase in DTC counts was asso‐ ciated with shortened progression free survival [15].

The cancer stem cell hypothesis holds that CSC are responsible for phenomena like drug re‐ sistance, tumor dormancy or minimal residual disease and may persist beyond chemothera‐ py and repopulate the tumor leading to relapse [11]. The stem cell subpopulation, but not the remaining differentiated cancer cells in the tumor, can sustain tumor formation and growth due to their high tumor-initiating potential. So far, such cells have been found in several solid tumors, such as colon [40], breast [41] and ovarian cancer [13, 42, 43]. Indeed, in accordance with recent studies, ovarian cancer cell lines feature ´side population´ cells (SP) with potential to differentiate into other morphological entities. Thus, this pluripotent sub‐ clone with stem cell-like features is considered a marker of CSC presence [14]. The detection of CSC is based on the presence of extracellular markers assumed to be stem cell-specific; commonly identified markers are CD44, CD133, and CD24, which are found in prostate, breast, pancreas, and ovarian cancer. It remains under discussion whether these parameters are universal markers relevant for CSC derived from all tumor types; for ovarian cancer,

Disseminated Tumor Cells and Cancer Stem Cells in Ovarian Cancer

http://dx.doi.org/10.5772/54391

365

multiple markers have been described for the stem cell-like tumor initiating cells.

independent predictor of poor progression-free survival [45].

characteristics have been described to target the subset of CSC.

in, and the stem cell factors Nestin, Nanong, and Oct-4.

**3.1. DTC and cancer stem cell model**

Owing to aggressive natural course of disease and emergence of multidrug resistance, an es‐ sential role of cancer stem cells has been postulated in ovarian cancer [13, 44]. In the study by Szotek et al. SP cells have been encountered in ovarian cancer cell lines as well as in pri‐ mary ascites cancer cells [13]. In the trial by Hosonuma et al. in 18 of 28 ovarian cancer pa‐ tients samples side population cells could be detected. SP cells occurred more often in relapsed and metastatic patients and SP positivity predicted significantly reduced overall survival [15]. A high proportion of CD44+ stem cells in ovarian cancer was reported to be an

As previously mentioned, CSC are considered responsible for high emergence of drug re‐ sistance in the natural history of ovarian cancer, since standard therapies fail to target pluri‐ potent tumor-initiating cells [13]. Latifi et al. could show in their recently published trial, the ability of cisplatin chemotherapy to generate residual tumor cells with mesenchymal stem cell-like characteristics *in vitro* [46]. Accordingly, new therapeutic strategies have to be de‐ veloped to target these cells by identifying their specific antigens. However, very few tumor

Based on an animal model, Bapat et al. reported the isolation and identification of ovarian cancer stem cells [42]. In an *in vitro* model comprised of 19 spontaneously immortalized clones derived from an advanced-stage patient, the authors demonstrated the ability of two clones with stem cell-like characteristics to differentiate to grow as spheroids and form xen‐ ografts in an animal model (nude mice). These cells were shown to express CD44, E-cadher‐

A currently discussed theory postulate DTC and CTC, the presumed precursor cells of sys‐ temic metastatic disease, to be in fact cancer stem cells. These observations have been so far reported in breast cancer studies. Balic et al. analyzed bone marrow specimens from 50 pri‐ mary breast cancer patients; 33-100% of DTC of every patient exhibited stem cell-like pheno‐ type: CD44+ / CD24 low/- [47]. This prevalence is estimated for less than 10% in primary

Evaluation of treatment efficacy in ovarian cancer patients after optimal surgical cytoreduc‐ tion and completion of first line systemic therapy is based on clinical, radiological and bio‐ logical (tumor marker CA125) criteria. However, a reliable tool to assess long-term prognosis has so far not been established. Therefore, a solid therapy monitoring tool could help to identify a group of high-risk patients who potentially benefit from additional treat‐ ment. New studies are required to evaluate whether persistent DTC indeed predict a worse prognosis and if these cells may be targeted by secondary adjuvant therapy.

