**Meet the editor**

Samir A. Farghaly is a Physician / Scientist and expert in Obstetrics and Gynecology at Joan and Sanford I. Weill College of Medicine, and the New York Presbyterian Hospital / Weill Cornell Medical Center, Cornell University, New York, USA. He received his M.D. and his PhD degree in molecular biology from London University. Dr. Farghaly received several national and international

clinical and research awards. He has been an invited speaker at several national and international conferences on Women's health, Molecular genetic of female cancers, and Gynecological cancer and oncologic radical surgical techniques. Dr. Farghaly is a member of several national and international societies, organizations, and foundations of Women health and cancer. He is the founder and Editor-in Chief of Enliven: Challenges in Cancer Detection and Therapy Journal, Current Trends in Gynecologic Oncology, and The International Journal of Gynecological, Obstetrical and Reproductive Medicine Research. He acts as Senior Editor / Editor and member of editorial boards, editorial advisory boards of 18 international medical journals on Gynecological Cancers, Gene Expression and Therapy, Women's Health and Gynecology. Dr. Farghaly has published 99 articles in reputed peer review journals, written several book chapters, and is an author and editor of 4 books.

## Contents

#### **Preface XI**


Carmen Rodríguez-Soriano, María-Luz Pombo, Earl Julie and Samir A. Farghaly

Chapter 6 **Ovarian Cancer Research in the Post Genomic Era — Challenges and Opportunities 149** Alicia A. Goyeneche and Carlos M. Telleria


## Preface

Chapter 7 **Analysing Molecular Mechanism Related to Therapy-**

Chapter 8 **Recurrent Ovarian Cancer — Basic Knowledge, Current Management, and Future Directions 189**

Chapter 9 **Management of Ovarian Cancer — Is There a Role for Cytoreductive Surgery (CRS) and Hyperthermic Intraperitoneal Chemotherapy (HIPEC)? 209** Melissa Ching Ching Teo and Grace Hwei Ching Tan

Chapter 10 **Anti-Angiogenic and Anti-Cancer Effects by Targeting the**

**Proteins, C-IAP1, Livin and Survivin 221**

Chapter 13 **Cervical Cancer in Human Immunodeficiency Virus (HIV)**

Chapter 15 **Adenocarcinoma of the Endometrium — The Art of Its**

Chapter 14 **Sentinel Lymph Node Detection in Early Stage**

**Protein Kinase G Type-Iα (PKG-Iα) Signaling Pathway and its Downstream Effects on Expression of Inhibitor of Apoptosis**

Janica C. Wong, Priyatham Gorjala, Benjamin Costantino and

Fernando Anschau and Manoel Afonso Guimarães Gonçalves

Mark Williams, John Villeneuve and Bernice Robinson-Bennett

Elisa Moreno-Palacios, Elsa Delgado, Javier De Santiago and Ignacio

Manoel Afonso Guimarães Gonçalves and Fernando Anschau

Valeri

**VI** Contents

Bernardo L. Rapoport

Ronald R. Fiscus

Chapter 11 **Cancer of the Vulva — A Review 237**

Chapter 12 **Robotic Surgery in the Management of Endometrial Cancer 253**

**Positive Patients 277**

**Cervical Cancer 295**

Atara Ntekim

Zapardiel

**Diagnosis 309**

**Resistance in In-vitro Models of Ovarian Cancer 167**

Jens C. Hahne, Arnd Honig, Jörg B. Engel, Andrea Lampis and Nicola

Worldwide, the number of new gynecological cancer cases in 2012, was 1,085,900 and 417,600 deaths from all cancers of the female genital tract. In the USA in 2015, there were 98,280 women diagnosed with gynecologic cancers and 30,440 died from it. In the United States, ovarian cancer is the leading cause of gynecologic cancer–related morbidity and mor‐ tality due to the difficulty in detecting early-stage disease. Early recognition of symptoms and regular gynecologic screening are the best tools for women to maintain gynecologic health. Over the last 10 years or so, the treatment of gynecologic cancers has evolved. New scientific and clinical advances have modified the standard of care and led to improved pa‐ tient oncologic outcomes. The treatment of gynecologic cancers requires comprehensive re‐ view and assessment of multiple issues including genetics, radiology, surgery, molecular diagnostics, and chemotherapy. A multidisciplinary team approach is crucial in providing the best care to patients and ensuring successful treatment, and optimal quality of life.

The purpose of this book is to provide a broad background of several aspects of basic scien‐ ces, clinical, and therapeutic aspects of gynecological cancers. It provides state-of-the-art in‐ formation on the molecular genetics and biology of cancers of the female genital tract and new approaches to its diagnosis and management. Better understandings of the molecular events that underlie gynecologic cancers development are very much needed.

The contributors of this book come from several renowned academic medical institutions in the USA, Germany, Italy, Spain, Romania, South Africa, Brazil, Singapore, India and Nigeria.

The role of BRCA1 gene mutation and EMR protein in the development of metastatic breast cancer is discussed in Chapter 1. The interaction between genetic and epigenetic alterations leading to the malignant phenotype presentation in gynecological cancers is presented in Chap‐ ter 2. The anti-angogenic therapy as a targeted molecular therapy for epithelial ovarian cancer is covered in Chapter 3. The comprehensive review and in depth analysis of peritonectomy and hyperthermic intraperitoneal chemotherapy (HIPEC), in patients with locally advanced epi‐ thelial ovarian cancer is discussed in Chapter 4. The molecular aspects, and the targeted molec‐ ular therapy of patients with BRCA1 and BRCA2 genes hereditary ovarian cancer is reviewed in Chapter 5. The outcome of the cancer Genome Atlas (TCGA) related to high grade serous ovari‐ an cancer (HGSOG) in reference to redefinition of ovarian cancer pathology, its propagation pattern, and the characteristics of histological subtypes of epithelial ovarian cancer is reviewed In Chapter 6. Overview of the PI3K/AKT/ mTOR signaling network in ovarian and endometrial cancer, in addition to the rationale for targeting this pathway is discussed in Chapter 7. Clinical, current management, and future directions in research related to recurrent ovarian cancer is covered in Chapter 8. The role of cytoreductive surgery and hyperthermic intraperitoneal che‐ motherapy in patients with recurrent ovarian cancer, who have peritoneal metastasis is pre‐ sented in Chapter 9. The anti-angiogenic effects by targeting the protein kinase G type- I- alpha signaling pathway and its downstream effects on expression of inhibition of apoptosis proteins in epithelial ovarian cancer is reviewed in Chapter 10. Analysis of risk factors, diagnostic meth‐ ods, and characteristics of women with endometrial cancer, and recommended current innova‐ tive therapy is presented in Chapter 11. The conventional surgical management of endometrial cancer, advantages of robotic surgery in those patients, comparison of outcome between open surgery, laparoscopic, and assisted robotic surgery for this disease is reviewed in Chapter 12. The pathophysiogy of Human Immunodeficiency Virus infection, clinical aspects, and man‐ agement of uterine cervical cancer in HIV positive patients is reviewed in Chapter 13. The feasi‐ bility of sentinel lymph node detection in uterine cervical cancer, its techniques and clinical advantages are discussed in Chapter 14. Clinical presentation, histopathological classification, prognosis, recurrence factors and treatment of cancer of the vulva is reviewed in Chapter 15.

This book volume is intended for all clinicians and basic medical scientists caring for women with gynecologic cancers, including attending surgeons and physicians, clinical fellows, and residents in the disciplines of gynecologic oncology, radiotherapy, surgical oncology, medi‐ cal oncology, and primary care. Also, PhD students and post-doctoral fellows in basic medi‐ cal sciences.

I would like to acknowledge the assistance of Janell Mensinger, PhD of the University of Drexel, Philadelphia, USA for her valuable contribution to the book, in reviewing the bio‐ statistical data of some of the book chapters.

I hope that you find this book very useful, and benefit from the extensive experience of the knowledgeable team of contributors who have authored its contents.

This book is dedicated to my beloved children Raied and Tamer, and the memory of my mother Amina, and father Aly, who had a great influence on me, and my academic and pro‐ fessional medical career. Also, to my sisters Sorya, Nadia, and brother Rafat and their fami‐ lies, my late siblings Nabil, and Magdy and their families. In addition to my late nephew, Islam.

#### **Samir A. Farghaly, M.D., PhD**

Joan and Sanford I. Weill Medical College / The Graduate School of Medical Sciences The New York Presbyterian Hospital / Weill Cornell Medical Center, Cornell University, New York, NY, USA

**Chapter 1**

## **Role of BRCA1 in Breast Cancer Metastasis**

sented in Chapter 9. The anti-angiogenic effects by targeting the protein kinase G type- I- alpha signaling pathway and its downstream effects on expression of inhibition of apoptosis proteins in epithelial ovarian cancer is reviewed in Chapter 10. Analysis of risk factors, diagnostic meth‐ ods, and characteristics of women with endometrial cancer, and recommended current innova‐ tive therapy is presented in Chapter 11. The conventional surgical management of endometrial cancer, advantages of robotic surgery in those patients, comparison of outcome between open surgery, laparoscopic, and assisted robotic surgery for this disease is reviewed in Chapter 12. The pathophysiogy of Human Immunodeficiency Virus infection, clinical aspects, and man‐ agement of uterine cervical cancer in HIV positive patients is reviewed in Chapter 13. The feasi‐ bility of sentinel lymph node detection in uterine cervical cancer, its techniques and clinical advantages are discussed in Chapter 14. Clinical presentation, histopathological classification, prognosis, recurrence factors and treatment of cancer of the vulva is reviewed in Chapter 15. This book volume is intended for all clinicians and basic medical scientists caring for women with gynecologic cancers, including attending surgeons and physicians, clinical fellows, and residents in the disciplines of gynecologic oncology, radiotherapy, surgical oncology, medi‐ cal oncology, and primary care. Also, PhD students and post-doctoral fellows in basic medi‐

I would like to acknowledge the assistance of Janell Mensinger, PhD of the University of Drexel, Philadelphia, USA for her valuable contribution to the book, in reviewing the bio‐

I hope that you find this book very useful, and benefit from the extensive experience of the

This book is dedicated to my beloved children Raied and Tamer, and the memory of my mother Amina, and father Aly, who had a great influence on me, and my academic and pro‐ fessional medical career. Also, to my sisters Sorya, Nadia, and brother Rafat and their fami‐ lies, my late siblings Nabil, and Magdy and their families. In addition to my late nephew,

Joan and Sanford I. Weill Medical College / The Graduate School of Medical Sciences

The New York Presbyterian Hospital / Weill Cornell Medical Center,

**Samir A. Farghaly, M.D., PhD**

Cornell University, New York, NY, USA

knowledgeable team of contributors who have authored its contents.

cal sciences.

VIII Preface

Islam.

statistical data of some of the book chapters.

S. Satheesh Kumar, K.H. Sreelatha, Revathy Nadhan and Priya Srinivas

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60787

#### **Abstract**

The role of BRCA1 in breast cancer metastasis is a less explored area that might have importance in increased aggressiveness of BRCA1 defective triple negative cancers. The possible influence of BRCA1 on apico basal polarity and ezrin, radixin and meosin (ERM) proteins are discussed in this review as a reason for cell metastasis. This might help in developing antimetastatic drugs that could help for better prognosis in BRCA1 defective breast cancers.

**Keywords:** BRCA1, ezrin, radixin, moesin, apicobasal polarity

#### **1. Introduction**

Breast cancer is the second largest cancer and the fifth major cause of death. There are many factors involved in breast cancer development and metastasis. Among the tumor suppressors that play a critical role in hereditary breast cancers, BRCA1 mutations are highly frequent, whereas loss of BRCA1 expression by promoter hyper methylation or allelic loss has frequently been noted in sporadic breast cancer [1, 2]. Mutation or loss of the functional BRCA1 expression in breast cancer is usually accompanied with TP53 mutations, ER/PR/HER2 negativity, and loss of ATM/CHK2, which, in turn, leads to a highly aggressive basal phenotype, which clearly possesses a therapeutic challenge [2, 3]. Specific malignant changes caused by BRCA1 muta‐ tions in the breast and the ovary remain a mystery till date.

© 2016 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. BRCA1**

BRCA1 is a multifunctional protein that is well known to be involved in multiple cellular processes by shuttling between nucleus and cytoplasm. Structurally, BRCA1 has three domains: (a) the RING domain; (b) the serine cluster domain (SCD); and (c) the BRCT domain. Potentially, four mutations are considered to be deleterious (5382insC, 5396 + 1G>A, 185delAG, and 2288delT) in the BRCA1 gene among the many mutations reported [4, 5]. The tumor suppressor function of BRCA1 is mainly attributed to the RING and BRCT domains of BRCA1, as women with hereditary breast cancer mainly possess mutations in one of the two domains.

Functionally, the RING domain of BRCA1 along with BARD1 possesses E3-ligase-mediated tumor suppressor activity, and any mutation in this domain would severely affect the heterodimerization and the stability of BRCA1 and BARD1, which, in turn, affects the tumor suppressor activity of BRCA1 [5]. The C-terminal BRCT domain is a phospho-protein binding domain known to interact with several partners and is reported to be critical for the localization of BRCA1 at the DNA damage site [6]. Furthermore, the tumor suppressor activity of the BRCT domain of BRCA1 has been reported using mouse models, although the exact mechanism is still debatable [7]. In short, functionally, BRCA1 is known to regulate multiple cellular processes such as DNA double strand repair, check point regulation, ubiquitination, and transcriptional regulation.

The RING domain, discovered as Really Interesting New Gene, spans from exon 2–7 of 24 exons of the BRCA1 gene. The RING domain of BRCA1 with a ring finger consists of seven cysteine and one histidine residues critically coordinate with Zn atoms, which actually stabilizes the RING structure [8, 9]. BARD1, a protein that is structurally homologous to the BRCA1 RING domain, interacts with the RING domain of BRCA1 and is critically important for the ubiquitin ligase activity, and it is reported to ubiquitinate several target proteins for degradation such as ER alpha, progesterone receptor, histone H2A, and CtIP [10-12]. It also modulates the nuclear import and export of BRCA1 [13, 14].

The core domain, which spans exons 11–13, is the largest domain of BRCA1 and is often called the serine cluster domain (SCD). It has two nuclear localization signals (NLSs), which control the nuclear import of BRCA1. Numerous proteins are reported to interact with this domain, and some of the notable binding partners are the retinoblastoma protein (RB), cMyc, PALB2, Rad50, and Rad51. The interaction between BRCA1 and RB is critically important for the BRCA1-mediated cell cycle regulation, as mutation in the binding region of BRCA1 failed to arrest the cell cycle progression [15]. PALB2, RAD50, and RAD51 interactions with BRCA1 are crucial for the BRCA1-mediated DNA repair. RAD50 and RAD51 mainly play a role in both homologous recombination (HR) and nonhomologous end joining (NHEJ) mediated by BRCA1, whereas PALB2 plays a role mainly in HR [16-18]. Mutations in any of the binding portions of this molecule severely affect the DNA repair capacity of BRCA1. Furthermore, BRCA1 is known to regulate the transcriptional activity of few oncogenic proteins reported till date. The well-studied example is that BRCA1 is known to downregulate the oncogenic transcriptional factor cMyc [19]. In addition, the serine clusters in this domain are reported to be phosphorylated by several kinases, including ATM/ATR during DNA damage, and this phosphorylation is mandatory for the assembly of BRCA1 to the DNA damage site, and again, any mutation affecting the phosphorylation of BRCA1 could severely affect the DNA repair ability [20].

Finally, the BRCA1 C terminal domain (BRCT) is reported to modulate its interactions with phosphoproteins that are critically important for the tumor suppressor activity of BRCA1 in particular nuclear localization and assembly at the DNA damage sites [21]. Several mutations have been reported in the BRCA1 gene portion that interfere with several cellular processes, and sometimes, they can be highly lethal [22]. Interestingly some of the mutations in the BRCA1 gene portion (for example, C61G RING mutation) are hypomorphic, i.e., it does not lose its complete DNA repair ability but still maintains the residual unknown DNA repair mechanism [5, 23]. From the therapeutic point of view, BRCA1 mutations with residual tumor suppressor activity clearly pose a complexity in the treatment compared with BRCA1-proficient or BRCA1-deficient breast/ovarian tumors.

#### **3. BRCA1 defect and pathological condition**

**2. BRCA1**

transcriptional regulation.

BRCA1 is a multifunctional protein that is well known to be involved in multiple cellular processes by shuttling between nucleus and cytoplasm. Structurally, BRCA1 has three domains: (a) the RING domain; (b) the serine cluster domain (SCD); and (c) the BRCT domain. Potentially, four mutations are considered to be deleterious (5382insC, 5396 + 1G>A, 185delAG, and 2288delT) in the BRCA1 gene among the many mutations reported [4, 5]. The tumor suppressor function of BRCA1 is mainly attributed to the RING and BRCT domains of BRCA1, as women with hereditary breast cancer mainly possess mutations in one of the two domains. Functionally, the RING domain of BRCA1 along with BARD1 possesses E3-ligase-mediated tumor suppressor activity, and any mutation in this domain would severely affect the heterodimerization and the stability of BRCA1 and BARD1, which, in turn, affects the tumor suppressor activity of BRCA1 [5]. The C-terminal BRCT domain is a phospho-protein binding domain known to interact with several partners and is reported to be critical for the localization of BRCA1 at the DNA damage site [6]. Furthermore, the tumor suppressor activity of the BRCT domain of BRCA1 has been reported using mouse models, although the exact mechanism is still debatable [7]. In short, functionally, BRCA1 is known to regulate multiple cellular processes such as DNA double strand repair, check point regulation, ubiquitination, and

The RING domain, discovered as Really Interesting New Gene, spans from exon 2–7 of 24 exons of the BRCA1 gene. The RING domain of BRCA1 with a ring finger consists of seven cysteine and one histidine residues critically coordinate with Zn atoms, which actually stabilizes the RING structure [8, 9]. BARD1, a protein that is structurally homologous to the BRCA1 RING domain, interacts with the RING domain of BRCA1 and is critically important for the ubiquitin ligase activity, and it is reported to ubiquitinate several target proteins for degradation such as ER alpha, progesterone receptor, histone H2A, and CtIP [10-12]. It also

The core domain, which spans exons 11–13, is the largest domain of BRCA1 and is often called the serine cluster domain (SCD). It has two nuclear localization signals (NLSs), which control the nuclear import of BRCA1. Numerous proteins are reported to interact with this domain, and some of the notable binding partners are the retinoblastoma protein (RB), cMyc, PALB2, Rad50, and Rad51. The interaction between BRCA1 and RB is critically important for the BRCA1-mediated cell cycle regulation, as mutation in the binding region of BRCA1 failed to arrest the cell cycle progression [15]. PALB2, RAD50, and RAD51 interactions with BRCA1 are crucial for the BRCA1-mediated DNA repair. RAD50 and RAD51 mainly play a role in both homologous recombination (HR) and nonhomologous end joining (NHEJ) mediated by BRCA1, whereas PALB2 plays a role mainly in HR [16-18]. Mutations in any of the binding portions of this molecule severely affect the DNA repair capacity of BRCA1. Furthermore, BRCA1 is known to regulate the transcriptional activity of few oncogenic proteins reported till date. The well-studied example is that BRCA1 is known to downregulate the oncogenic transcriptional factor cMyc [19]. In addition, the serine clusters in this domain are reported to be phosphorylated by several kinases, including ATM/ATR during DNA damage, and this

modulates the nuclear import and export of BRCA1 [13, 14].

2 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

Clinically, BRCA1 is reported to be functional in different organs of the body apart from its cardinal role in the breast and the ovary. Recently, BRCA1 has been reported to play an immense role in brain development [24]. In addition, its role as a regulator of metabolic function in skeletal muscles has been reported [25]. It also plays a huge role in Alzheimer disease, although the exact role is still unclear [26]. Recently, BRCA1 has been reported to act as a transcriptional cofactor during HIV infection [27]; again, the evidence is still preliminary. The function of BRCA1 as a tumor suppressor is crucial in the breast and ovarian tissue, and mutations in BRCA1 usually predispose to breast or ovarian cancer as discussed earlier. Apart from that, BRCA1 mutations are also reported to develop cancers in the prostate, fallopian tube, peritoneal, and pancreas, the specificity being unclear [28, 29]. Acute myeloid leukemia and the Fanconi anemia subtype has been reported if BRCA1 mutations are inherited from both parents [30-32]. Although BRCA1 has been studied for 20 years, its multiple facets are still undiscovered to a larger extent, which makes BRCA1 the molecule of attraction in current research. The role of BRCA1 in metastasis is one of the novel functions evidenced very recently.