#### **3. Cancer stem cell model**

An important hypothesis on tumor initiation and progression has attracted much attention in the ovarian cancer research in the past decade. In contrast to stochastic model that postu‐ lates every cell as a potential source of malignant transformation, the cancer stem cell model, a theory introduced over a century ago, proposes that tumors are organized in a cellular hi‐ erarchy, in which cancer stem cell (CSC) are the only cells with tumorigenic potential. Ac‐ cordingly, tumors are initiated in cancer stem cells or their immediate progeny through imbalance of self-renewal and apoptosis; these tumors contain a cellular subpopulation that retains key stem cell feature [39]. This small cell group with unlimited proliferative potential is assumed to play a marked role in initiation and development of several tumor entities like retinoblastoma, gastrointestinal cancer as well as breast and ovarian cancer [12]. The cancer stem cell concept has important implications for understanding the process of carcinogene‐ sis as well as for designing new treatment strategies. Due to their long life, CSC are more likely to acquire transforming mutations; further, they seem more resistant to apoptosis and DNA damage and are therefore able to persist beyond therapy. New evidence in support of the cancer stem cell model has arisen due to advances in stem cell biology and the introduc‐ tion of novel animal models to assess self-renewal and challenge the validity of this concept.

The cancer stem cell hypothesis holds that CSC are responsible for phenomena like drug re‐ sistance, tumor dormancy or minimal residual disease and may persist beyond chemothera‐ py and repopulate the tumor leading to relapse [11]. The stem cell subpopulation, but not the remaining differentiated cancer cells in the tumor, can sustain tumor formation and growth due to their high tumor-initiating potential. So far, such cells have been found in several solid tumors, such as colon [40], breast [41] and ovarian cancer [13, 42, 43]. Indeed, in accordance with recent studies, ovarian cancer cell lines feature ´side population´ cells (SP) with potential to differentiate into other morphological entities. Thus, this pluripotent sub‐ clone with stem cell-like features is considered a marker of CSC presence [14]. The detection of CSC is based on the presence of extracellular markers assumed to be stem cell-specific; commonly identified markers are CD44, CD133, and CD24, which are found in prostate, breast, pancreas, and ovarian cancer. It remains under discussion whether these parameters are universal markers relevant for CSC derived from all tumor types; for ovarian cancer, multiple markers have been described for the stem cell-like tumor initiating cells.

Owing to aggressive natural course of disease and emergence of multidrug resistance, an es‐ sential role of cancer stem cells has been postulated in ovarian cancer [13, 44]. In the study by Szotek et al. SP cells have been encountered in ovarian cancer cell lines as well as in pri‐ mary ascites cancer cells [13]. In the trial by Hosonuma et al. in 18 of 28 ovarian cancer pa‐ tients samples side population cells could be detected. SP cells occurred more often in relapsed and metastatic patients and SP positivity predicted significantly reduced overall survival [15]. A high proportion of CD44+ stem cells in ovarian cancer was reported to be an independent predictor of poor progression-free survival [45].

As previously mentioned, CSC are considered responsible for high emergence of drug re‐ sistance in the natural history of ovarian cancer, since standard therapies fail to target pluri‐ potent tumor-initiating cells [13]. Latifi et al. could show in their recently published trial, the ability of cisplatin chemotherapy to generate residual tumor cells with mesenchymal stem cell-like characteristics *in vitro* [46]. Accordingly, new therapeutic strategies have to be de‐ veloped to target these cells by identifying their specific antigens. However, very few tumor characteristics have been described to target the subset of CSC.

Based on an animal model, Bapat et al. reported the isolation and identification of ovarian cancer stem cells [42]. In an *in vitro* model comprised of 19 spontaneously immortalized clones derived from an advanced-stage patient, the authors demonstrated the ability of two clones with stem cell-like characteristics to differentiate to grow as spheroids and form xen‐ ografts in an animal model (nude mice). These cells were shown to express CD44, E-cadher‐ in, and the stem cell factors Nestin, Nanong, and Oct-4.