#### **4. BRCA1 in migration/invasion**

Although there are no evidencing reports regarding the role of BRCA1 in ovarian cancer metastasis, its role in breast cancer metastasis is clearly an emerging subject with a few reports. The RING and BRCT domain of BRCA1 has been reported to be critically important for controlling the cancer cell migration and motility in the breast or the ovary [7, 33], although the complete mechanism is ill understood. In addition, restoration of full-length BRCA1 in 3450delCAAG mutated breast cancer cells is reported to block the cell invasion and motility induced by that particular mutation [34]. Furthermore, BRCA1 has been implicated to play a key role in epithelial to mesenchymal transition, again the exact mechanism being unclear [35]. In this review, we discuss the possible mechanistic role of BRCA1 in the migration and invasion of BRCA1-defective breast tumors, which is less explored till date. Currently, there are two evident mechanisms through which BRCA1 could control the migration and invasion of breast cancers that will have immense potential in the futuristic breast cancer treatments. First, the mechanism deals with the role of BRCA1 in maintaining the apicobasal polarity. The second mechanism deals with the role of BRCA1 in regulating the ERM complex that maintain the cytoskeleton.

#### **4.1. BRCA1 and apicobasal polarity**

Apicobasal polarity is a unique polarity feature of the epithelial cells that refers to the apical membrane on one side and the basolateral membrane on other side, separated by tight junctions. It is a critical feature of cytoskeletal reorganization in the epithelium of the breast and plays a key role in maintaining the integrity of cell-cell connections by maintaining the adherent junctions through microtubule organization [36]. In addition, it is known to be regulated by several signaling pathways such as WNT signaling, TGFβ, and integrin-mediated signaling. Furthermore, it is critically important for the differentiation of the breast epithelium, whereas the loss of epithelial polarity is often considered a hallmark of EMT and cancer [37-39]. Frequently, the loss of expression or mislocalization of the molecules of polarity complex such as SCRIB, Crumbs, and PAR has been implicated in the carcinogenesis of the breast [40-42]. Recently, BRCA1 has been reported to play a key role in the cytoskeletal organization and polarity of the breast tissue [43]. Probably, the loss of polarity in BRCA1-mutated breast tumors results in the loss of cell–cell adhesion and, hence, the movement of cancer cells from the primary to the distant site.

Mechanistically, BRCA1 regulates the polarity and, hence, the differentiation of breast cancer cells by regulating the expression of Hyaluronan-Mediated Motility Receptor (HMMR), a lowpenetrance breast cancer susceptibility gene product that is usually over expressed in BRCA1 related tumors and results in poor prognosis [43-45]. The early report comes from a linkage association study where the genetic variation at chromosome 5q33–34, which is actually the gene location of HMMR, is clearly associated with the risk of breast cancer among BRCA1 mutation carriers [46]. Furthermore, it was confirmed by a pilot study conducted in Spain and Italy, where HMMR rs299290 variation among BRCA1 mutation carriers clearly posed a risk of breast cancer [47]. In addition, BRCA1-related breast cancers, which are generally ER negative but not ER positive, are associated with the HMMR genetic variation. Further knockdown of BRCA1 has clearly impaired the polarization by modulating the cytoskeletal components and its organization. For instance, the cytoskeletal molecule vimentin is increased, and CCD49f is decreased upon BRCA1 knockdown. Maxwell et al. (2011) have shown that BRCA1, through the non-centrosome-dependent assembly of microtubules, maintains the polarity of the breast epithelium and the loss of BRCA1 clearly impair the cytoskeletal reorganization, as observed by increased levels of intermediate filament proteins such as vimentin. Furthermore, BRCA1 is reported to maintain the polarization of breast epithelium by directing the proteosome-mediated degradation of the BRCA1 target, HMMR [47]. Sup‐ porting evidence shows that proteosome inhibition and BRCA1 depletion clearly induced the expression of HMMR, which might be the probable reason why an overexpression of HMMR and polarity loss is frequently observed in BRCA1-related breast cancer than BRCA1-unrelated breast cancer [47]. Further accumulation of microtubule-associated factors TUBG1 by HMMR at the centromere in BRCA1-mutated breast tumors was reported to have impaired the polarity and hence induced the basal phenotype [43, 48]. Overexpression of TUBG1 and HMMR has clearly impaired the polarization, even in the presence of BRCA1, suggesting that the upre‐ gulation of microtubule-associated factors together with the depletion or mutation of BRCA1 and proteosome inhibition is the prime event in the loss of polarity in BRCA1-related breast tumors. Further overexpression of Aurora kinase A (AURKA) is reported to regulate the HMMR-mediated polarity loss, and HMMR is shown to negatively regulate AURKA. The depletion of AURKA is also known to reduce the abundance of HMMR, and the abundance is restored to the normal level in AURKA- and BRCA1-depleted conditions [43]. Clearly, a strict balance exists between BRCA1, HMMR, and AURKA, and probably, the polarity is completely dependent on the interactions between these molecules.

PAR is a polarity complex of par3, par6, and aPKC known to regulate cell plasticity by localizing at the tight junction [14]. Par6 is critically regulated by TGFβ signaling, and its misregulation leads to the highly aggressive breast tumorigenesis. Further correlation of the par6 expression and BRCA1 mutation has recently been reported. Although no direct regula‐ tion has been established between par6 and BRCA1, par6 has been shown to be over expressed in BRCA1-mutated breast tumors, which, in turn, have been linked with the high expression of basal markers such as cytokeratin 5/14 and vimentin. Alternatively, a positive association was reported between the activation of PAR6 pathway and the expression of basal cytokeratins in BRCA1-mutated breast tumors [40, 49, 50].

Starita et al. (2004) have shown that BRCA1 inhibits the expression of gamma tubulin by direct ubiquitination and is reported to maintain the centrosome number, and probably, the mutation in BRCA1 has readily increased the tubulin expression and polymerization and, hence, might induce the metastasis of breast cancer cells.

It clearly shows that BRCA1 sustains the polarity of breast cancer cells by maintaining a tight regulation with centrosome pathway components and the loss of BRCA1 in BRCA1-mutated breast tumors, leading to impaired polarization, which, in turn, results in the basal-like phenotype of breast cancer cells. Furthermore, the loss of polarity induces the EMT process [51-53], which might promote the migration and invasion of BRCA1-related breast cancer cells. Here comes the question of how the cancer cell migrates in a condition where BRCA1 is a wild type. Probably, the epigenetic silencing of BRCA1 as reported in many sporadic breast tumors might prevail in such situations, which needs future experimentations.

#### **4.2. BRCA1 and ERM complex**

key role in epithelial to mesenchymal transition, again the exact mechanism being unclear [35]. In this review, we discuss the possible mechanistic role of BRCA1 in the migration and invasion of BRCA1-defective breast tumors, which is less explored till date. Currently, there are two evident mechanisms through which BRCA1 could control the migration and invasion of breast cancers that will have immense potential in the futuristic breast cancer treatments. First, the mechanism deals with the role of BRCA1 in maintaining the apicobasal polarity. The second mechanism deals with the role of BRCA1 in regulating the ERM complex that maintain the

Apicobasal polarity is a unique polarity feature of the epithelial cells that refers to the apical membrane on one side and the basolateral membrane on other side, separated by tight junctions. It is a critical feature of cytoskeletal reorganization in the epithelium of the breast and plays a key role in maintaining the integrity of cell-cell connections by maintaining the adherent junctions through microtubule organization [36]. In addition, it is known to be regulated by several signaling pathways such as WNT signaling, TGFβ, and integrin-mediated signaling. Furthermore, it is critically important for the differentiation of the breast epithelium, whereas the loss of epithelial polarity is often considered a hallmark of EMT and cancer [37-39]. Frequently, the loss of expression or mislocalization of the molecules of polarity complex such as SCRIB, Crumbs, and PAR has been implicated in the carcinogenesis of the breast [40-42]. Recently, BRCA1 has been reported to play a key role in the cytoskeletal organization and polarity of the breast tissue [43]. Probably, the loss of polarity in BRCA1-mutated breast tumors results in the loss of cell–cell adhesion and, hence, the movement of cancer cells from the

Mechanistically, BRCA1 regulates the polarity and, hence, the differentiation of breast cancer cells by regulating the expression of Hyaluronan-Mediated Motility Receptor (HMMR), a lowpenetrance breast cancer susceptibility gene product that is usually over expressed in BRCA1 related tumors and results in poor prognosis [43-45]. The early report comes from a linkage association study where the genetic variation at chromosome 5q33–34, which is actually the gene location of HMMR, is clearly associated with the risk of breast cancer among BRCA1 mutation carriers [46]. Furthermore, it was confirmed by a pilot study conducted in Spain and Italy, where HMMR rs299290 variation among BRCA1 mutation carriers clearly posed a risk of breast cancer [47]. In addition, BRCA1-related breast cancers, which are generally ER negative but not ER positive, are associated with the HMMR genetic variation. Further knockdown of BRCA1 has clearly impaired the polarization by modulating the cytoskeletal components and its organization. For instance, the cytoskeletal molecule vimentin is increased, and CCD49f is decreased upon BRCA1 knockdown. Maxwell et al. (2011) have shown that BRCA1, through the non-centrosome-dependent assembly of microtubules, maintains the polarity of the breast epithelium and the loss of BRCA1 clearly impair the cytoskeletal reorganization, as observed by increased levels of intermediate filament proteins such as vimentin. Furthermore, BRCA1 is reported to maintain the polarization of breast epithelium by directing the proteosome-mediated degradation of the BRCA1 target, HMMR [47]. Sup‐

cytoskeleton.

**4.1. BRCA1 and apicobasal polarity**

4 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

primary to the distant site.

Ezrin, radixin, and moesin, together known as ERM, are three functionally homologous adapter proteins consisting of an N-terminal FERM domain and a C-terminal ERM associated the F-actin-binding domain (C-ERMAD) that is linked to the N-terminal FERM domain through the intermediate alpha helical region. Activation of ERM has been reported as an important process in the functioning of ERM. ERM remains in the closed conformation until it is activated by the phosphorylation of threonine residues in ezrin, moesin, and radixin [54]. Activated ERM helps in linking the actin cytoskeleton to the plasma membrane through the FERM and F-actin-binding domain [55]. Further activated ERM is reported to interact with transmembrane proteins such as receptor kinases, CD43, and CD44 [56]. Functionally, ERM has been critically implicated in the normal physiological as well as in the cancer conditions. In particular, ERM is known to be involved in three key events: (a) epithelial morphogenesis; (b) migration; and (c) adhesion. Changes in the above-mentioned events are observed during cancer, and it is a clear indication of cancer. Basically, polarity is maintained by ERM in normal physiological conditions and the overexpression of ERM during cancerous conditions leads to a more mesenchymal nature of the cells, and hence, it promotes the event of metastasis by probably interacting with EGFR, CD44, and HGFR [57, 58].

Abnormal expression and localization of ERM has been reported in different types of cancer, and it is clearly known to regulate the cellular signaling and the cytoskeleton during cancer progression, which, in turn, affects the migration and motility behavior of cancer cells. The ERM complex is known to be directly or indirectly phosphorylated by many kinases, which, in turn, activate many signaling pathways involved in cell adhesion, migration, morphology, and proliferation during tumorigenesis [58, 59]. Further overexpression of ERM molecules together or individually was reported to be a clear indication of the EMT process [60, 61]. All the above-mentioned activities of ERM were also highly pronounced in breast cancer [62-64]. In addition, moesin was associated with poor relapse-free survival in breast cancer patients. Although the role of ERM in breast cancer progression was well studied over the years, its relation with BRCA1 during metastasis was ill understood, with only a few recent reports [33]. ERM has been reported to be associated with the ER-negative basal phenotype, and the expression of ERM was also reported to be high in BRCA1-related basal breast tumors compared with BRCA1-unrelated or sporadic breast tumors [65], which, in turn, could contribute to the migration and invasion of cancer cells. Recently, its relation with BRCA1 during migration has been revealed in breast cancer cell lines. Although ERM acts through multiple pathways to promote cancer cell migration and invasion, the presence or absence of BRCA1 was found to be highly significant in ERM-mediated cell motility and migration of breast cancer cells [33]. As previously discussed, the tumor suppressor activity of BRCA1 mainly lies in the BRCT domain, as the mutation of BRCA1 leading to the expression of truncated protein is frequently associated with breast and ovarian cancers [7]. Interestingly, BRCA1 was found to localize at the leading edges and focal adhesion sites of the plasma membrane and reported to control the breast cancer cell spreading and cell motility [33]. Furthermore, the BRCT domain of wild-type BRCA1 was found to co-localize with F-actin, ezrin, moesin, and radixin in the plasma membrane of breast cancer cells and hence controls the breast cancer cell motility in an unknown manner. In addition, a detailed study on this will give an idea on the exact localization of BRCA1 in the plasma membrane and its contributions to inhibit metastasis. Further stable expression of the BRCT coding domain of BRCA1 in breast cancer cells was found to co-localize with ERM and F-actin along with wild-type BRCA1. The BRCT coding domain acts as a dominant negative factor by gradually displacing the endoge‐ nous wild-type BRCA1 at leading edges and focal adhesion sites, thus promoting the motility and migratory capacity of breast cancer cells. Probably, BRCA1, by interacting through its BRCT domain, might reduce the ERM protein levels by ubiquitinating it through the E3 ubiquitin ligase activity and hence reduce the motility of breast cancer cells. Alternatively, mutation in the BRCA1 gene at the BRCT domain fails to reduce the levels of ERM at the leading edges, and hence, the motility behavior of breast cancer cells was increased. In addition, this might be one of the reasons why ERM is highly overexpressed in BRCA1-related basal breast tumors than BRCA1-unrelated tumors [65]. It was also reported that not only the BRCT domain but also the E3 ubiquitin ligase activity of BRCA1 are required for complete tumor suppressor function [33], further supporting the above-mentioned speculation, although it was contra‐ dictory to previous reports. This will have immense potential in tumor metastasis in BRCA1 defective cancers which the researchers have overlooked. Further studies are warranted to elucidate the exact signaling pathways and the biological consequences associated with ERM in BRCA1-related and BRCA1-unrelated breast tumors.

important process in the functioning of ERM. ERM remains in the closed conformation until it is activated by the phosphorylation of threonine residues in ezrin, moesin, and radixin [54]. Activated ERM helps in linking the actin cytoskeleton to the plasma membrane through the FERM and F-actin-binding domain [55]. Further activated ERM is reported to interact with transmembrane proteins such as receptor kinases, CD43, and CD44 [56]. Functionally, ERM has been critically implicated in the normal physiological as well as in the cancer conditions. In particular, ERM is known to be involved in three key events: (a) epithelial morphogenesis; (b) migration; and (c) adhesion. Changes in the above-mentioned events are observed during cancer, and it is a clear indication of cancer. Basically, polarity is maintained by ERM in normal physiological conditions and the overexpression of ERM during cancerous conditions leads to a more mesenchymal nature of the cells, and hence, it promotes the event of metastasis by

Abnormal expression and localization of ERM has been reported in different types of cancer, and it is clearly known to regulate the cellular signaling and the cytoskeleton during cancer progression, which, in turn, affects the migration and motility behavior of cancer cells. The ERM complex is known to be directly or indirectly phosphorylated by many kinases, which, in turn, activate many signaling pathways involved in cell adhesion, migration, morphology, and proliferation during tumorigenesis [58, 59]. Further overexpression of ERM molecules together or individually was reported to be a clear indication of the EMT process [60, 61]. All the above-mentioned activities of ERM were also highly pronounced in breast cancer [62-64]. In addition, moesin was associated with poor relapse-free survival in breast cancer patients. Although the role of ERM in breast cancer progression was well studied over the years, its relation with BRCA1 during metastasis was ill understood, with only a few recent reports [33]. ERM has been reported to be associated with the ER-negative basal phenotype, and the expression of ERM was also reported to be high in BRCA1-related basal breast tumors compared with BRCA1-unrelated or sporadic breast tumors [65], which, in turn, could contribute to the migration and invasion of cancer cells. Recently, its relation with BRCA1 during migration has been revealed in breast cancer cell lines. Although ERM acts through multiple pathways to promote cancer cell migration and invasion, the presence or absence of BRCA1 was found to be highly significant in ERM-mediated cell motility and migration of breast cancer cells [33]. As previously discussed, the tumor suppressor activity of BRCA1 mainly lies in the BRCT domain, as the mutation of BRCA1 leading to the expression of truncated protein is frequently associated with breast and ovarian cancers [7]. Interestingly, BRCA1 was found to localize at the leading edges and focal adhesion sites of the plasma membrane and reported to control the breast cancer cell spreading and cell motility [33]. Furthermore, the BRCT domain of wild-type BRCA1 was found to co-localize with F-actin, ezrin, moesin, and radixin in the plasma membrane of breast cancer cells and hence controls the breast cancer cell motility in an unknown manner. In addition, a detailed study on this will give an idea on the exact localization of BRCA1 in the plasma membrane and its contributions to inhibit metastasis. Further stable expression of the BRCT coding domain of BRCA1 in breast cancer cells was found to co-localize with ERM and F-actin along with wild-type BRCA1. The BRCT coding domain acts as a dominant negative factor by gradually displacing the endoge‐ nous wild-type BRCA1 at leading edges and focal adhesion sites, thus promoting the motility and migratory capacity of breast cancer cells. Probably, BRCA1, by interacting through its BRCT domain, might reduce the ERM protein levels by ubiquitinating it through the E3

probably interacting with EGFR, CD44, and HGFR [57, 58].

6 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

#### **5. Screening and diagnosis of BRCA1 mutated breast and ovarian cancers**

The major annoying fact about BRCA1/2 mutation is that the inheritance of BRCA1/2 mutation increases the risk for breast cancer by about 20–25% [66, 67]. Women who inherit BRCA1 mutation have 55–65% risk of getting breast cancer [68, 69]. In addition, BRCA1 mutations are quite frequent among a particular ethnic population; e.g. Ashkenazi Jews have a high preva‐ lence of BRCA1 mutation than any other population. Particularly, 2288delT and 5382insC mutation in the gene portion of BRCA1 is highly prevalent in Ashkenazi Jews, with a frequency of 1.1% and 0.1–0.15%, respectively [22, 70]. High prevalence has also been reported among the Dutch and Norwegian populations. In addition, the prevalence highly varies within the population, e.g., the US population based on their different ethnic origin [22, 71, 72]. Early clinical breast examination is the best possible method of diagnosing and treating breast tumors [73]. There are many screening tools in the current scenario that particularly assess the family history and its probable association with BRCA1 mutation [74]. However, the screening is mainly recommended for those who have a family history of breast/ovarian cancers [74]. The other specific tissue where inherited BRCA1 mutations usually predispose cancer is the ovary. Estimated data show that women who inherit BRCA1 mutations have 39% of devel‐ oping ovarian cancer [68, 69]. Women with Ashkenazi Jewish heritage or familial history of breast cancer have increased risk of three to six times than the general public, and women with BRCA1 mutations have more than six times greater risk than the general population to develop ovarian cancer. Screening is usually done by analyzing serum markers such as CA-125 and/or transvaginal ultrasound, and in the case of BRCA1-related ovarian cancer, the screening starts early at the age of 30.