#### **3.1. DTC and cancer stem cell model**

ter available after surgical removal of the primary tumor. While tumor markers are estab‐ lished tools for the evaluation of treatment efficacy in patients with advanced ovarian cancer, CA 125 levels fall during adjuvant chemotherapy and remain often below cut-off values after completion of first line systemic treatment even though significant number of patients will suffer from relapse within five years. Furthermore, the clinical relevance of se‐ rial CA125 measurements for early detection and treatment of disease recurrence is current‐ ly being controversially discussed [37]. In this regard, the detection of isolated tumor cells in bone marrow or peripheral blood might serve as a parameter for occult tumor load after completion of first line therapy. DTC persistence despite adjuvant treatment is so far an in‐ dependent prognostic factor in patients with primary breast cancer [38]. Whether persistent DTC influence prognosis in ovarian cancer patients, is currently being investigated. In the study by Wimberger et al. DTC counts before and after the first line systemic treatment were correlated to clinical course of disease in 30 ovarian cancer patients; 54% of these patients presented with DTC after first-line chemotherapy. Marked increase in DTC counts was asso‐

Evaluation of treatment efficacy in ovarian cancer patients after optimal surgical cytoreduc‐ tion and completion of first line systemic therapy is based on clinical, radiological and bio‐ logical (tumor marker CA125) criteria. However, a reliable tool to assess long-term prognosis has so far not been established. Therefore, a solid therapy monitoring tool could help to identify a group of high-risk patients who potentially benefit from additional treat‐ ment. New studies are required to evaluate whether persistent DTC indeed predict a worse

An important hypothesis on tumor initiation and progression has attracted much attention in the ovarian cancer research in the past decade. In contrast to stochastic model that postu‐ lates every cell as a potential source of malignant transformation, the cancer stem cell model, a theory introduced over a century ago, proposes that tumors are organized in a cellular hi‐ erarchy, in which cancer stem cell (CSC) are the only cells with tumorigenic potential. Ac‐ cordingly, tumors are initiated in cancer stem cells or their immediate progeny through imbalance of self-renewal and apoptosis; these tumors contain a cellular subpopulation that retains key stem cell feature [39]. This small cell group with unlimited proliferative potential is assumed to play a marked role in initiation and development of several tumor entities like retinoblastoma, gastrointestinal cancer as well as breast and ovarian cancer [12]. The cancer stem cell concept has important implications for understanding the process of carcinogene‐ sis as well as for designing new treatment strategies. Due to their long life, CSC are more likely to acquire transforming mutations; further, they seem more resistant to apoptosis and DNA damage and are therefore able to persist beyond therapy. New evidence in support of the cancer stem cell model has arisen due to advances in stem cell biology and the introduc‐ tion of novel animal models to assess self-renewal and challenge the validity of this concept.

prognosis and if these cells may be targeted by secondary adjuvant therapy.

ciated with shortened progression free survival [15].

364 Ovarian Cancer - A Clinical and Translational Update

**3. Cancer stem cell model**

A currently discussed theory postulate DTC and CTC, the presumed precursor cells of sys‐ temic metastatic disease, to be in fact cancer stem cells. These observations have been so far reported in breast cancer studies. Balic et al. analyzed bone marrow specimens from 50 pri‐ mary breast cancer patients; 33-100% of DTC of every patient exhibited stem cell-like pheno‐ type: CD44+ / CD24 low/- [47]. This prevalence is estimated for less than 10% in primary tumor suggesting much higher, stem cell like self-renewal and tumorigenic potential in DTC. Aktas et al. detected stem cells markers on CTC in peripheral blood of metastatic breast cancer patients [41]. Moreover, Abraham et al. reported that high proportion of stem cell-like subpopulation in primary breast cancer correlate with a higher prevalence of dis‐ tant metastases [48]. As breast cancer stem cells have been shown to be generally triple-neg‐ ative, basal-like CTC, independent of the phenotype of the primary tumor, support the cancer stem cell theory [20, 49, 50]. However, this aspect has not been researched in ovarian cancer so far. Therefore, futher studies have to be performed to evaluate whether isolated tumor cells in extraperitoneal sites, such as blood and bone marrow, may reflect ovarian cancer stem cell population.

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#### **4. Conclusions**

Despite advances in surgical and systemic therapy, ovarian cancer leads to relapse in 60% of patients within 5 years, resulting in impaired clinical outcome. In the last decades, novel bio‐ markers have been introduced for better prediction and prognostication [51]. Early hema‐ togenous dissemination of single tumor cells is a general phenomenon observed in most solid tumors of epithelial origin; recent data supports the clinical relevance of disseminated and circulating tumor cells (DTC/CTC) in ovarian cancer. A currently discussed hypothesis postulates isolated tumor cells in secondary sites to be not only the presumed precursors of systemic metastatic disease but in fact pluripotent 'cancer stem cells'. Future research will clarify the implications of these findings for clinical management of ovarian cancer patients. While the published data do not support the use of DTC/CTC detection for early detection or screening purposes, its role as an important prognostic factor has been confirmed in sev‐ eral studies. One of the most promising applications of DTC detection is their use as a thera‐ py monitoring tool. DTC persistence beyond surgery and adjuvant chemotherapy may help to identify patients at risk of developing a relapse.

#### **Author details**

Malgorzata Banys1,2, Natalia Krawczyk1 , Andreas D. Hartkopf1 and Tanja Fehm1\*

\*Address all correspondence to: tanja.fehm@t-online.de

1 Department of Obstetrics and Gynecology, University of Tuebingen, Tuebingen, Germany

2 Department of Obstetrics and Gynecology, Marienkrankenhaus Hamburg, Hamburg, Ger‐ many

#### **References**

tumor suggesting much higher, stem cell like self-renewal and tumorigenic potential in DTC. Aktas et al. detected stem cells markers on CTC in peripheral blood of metastatic breast cancer patients [41]. Moreover, Abraham et al. reported that high proportion of stem cell-like subpopulation in primary breast cancer correlate with a higher prevalence of dis‐ tant metastases [48]. As breast cancer stem cells have been shown to be generally triple-neg‐ ative, basal-like CTC, independent of the phenotype of the primary tumor, support the cancer stem cell theory [20, 49, 50]. However, this aspect has not been researched in ovarian cancer so far. Therefore, futher studies have to be performed to evaluate whether isolated tumor cells in extraperitoneal sites, such as blood and bone marrow, may reflect ovarian

Despite advances in surgical and systemic therapy, ovarian cancer leads to relapse in 60% of patients within 5 years, resulting in impaired clinical outcome. In the last decades, novel bio‐ markers have been introduced for better prediction and prognostication [51]. Early hema‐ togenous dissemination of single tumor cells is a general phenomenon observed in most solid tumors of epithelial origin; recent data supports the clinical relevance of disseminated and circulating tumor cells (DTC/CTC) in ovarian cancer. A currently discussed hypothesis postulates isolated tumor cells in secondary sites to be not only the presumed precursors of systemic metastatic disease but in fact pluripotent 'cancer stem cells'. Future research will clarify the implications of these findings for clinical management of ovarian cancer patients. While the published data do not support the use of DTC/CTC detection for early detection or screening purposes, its role as an important prognostic factor has been confirmed in sev‐ eral studies. One of the most promising applications of DTC detection is their use as a thera‐ py monitoring tool. DTC persistence beyond surgery and adjuvant chemotherapy may help

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366 Ovarian Cancer - A Clinical and Translational Update

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\*Address all correspondence to: tanja.fehm@t-online.de

Malgorzata Banys1,2, Natalia Krawczyk1

**4. Conclusions**

**Author details**

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### *Edited by Ivan Diaz Padilla*

"Ovarian Cancer: A Clinical and Translational Update" embraces the most recent advances in diagnosis and treatment of ovarian cancer. With the valuable collaboration of international experts in the field, this book is intended to provide the readership with a comprehensive update in the subject of epithelial ovarian cancer.

Ovarian Cancer - A Clinical and Translational Update

Ovarian Cancer

A Clinical and Translational Update

*Edited by Ivan Diaz Padilla*

Photo by arcoss / iStock