#### **6. Management and therapy of BRCA1 mutated cancers**

Surprisingly, a survival advantage for BRCA1 mutation carriers is growing now, although it is still under controversy. An improvement in the survival rate was observed in BRCA1 mutation carriers of the Ashkenazi ethnicity upon platinum-based chemotherapy compared with BRCA1 non-mutated patients [75]. In addition, many studies from different parts of the world have substantiated the survival advantage among BRCA1 mutation carriers of ovarian origin, although the exact reason is not yet clear [76-78]. However, there are reports which indicates in the case of breast cancer, BRCA1 mutation does not pose any survival advantage; instead, it poses a clear challenge to chemotherapy. In the treatment point of view, the hormone therapy usually fails as BRCA1-mutated breast and ovarian cancer tends to be triple negative in general. The commonly used drugs are either insensitive or have developed resistance in BRCA1-mutated conditions. The only promising drug that is effective in treating BRCA1 mutated breast and ovarian cancer is the PARP inhibitor. The well-studied PARP inhibitor, Olaparib, has been found to be effective in treating BRCA1-mutated breast, ovarian, pancreatic, and prostate cancer. The loss of DNA repair by homologous recombination in BRCA1-mutated conditions activates the alternate method of single-strand DNA repair by poly(ADP-ri‐ bose)polymerase [79-81]. The rescue of DNA repair by PARP clearly imposes a chemothera‐ peutic challenge, and the inhibition of PARP during this condition has improved the benefit rate by 63% [82, 83]. Although it is not completely evaluated as a drug for treating BRCArelated cancers, significant clinical activity has been demonstrated in BRCA1-mutated breast and ovarian cancer during phase trials. There was suspicion that the PARP inhibitor alone or in combination could be an effective drug alternative in treating BRCA1-mutated breast and ovarian cancer. However, recent information has shown that the PARP inhibitor may not be clinically successful as drug resistance against the PARP inhibitor is also observed. As of now, we do not have an effective treatment for BRCA1-related cancers. Designing drugs considering BRCA1 interaction with metastasis-related proteins would be an effective strategy to treat BRCA1-related cancers.

#### **7. Conclusions**

It is very clear that BRCAl is a multifunctional protein that exerts its function from the nucleus to the cytoplasm to the plasma membrane. BRCA1, by controlling apicobasal polarity and by interacting with ERM proteins, is supposed to be involved in cancer cell metastasis. If the link between BRCA1 and migration/invasion is completely unraveled, then we could revolutionize the treatment modalities for controlling metastasis in BRCA1-defective breast tumors.

#### **Acknowledgements**

This work was supported by an intramural grant from the Rajiv Gandhi Centre for Biotech‐ nology, Kerala State Council for Science Technology and Environment (No. 016/SRSHS/2011/ CSTE), a grant-in-aid from the Board of Research in Nuclear Sciences *(No. 2009/37/5/BRNS/ 1620 and No.37(1)/14/16/2014)* and Indian Council of Medical Research (*No.3/2/2/215/2013/ NCD-III*) to P. S. The authors also acknowledge the University Grants Commission (UGC), Government of India, for the Senior Research Fellowship to Satheesh Kumar S., the Indian Council for Medical Research (ICMR), Government of India, for the Senior Research Fellow‐ ship to Sreelatha K H; and the Council for Scientific and Industrial Research (CSIR), Govern‐ ment of India, for the Senior Research Fellowship to Revathy Nadhan.

#### **Author details**

with BRCA1 non-mutated patients [75]. In addition, many studies from different parts of the world have substantiated the survival advantage among BRCA1 mutation carriers of ovarian origin, although the exact reason is not yet clear [76-78]. However, there are reports which indicates in the case of breast cancer, BRCA1 mutation does not pose any survival advantage; instead, it poses a clear challenge to chemotherapy. In the treatment point of view, the hormone therapy usually fails as BRCA1-mutated breast and ovarian cancer tends to be triple negative in general. The commonly used drugs are either insensitive or have developed resistance in BRCA1-mutated conditions. The only promising drug that is effective in treating BRCA1 mutated breast and ovarian cancer is the PARP inhibitor. The well-studied PARP inhibitor, Olaparib, has been found to be effective in treating BRCA1-mutated breast, ovarian, pancreatic, and prostate cancer. The loss of DNA repair by homologous recombination in BRCA1-mutated conditions activates the alternate method of single-strand DNA repair by poly(ADP-ri‐ bose)polymerase [79-81]. The rescue of DNA repair by PARP clearly imposes a chemothera‐ peutic challenge, and the inhibition of PARP during this condition has improved the benefit rate by 63% [82, 83]. Although it is not completely evaluated as a drug for treating BRCArelated cancers, significant clinical activity has been demonstrated in BRCA1-mutated breast and ovarian cancer during phase trials. There was suspicion that the PARP inhibitor alone or in combination could be an effective drug alternative in treating BRCA1-mutated breast and ovarian cancer. However, recent information has shown that the PARP inhibitor may not be clinically successful as drug resistance against the PARP inhibitor is also observed. As of now, we do not have an effective treatment for BRCA1-related cancers. Designing drugs considering BRCA1 interaction with metastasis-related proteins would be an effective strategy to treat

8 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

It is very clear that BRCAl is a multifunctional protein that exerts its function from the nucleus to the cytoplasm to the plasma membrane. BRCA1, by controlling apicobasal polarity and by interacting with ERM proteins, is supposed to be involved in cancer cell metastasis. If the link between BRCA1 and migration/invasion is completely unraveled, then we could revolutionize the treatment modalities for controlling metastasis in BRCA1-defective breast tumors.

This work was supported by an intramural grant from the Rajiv Gandhi Centre for Biotech‐ nology, Kerala State Council for Science Technology and Environment (No. 016/SRSHS/2011/ CSTE), a grant-in-aid from the Board of Research in Nuclear Sciences *(No. 2009/37/5/BRNS/ 1620 and No.37(1)/14/16/2014)* and Indian Council of Medical Research (*No.3/2/2/215/2013/ NCD-III*) to P. S. The authors also acknowledge the University Grants Commission (UGC), Government of India, for the Senior Research Fellowship to Satheesh Kumar S., the Indian Council for Medical Research (ICMR), Government of India, for the Senior Research Fellow‐

BRCA1-related cancers.

**Acknowledgements**

**7. Conclusions**


Cancer Research Program , Rajiv Gandhi Centre for Biotechnology3, Thycaud PO, Thiruvananthapuram, Kerala, India

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## **Interplay of Epigenetics with Gynecological Cancer**

Coralia Bleotu, Demetra Socolov, Mariana Anton, Anca Botezatu, Adriana Plesa, Iulia Virginia Iancu, Lorelei Irina Brasoveanu, Gabriela Anton and Carmen Cristina Diaconu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61032

#### **Abstract**

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Recent data on the cell deregulation that occurs during the progression to cancer un‐ derlines the cooperation between genetic and epigenetic alterations leading to a ma‐ lignant phenotype. Unlike genetic alterations, the epigenetic changes do not affect the DNA sequence of the genes, but determine the regulation of gene expression acting upon the genome. Moreover, unlike genetic changes, epigenetic ones are reversible, making them therapeutic targets in various conditions in general and in cancer dis‐ ease in particular. The term *epigenetics* includes a series of covalent modifications that regulate the methylation pattern of DNA and posttranslational modifications of histo‐ nes. Gene expression can also be regulated at the posttranscriptional level by micro‐ RNAs (miRNAs), a family of small noncoding RNAs that inhibit the translation of mRNA to protein. miRNAs can act as 'oncomiRs', as tumor suppressors, or both. In this chapter, we will (1) summarize the current literature on the key processes respon‐ sible for epigenetic regulation: DNA methylation, histone modifications and posttran‐ scriptional gene regulation by miRNAs; (2) evaluate aberrant epigenetic modifications as essential players in cancer progression; (3) establish the roles of microenvironmentmediated epigenetic perturbations in the development of gynecological neoplasia; (4) evaluate epigenetic factors involved in drug resistance.

**Keywords:** Epigenetic, biomarker, gynecological cancer

#### **1. Introduction**

#### **1.1. Key processes responsible for epigenetic regulation**

Epigenetics could be broadly defined as the sum of cellular and physiological trait variations that are not caused by changes in the DNA sequence. Epigenetic mechanisms are essential for

© 2016 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.

the normal development and maintenance of tissue-specific gene expression patterns in mammals. Disruption of epigenetic processes can lead to altered gene function resulting in imprinting disorders, developmental abnormalities and cancer. The epigenetic mechanisms that will be presented in this chapter are (1) DNA methylation, (2) chromatin and histone modifications, and (3) regulatory noncoding RNAs.

#### *1.1.1. DNA methylation*

DNA methylation is a biochemical process characterized by the addition of a methyl group especially at the C5 position of cytosine from CpG dinucleotides and is accomplished by two classes of DNA methyltransferases involved in maintenance and *de novo* methylation [1]. CpG dinucleotides are not randomly distributed across the human genome but are found in short CpG-rich DNA sequences called 'CpG islands.' CpG islands are found in regions of large repetitive sequences (e.g. centromeric repeats, retrotransposon elements, rDNA) [2, 3] and in 60% of human gene promoters [4]. Some CpG islands are methylated, whereas the majority of them usually remain unmethylated during development and in differentiated tissues [5]. CpG islands' promoters become methylated during development (imprinted genes, chromosome X inactivation) [2]. Another role of CpG island methylation is to silence noncoding DNA and transposable DNA elements to prevent chromosomal instability by heavy methylation of repetitive sequences [5]. DNA methylation leads to gene silencing by either preventing or promoting the recruitment of regulatory proteins to DNA. Methylation of CpG islands can block the access of transcription factors to the transcription sites [6, 7], or by recruiting methylbinding domain proteins (MBDs), which can mediate gene repression through interactions with histone deacetylases (HDACs) [8, 9]. This epigenetic modification does not change the DNA sequence, but enhances the stability and chromosome integrity and promotes genome organization into transcriptionally active or silenced regions. DNA methylation at the whole genome level provides a specific global methylation pattern [2, 10] that plays an important role in regulating gene expression (e.g. development and cell-specific gene expression) in associa‐ tion with chromatin-associated proteins. The maintenance of a cell-specific methylation pattern after every cellular DNA replication cycle provides a stable gene-silencing mechanism that plays an important role in regulating gene expression. The maintenance methyltransferase DNMT1 is responsible for copying DNA methylation patterns to the daughter strands during DNA replication, whereas DNMT3a and DNMT3b are *de novo* methyltransferases that establish the methylation patterns early in development [11]. DNMT3L, a homologous protein to other DNMT3s, increases the ability of DNM3a and 3b to bind to DNA, stimulating their activity. Some problems in the establishment of methylation biomarkers in gynecologic cancers, especially in cervical cancer [12], come from the fact that: (1) the extent of methylation across the various CpG sites in a promoter can be rather heterogeneous and consequently, the assay outcome is likely to be influenced by the region of CpGs that is targeted; (2) the distinct levels of background methylation due to differences in cell type composition between cervical tissue samples that can contain substantial amounts of nonepithelial (stromal) cells and cervical scrapings that are enriched in superficial epithelial cells. For this reason, the methylation results obtained from tissue samples may not be directly extrapolated to cervical scrapings [13]. In addition, while the methylation of tumor suppressor' promoters is an early and frequent alteration in carcinogenesis [14] and, on the other hand, is widespread in the human genome, only a subset of affected loci play critical roles in tumorigenesis [15]. CpG hyperme‐ thylation is gene- and cancer type–specific [16, 17, 18, 19], providing a useful signature for tumor diagnosis and prognosis [18] that must be established accurately.

#### *1.1.2. Covalent histone modifications*

the normal development and maintenance of tissue-specific gene expression patterns in mammals. Disruption of epigenetic processes can lead to altered gene function resulting in imprinting disorders, developmental abnormalities and cancer. The epigenetic mechanisms that will be presented in this chapter are (1) DNA methylation, (2) chromatin and histone

DNA methylation is a biochemical process characterized by the addition of a methyl group especially at the C5 position of cytosine from CpG dinucleotides and is accomplished by two classes of DNA methyltransferases involved in maintenance and *de novo* methylation [1]. CpG dinucleotides are not randomly distributed across the human genome but are found in short CpG-rich DNA sequences called 'CpG islands.' CpG islands are found in regions of large repetitive sequences (e.g. centromeric repeats, retrotransposon elements, rDNA) [2, 3] and in 60% of human gene promoters [4]. Some CpG islands are methylated, whereas the majority of them usually remain unmethylated during development and in differentiated tissues [5]. CpG islands' promoters become methylated during development (imprinted genes, chromosome X inactivation) [2]. Another role of CpG island methylation is to silence noncoding DNA and transposable DNA elements to prevent chromosomal instability by heavy methylation of repetitive sequences [5]. DNA methylation leads to gene silencing by either preventing or promoting the recruitment of regulatory proteins to DNA. Methylation of CpG islands can block the access of transcription factors to the transcription sites [6, 7], or by recruiting methylbinding domain proteins (MBDs), which can mediate gene repression through interactions with histone deacetylases (HDACs) [8, 9]. This epigenetic modification does not change the DNA sequence, but enhances the stability and chromosome integrity and promotes genome organization into transcriptionally active or silenced regions. DNA methylation at the whole genome level provides a specific global methylation pattern [2, 10] that plays an important role in regulating gene expression (e.g. development and cell-specific gene expression) in associa‐ tion with chromatin-associated proteins. The maintenance of a cell-specific methylation pattern after every cellular DNA replication cycle provides a stable gene-silencing mechanism that plays an important role in regulating gene expression. The maintenance methyltransferase DNMT1 is responsible for copying DNA methylation patterns to the daughter strands during DNA replication, whereas DNMT3a and DNMT3b are *de novo* methyltransferases that establish the methylation patterns early in development [11]. DNMT3L, a homologous protein to other DNMT3s, increases the ability of DNM3a and 3b to bind to DNA, stimulating their activity. Some problems in the establishment of methylation biomarkers in gynecologic cancers, especially in cervical cancer [12], come from the fact that: (1) the extent of methylation across the various CpG sites in a promoter can be rather heterogeneous and consequently, the assay outcome is likely to be influenced by the region of CpGs that is targeted; (2) the distinct levels of background methylation due to differences in cell type composition between cervical tissue samples that can contain substantial amounts of nonepithelial (stromal) cells and cervical scrapings that are enriched in superficial epithelial cells. For this reason, the methylation results obtained from tissue samples may not be directly extrapolated to cervical scrapings [13]. In addition, while the methylation of tumor suppressor' promoters is an early and

modifications, and (3) regulatory noncoding RNAs.

16 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

*1.1.1. DNA methylation*

Mammalian genome represents a highly structured complex comprised of compacted DNA and proteins that can adopt different three-dimensional conformations dependent of nuclear context and biochemical changes present in the genome and at the histone level [20]. At first glance, the chromatin is present in two forms: transcriptionally active euchromatin and more condensed and transcriptionally inactive heterochromatin. In the genome, there are some structural regions (such as centromeres) containing constitutive heterochromatin; others may go through an open conformation to a compact one—optional heterochromatin. These transitions, vital to the establishment of necessary transcriptional various models of embryonic development, growth, and adult life, are under epigenetic control. Nucleosomes form the repetitive fundamental units of the chromatin and are designed to pack the huge eukaryotic genome in the nucleus (mammalian cells contain approximately 2 m of linear DNA wrapped in a core size of 10 µm in diameter) [20]. The nucleosomes in turn are compacted and form the chromosomes. The nucleosomal core consists of approximately 147 base pairs wrapped around a histone octamer made up of two copies of the histones H2A, H2B, H3, and H4. Histone H1 (linker histone) and its isoforms are involved in chromatin compaction underlying nucleosome condensation. Decondensed nucleosomes look like a bead wrapping a DNA molecule [21]. Histone covalent modifications (epigenetic changes) represent important regulatory elements that influence chromatin interactions by structural changes either by electrostatic interactions and recruitment of nonhistone proteins [22].

Histones can undergo a variety of posttranslational modifications at the N-terminus (like acetylation, methylation, phosphorylation, sumoylation, ubiquitination, and ADP-ribosyla‐ tion) that can alter the DNA–histone interaction, with a major impact on chromatin structure and key cellular processes such as transcription, replication, and repair [20]. The histone code may be transient or stable. The mechanism of inheritance of this histone code is not fully understood. The patterns of histone modifications are specific to each cell type and play a key role in determining cellular identity [23, 24]. In contrast with stem cells, differentiated cells acquire a more rigid chromatin structure, which is important for maintaining cell specialization [23]. Epigenetic regulation mediated by histone modification is a dynamic process. Lysine residue methylation using histone methyltransferase (HMT) is correlated either with tran‐ scriptional activation or repression, whereas lysine acetylation correlates with transcriptional activation [25]. Histone methyltransferases (HMTs) and demethylases (HDMs) work in tandem to determine the degree of methylation of the lysine residue [26]. Histone H3 lysine 4 trimethylation (H3K4me3) correlates with euchromatin and gene transcription activation. Histone H3 lysine 27 trimethylation and/or lysine 9 (H3K27me3/H3K9me3) is correlated with the transcriptional repression of heterochromatin and H3K27me3 modification is critical for stem cells; demethylation at this level is correlated with differentiation [27, 28, 29, 30, 31]. These two modifications represent the main silencing mechanisms in mammalian cells, H3K9me3 working in concert with DNA methylation and H3K27me3 largely working exclusive of DNA methylation [32]. Histone acetylation is one of the histone modifications that have been studied extensively. The two homonymous enzymes that are involved in maintaining a specific profile are histone acetyltransferases (HATs) and histone deacetylases (HDACs) [26]. Generally, the level of histone acetylation correlated with transcriptional activation and deacetylation correlates with transcriptional repression. H3 histone acetylations at lysine 9 (H3K9ac) and lysine 4 to 16 are characteristic euchromatin changes located in regions where genes are actively transcribed. Although histone modifications act mainly by altering the architecture of some modifications (H3K4me3 and H3K9ac) mediates gene regulation by recruiting other proteins involved in chromatin remodeling [33, 34]. Histone modifications and DNA methyl‐ ation interact with each other at multiple levels to determine gene expression status, chromatin organization, and cellular identity [35]. Several HMTs, including G9a, SUV39H1, and PRMT5, methylate DNA to specific genomic targets recruiting DNA methyltransferases (DNMTs) [36, 37, 38]. In addition, DNMTs may recruit HDACs and methyl-binding proteins to achieve gene silencing and chromatin condensation [8, 9]. DNA methylation can also be established via H3K9 methylation, such as MeCP2, thereby establishing a repressive chromatin state [39]. Recent studies showed that the main chromatin changes that occurs during tumorigenesis are characterized by a global loss of acetylated H4 lysine 16 (H4K16ac) and H4 lysine 20 trime‐ thylation (H4K20me3) [40]. HDACs were found overexpressed in various types of cancer [41, 42] (becoming a major target for epigenetic therapy), along with HATs, whose expression can also be altered in cancer. MOZ, MORF, CBP, and p300 (HATs) may be targets for chromosomal translocations, especially in leukemia [43]. Changes in histone methylation patterns (deregu‐ lation of HMTs) are associated with aberrant gene silencing in cancer, and an effective cancer treatment strategy targeting HDMs represents a promising treatment option.

#### **1.2. Posttranscriptional gene regulation by noncoding RNAs**

Noncoding RNAs are involved in fundamental processes, such as chromatin dynamics and gene silencing, and their transcripts outnumber the group of protein transcripts. It is well known that the initiation of X-chromosome inactivation is regulated by noncoding RNAs (Xist function) and the noncoding RNAs molecules are also involved in imprinting, suggesting that antisense RNA can induce transcriptional silencing [44, 45, 46]. The characterized noncoding RNA family consists of a large group of small regulatory microRNAs (about 1400 microRNAs in humans) [47].MicroRNAs (miRNAs) are short noncoding RNAs of 20–24 nucleotides that play important roles in virtually all biological pathways in mammals like differentiation and growth control. Based on computer predictions, it was proposed that miRNAs may regulate many cell cycle control genes [48]. miRNAs influence numerous cancer-relevant processes such as proliferation, cell cycle control, apoptosis, differentiation, migration, and metabolism. The key processes of miRNA biogenesis pathways have been characterized. Primary miRNA transcripts are transcribed from separate transcriptional units or embedded within the introns of protein coding genes by RNA polymerase II. Primary miRNA transcripts are processed by a complex formed by RNase III enzyme and Drosha, resulting in a pre-miRNA hairpin that is subsequently exported from the nucleus to the cytoplasm by exportin 5 (XPO5). Further premiRNA molecules are processed by another protein complex, including DICER and TRBP, to produce the single-stranded mature miRNA (ssmiRNA). ssmiRNA is subsequently incorpo‐ rated in RNA induced silencing complex (RISC), along with key proteins such as AGO2 and GW182. The role of mature miRNA (as part of the RISC) is to induce posttranscriptional gene silencing by complementary sequence motifs to the target mRNAs predominantly found within the 3′ untranslated regions (UTRs) [47, 49, 50]. One specific miRNA may target up to several hundred mRNAs; therefore, a miRNA may silence various genes while a specific mRNA may be targeted by several miRNAs. Aberrant miRNA expression may interfere with gene transcription and influence cancer-related signaling pathways [51, 52, 53].New data are added to decipher the role of miRNAs in normal physiology and pathology. Several microar‐ ray expression studies performed on a wide spectrum of cancer types have proved that deregulated miRNAs expression is the rule rather than the exception in cancer [54, 55, 56, 57]. Animal models featuring miRNA overexpression or knock-down have demonstrated the relation between miRNAs and cancer development, thus proposing miRNAs as potential biomarkers and putative therapeutic targets [58]. In addition, since miRNAs were discovered, many researchers focused their interest on identifying miRNAs generated by viruses. Several data support this hypothesis mainly based on miRNA size, which allows them to avoid the immune system but also to be supported by the small size of viral genome. It is not unexpected that many miRNAs encoded by viruses have been discovered, most of them transcribed from double-stranded DNA viruses [59]. miRNAs can regulate the expression of viral genes that are involved in controlling viral replication. It is supposed that these miRNAs might influence viral gene expression in a differentiation-dependent manner by targeting viral transcripts. On the other hand, different hrHPV types have different oncogenic potentials, viral miRNA being considered one of the factors involved in oncogenic regulation; some conserved miRNAs are involved in the switch from HPV productive to transforming infections.

two modifications represent the main silencing mechanisms in mammalian cells, H3K9me3 working in concert with DNA methylation and H3K27me3 largely working exclusive of DNA methylation [32]. Histone acetylation is one of the histone modifications that have been studied extensively. The two homonymous enzymes that are involved in maintaining a specific profile are histone acetyltransferases (HATs) and histone deacetylases (HDACs) [26]. Generally, the level of histone acetylation correlated with transcriptional activation and deacetylation correlates with transcriptional repression. H3 histone acetylations at lysine 9 (H3K9ac) and lysine 4 to 16 are characteristic euchromatin changes located in regions where genes are actively transcribed. Although histone modifications act mainly by altering the architecture of some modifications (H3K4me3 and H3K9ac) mediates gene regulation by recruiting other proteins involved in chromatin remodeling [33, 34]. Histone modifications and DNA methyl‐ ation interact with each other at multiple levels to determine gene expression status, chromatin organization, and cellular identity [35]. Several HMTs, including G9a, SUV39H1, and PRMT5, methylate DNA to specific genomic targets recruiting DNA methyltransferases (DNMTs) [36, 37, 38]. In addition, DNMTs may recruit HDACs and methyl-binding proteins to achieve gene silencing and chromatin condensation [8, 9]. DNA methylation can also be established via H3K9 methylation, such as MeCP2, thereby establishing a repressive chromatin state [39]. Recent studies showed that the main chromatin changes that occurs during tumorigenesis are characterized by a global loss of acetylated H4 lysine 16 (H4K16ac) and H4 lysine 20 trime‐ thylation (H4K20me3) [40]. HDACs were found overexpressed in various types of cancer [41, 42] (becoming a major target for epigenetic therapy), along with HATs, whose expression can also be altered in cancer. MOZ, MORF, CBP, and p300 (HATs) may be targets for chromosomal translocations, especially in leukemia [43]. Changes in histone methylation patterns (deregu‐ lation of HMTs) are associated with aberrant gene silencing in cancer, and an effective cancer

18 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

treatment strategy targeting HDMs represents a promising treatment option.

Noncoding RNAs are involved in fundamental processes, such as chromatin dynamics and gene silencing, and their transcripts outnumber the group of protein transcripts. It is well known that the initiation of X-chromosome inactivation is regulated by noncoding RNAs (Xist function) and the noncoding RNAs molecules are also involved in imprinting, suggesting that antisense RNA can induce transcriptional silencing [44, 45, 46]. The characterized noncoding RNA family consists of a large group of small regulatory microRNAs (about 1400 microRNAs in humans) [47].MicroRNAs (miRNAs) are short noncoding RNAs of 20–24 nucleotides that play important roles in virtually all biological pathways in mammals like differentiation and growth control. Based on computer predictions, it was proposed that miRNAs may regulate many cell cycle control genes [48]. miRNAs influence numerous cancer-relevant processes such as proliferation, cell cycle control, apoptosis, differentiation, migration, and metabolism. The key processes of miRNA biogenesis pathways have been characterized. Primary miRNA transcripts are transcribed from separate transcriptional units or embedded within the introns of protein coding genes by RNA polymerase II. Primary miRNA transcripts are processed by a complex formed by RNase III enzyme and Drosha, resulting in a pre-miRNA hairpin that is subsequently exported from the nucleus to the cytoplasm by exportin 5 (XPO5). Further pre-

**1.2. Posttranscriptional gene regulation by noncoding RNAs**

#### **2. Evaluation of aberrant epigenetic modifications as essential players in cancer progression**

Normally, evolution and morphological state of genital organs are in close interdependence with hormonal status that is different in different periods: childhood, sexual maturity, climacterium, and menopause. On the other hand, there is an increasing interest in the identification of diagnostic biomarkers and biomarkers able to predict both response to treatment and survival. For an optimal planning of therapeutic strategy in high-risk patients, a close association between biological variables and (epi)genetic profiles associated with aggressive clinical behavior could be useful. Therefore, many cellular changes should be analysed in this context.

*Benign tumors of the vulva* can be developed from epithelial components (papillomas and warts) mezenchimatos tissue (fibroma, leiomyoma, lipoma, hemangioma, and lymphangioma), and local glands (Bartholin gland cysts or cysts of sweat glands). *Vulvar cancer* is a rare malignant disease accounting for less than 5% of gynecological malignancies [60, 61, 62]. The most common vulvar cancers are epidermoid carcinoma and rarely adenocarcinomas that are developed in the Bartholin glands or sweat glands. Approximately 20%–40% of vulvar squamous carcinomas are often associated with papilloma virus infection [60 - 66] and are more frequent in young people. Non-HPV vulvar cancers occur in the elderly and are associ‐ ated with somatic mutations, especially in TP53 [60 - 63, 65, 66]. Tumors harbouring a mutation have a worse prognosis than vulvar squamous cancers without (epi)genetic changes [67 - 70]. However, allelic imbalances seem to occur in both groups and the cumulative number of epigenetic changes increases from dysplasia to cancer [71]. The data with respect to epigenetic changes in vulvar cancer progression is limited to a few articles on DNA hypermethylation but not to chromatin remodeling or histone modifications. This data is presented in Table 1. Hypermethylation seems to be more frequent in vulvar squamous cancers than in vulvar intraepithelial neoplasia, but more studies are needed. Taking into account the existence of two etiological categories of vulvar carcinomas (related or not to HPV), the miRNA signature in these two types of vulvar carcinomas were evaluated [72]. Some miRNAs had lower expression in HPV-positive tumors (miR-1291, miR-342-3p, miR-193a-5p, miR-29c#-, miR-106b#, miR-22#, miR-365, miR-151-5P, miR-144#, miR-125b-1#, miR-519b-3p, miR-26b, miR-19b-1, and miR-1254) and other microRNAs had higher expression in HPV-positive tumors (miR-1274B, miR-142-3p, miR-21, miR-708, miR-16, miR-660, miR-29c, miR-1267, miR-454, and miR-186) [72]. In HPV-negative samples, we observed an association between lymph node metastases with decreased expression of miR-223-5p and miR-19b-1-5p, vascular invasion with decreased expression of miR-100-3p and miR-19b-5p-1, and advanced tumor staging (FIGO IIIA, IIIB, and IIIC) with expression of microRNAs miR-519b-1-5p and miR-133a. In addition, de Melo Maia and collaborators (2013) built a network between miRNA expression profiles and putative target mRNAs (TP53, RB, PTEN, and EGFR) based on prediction algorithms, demonstrating that the evaluated miRNAs can be involved in vulvar cancer progression, thereby providing biomarkers for the establishment of prognostic and predictive values of response to novel targeting therapies in vulvar cancer [72].

*The vagina* is a fibromuscular tubular organ, which histologically consists of three layers of tissue: (1) an outer layer consisting of fibro-elastic connective tissue; (2) vaginal muscles with a longitudinal outer layer and an inner layer of fibers circularly arranged in a spiral; and (3) Malpighian mucosa, covered by squamous epithelium. The vaginal epithelium undergoes changes in relation to the period of the woman's life and depending on hormonal stimulus. Histological changes are reflected in vaginal cytology. Vaginal epithelium responds to ovarian stimuli through proliferation, differentiation and desquamation. Thus, in adult women, under the action of estrogen during the proliferative phase, vaginal mucosa proliferates and differ‐ entiates morphologically and functionally, and later, during the luteal phase, under the action of progesterone, superficial cell layer desquamation occurs. The action of estrogen on the vaginal mucosa is exercised on the epithelium as well as on the subjacent stroma.

Vaginal cancer is also a rare malignancy, accounting for about 2%–3% of all gynecologic cancers [73, 74]. The squamous cell carcinomas (SCC) are more frequent (80%–90%) than adenocarci‐ nomas. If the risk factors linked to vaginal squamous cell carcinoma are smoking, immuno‐ suppression, high number of sexual partners, papillomavirus and history of cervical precancerous and cancerous lesions [75, 76, 77], in the case of the vaginal adenocarcinomas, particularly clear cell adenocarcinomas, exposure to an antiabortive drug diethylstilbestrol (DES) was incriminated [78, 79, 80]. On the other hand, if squamous vaginal cancer tends to occur more commonly in the proximal third of the vagina, especially the posterior vaginal wall, the adenocarcinomas are mostly seen in the anterior upper vaginal wall [74]. Human papillomaviruses have been also linked to vaginal cancers, HPV prevalence in 2/3 lesions of vaginal intraepithelial neoplasia and invasive vaginal cancer being over 90% and 70%, respectively [81, 82]. The HPV oncogenic transformation has been associated with high levels of E6 and E7 viral oncoproteins in the epithelia that can be achieved by two mechanisms: (1) increased production of E6 and E7 after the loss of E2 (the normal regulator of E6 and E7 expression) during viral integration [83]; (2) methylation of the E2-binding sites (E2BS) in the viral LCR in the region close to the early promoter that could inhibit E6 and E7 transcription [84]. Therefore, HPV16-related integration, methylation in E2BS3 and 4, and viral load may represent different viral characteristics driving vaginal and vulvar carcinogenesis [85].The adverse health outcomes induced by DES exposure during fetal development include infer‐ tility, early menopause, and breast cancer, along with a rare form of vaginal adenocarcinoma in adolescent girls [86, 87]. While animal models show an association of early exposure to estrogens with the expression levels of several genes [88, 89, 90] and epigenetic changes, including DNA methylation and histone modifications [91, 92, 93], the first study that evaluates the possible effects of *in utero* DES exposure on genome wide DNA methylation in humans cannot find evidence of large persistent effects of *in utero* DES exposure on blood DNA methylation [94].

developed in the Bartholin glands or sweat glands. Approximately 20%–40% of vulvar squamous carcinomas are often associated with papilloma virus infection [60 - 66] and are more frequent in young people. Non-HPV vulvar cancers occur in the elderly and are associ‐ ated with somatic mutations, especially in TP53 [60 - 63, 65, 66]. Tumors harbouring a mutation have a worse prognosis than vulvar squamous cancers without (epi)genetic changes [67 - 70]. However, allelic imbalances seem to occur in both groups and the cumulative number of epigenetic changes increases from dysplasia to cancer [71]. The data with respect to epigenetic changes in vulvar cancer progression is limited to a few articles on DNA hypermethylation but not to chromatin remodeling or histone modifications. This data is presented in Table 1. Hypermethylation seems to be more frequent in vulvar squamous cancers than in vulvar intraepithelial neoplasia, but more studies are needed. Taking into account the existence of two etiological categories of vulvar carcinomas (related or not to HPV), the miRNA signature in these two types of vulvar carcinomas were evaluated [72]. Some miRNAs had lower expression in HPV-positive tumors (miR-1291, miR-342-3p, miR-193a-5p, miR-29c#-, miR-106b#, miR-22#, miR-365, miR-151-5P, miR-144#, miR-125b-1#, miR-519b-3p, miR-26b, miR-19b-1, and miR-1254) and other microRNAs had higher expression in HPV-positive tumors (miR-1274B, miR-142-3p, miR-21, miR-708, miR-16, miR-660, miR-29c, miR-1267, miR-454, and miR-186) [72]. In HPV-negative samples, we observed an association between lymph node metastases with decreased expression of miR-223-5p and miR-19b-1-5p, vascular invasion with decreased expression of miR-100-3p and miR-19b-5p-1, and advanced tumor staging (FIGO IIIA, IIIB, and IIIC) with expression of microRNAs miR-519b-1-5p and miR-133a. In addition, de Melo Maia and collaborators (2013) built a network between miRNA expression profiles and putative target mRNAs (TP53, RB, PTEN, and EGFR) based on prediction algorithms, demonstrating that the evaluated miRNAs can be involved in vulvar cancer progression, thereby providing biomarkers for the establishment of prognostic and

20 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

predictive values of response to novel targeting therapies in vulvar cancer [72].

vaginal mucosa is exercised on the epithelium as well as on the subjacent stroma.

Vaginal cancer is also a rare malignancy, accounting for about 2%–3% of all gynecologic cancers [73, 74]. The squamous cell carcinomas (SCC) are more frequent (80%–90%) than adenocarci‐ nomas. If the risk factors linked to vaginal squamous cell carcinoma are smoking, immuno‐ suppression, high number of sexual partners, papillomavirus and history of cervical precancerous and cancerous lesions [75, 76, 77], in the case of the vaginal adenocarcinomas,

*The vagina* is a fibromuscular tubular organ, which histologically consists of three layers of tissue: (1) an outer layer consisting of fibro-elastic connective tissue; (2) vaginal muscles with a longitudinal outer layer and an inner layer of fibers circularly arranged in a spiral; and (3) Malpighian mucosa, covered by squamous epithelium. The vaginal epithelium undergoes changes in relation to the period of the woman's life and depending on hormonal stimulus. Histological changes are reflected in vaginal cytology. Vaginal epithelium responds to ovarian stimuli through proliferation, differentiation and desquamation. Thus, in adult women, under the action of estrogen during the proliferative phase, vaginal mucosa proliferates and differ‐ entiates morphologically and functionally, and later, during the luteal phase, under the action of progesterone, superficial cell layer desquamation occurs. The action of estrogen on the *The uterus* is a hollow organ, in which the product of conception is developed. It consists of three parts: body, isthmus, and cervix. The corpus presents a mucosa (endometrium), muscular wall (myometrium), and serous peritoneal surface. The endometrium is a specialized tissue, particularly receptive to the influence of sex hormones that differs from a histological point of view at prepubertal periods, sexual maturity, and menopause. Also, the uterine mucosa is in constant transformation during menstrual cycles, sexual maturity, growth processes, func‐ tional maturation, and regression. Similar risk factors for endometrial cancers were incrimi‐ nated: adult obesity [95], first-degree family history of endometrial cancer, or colorectal cancer [96]. Nulliparity and infertility appeared to independently contribute to endometrial cancer risk [97]. The endometrium is extremely sensitive to hormones, the estrogen and progesterone being two key regulators of proliferation and differentiation in reproductive tissues [98]. The two isoforms of the progesterone receptor, PRA and PRB, required for endometrial differen‐ tiation [99], are generated by alternative transcription and translation from the same gene with the addition of 164 amino acids in the N-terminus sequences of PRB [98] that makes them functionally different [99]. A shift in the estrogen–progesterone balance is the major cause for the development of endometrial cancer [100]. Progesterone is an important inducer of endometrial differentiation and an inhibitor of tumorigenesis because the addition of progestin (synthetic progesterone) can prevent endometrial cancer induced by an excess of estrogens from endogenous sources (e.g., adipose tissue storage of estrogen and with polycystic ovarian syndrome) or from exogenous sources in therapeutic administration [100]. While progestin therapy achieves promising outcomes with early stage endometrial cancer, advanced and recurrent disease has only minor effects. This is due to the fact that in advanced endometrial cancer, the progesterone receptor is lost but it has been demonstrated that reestablishing progesterone signaling in these cells can inhibit endometrial cancer cell proliferation and invasion and increase sensitivity to apoptotic stimuli [100]. The epigenetic restoration of progesterone receptor expression could result in resensitization of endometrial tumors to progestin therapy. The functional role of epigenetic factors in endometrial cancer development began to be evaluated. A study by Jones and collaborators (2013) emphasizes the role of HAND2 hypermethylation, which is a key step in endometrial carcinogenesis [101]. HAND2 is a basic helix-loop-helix transcription factor and developmental regulator [102], expressed in the normal endometrial stroma. The physiological function of HAND2 is to suppress the production of fibroblast growth factors that mediate the paracrine mitogenic effects of estrogen on the endometrial epithelium [103]. HAND2 is under progesterone regulation [104, 105], entering in the progesterone-mediated suppression of estrogen-induced pathways. Conse‐ quently, the methylation of HAND2 is able to predict the response to progesterone [101]. HAND2 methylation is the most common molecular alteration in endometrial cancer and, on the other hand, is an early event in endometrial carcinogenesis that makes it a sensitive test to correctly identify endometrial cancer patients amongst those women who present with postmenopausal bleeding [101].

Histologically, *the cervix* shows mucosa, muscle wall, and the peritoneal serosa. The mucosa of the cervix has an exocervical portion (covered by squamous epithelium, nonkeratinized) and one endocervical (covered by a single-layered cylindrical epithelium, mucus secreting, which contains a small number of ciliated cells, basal stem cells and racemic, tubular, or branched type glands). Cancer of the uterine cervix is the major cause of death from gyneco‐ logical cancers and in over 90% of cases is associated with high-risk human papilloma virus (hrHPV). Etiological factors include cigarette smoking, impairment of cell-mediated immun‐ ity, and long-term estrogen–progestin use [106, 107, 108]. But the main etiological factor of squamous cell carcinoma (that accounts for about 80% of the cases) as well as adenocarcinoma are human papilloma virus infections [109]. The role of other sexually transmitted infections (*Chlamydia trachomatis* and herpes simplex virus) is still unclear [108, 110].In cervical cancer, tumorigenesis of both squamous cell carcinoma and adenocarcinoma is HPV-related [109]. The transforming potential of E6 and E7 viral oncoproteins is based on their numerous actions on cellular proteins, mainly on p53 and pRB tumor suppressors, which are degraded and inactivated, respectively. In addition to the already reported genomic alterations in cervical cancer development by hrHPV, many studies underline the involvement of epigenetic alteration in host cell genes or at the levels of RNA. In order to find some diagnostic and prognostic biomarkers, the methylation of host cell genes and methylation of viral genes were evaluated [12]. The CpG hypermethylation of promoters of tumor suppressor genes, an early and frequent alteration in carcinogenesis, affects all important pathways: cell adhesion (cell adhesion molecule 1 (CADM1)) [13], E-cadherin [111, 112], apoptosis (DAPK, a proapoptotic serine/threonine kinase [113, 114]), cell cycle (cyclin A1 methylation [114, 115]), fragile histidine triad (FHIT) [116], cell signaling pathways (retinoic acid receptor [117], Ras association domain family 1 isoform A (RASSF1) [118]), Wnt/β catenin pathway (adenomatous polyposis coli (APC) [119] and PTEN [120]), p53 signaling pathway (p73 [121]), and DNA repair (O6 methylguanine DNA methyltransferase (MGMT) [113, 122]).For cervical scrapings, some methylation marker panels of host genes, with sensitivities of over 80% for CIN3+ were evaluated: SOX1/PAX1, SOX1/LMX1A, SOX1/NKX6-1, PAX1/LMX1A; PAX1/NKX6-1, LMX1A/NKX6-1 [123], JAM3/EPB41L3/TERT/C13ORF18 [124], and CADM1/MAL [13, 125], etc. Host gene methylation analysis might be an alternative for hrHPV DNA detection because aberrant methylation can be detected in cervical smears up to 7 years prior to the diagnosis of cervical cancer [126]. On the other hand, for methylation analysis, cervical scrape samples as well as self-collected cervico-vaginal lavage samples can be used [127].As accurate predictor tests, the measurement of DNA methylation in HPV genomes, in certain early (E) and late (L) open reading frames (ORF) as well as in parts of the upstream regulatory region (URR), may have diagnostic value. The hypermethylation in the L1 region was a common feature of cervical cancer but not of CIN induced by HPV16 [128], or HPV18 [129]. But the DNA methylation on multiple CG sites in the L1, L2, E2, and E4 ORFs were significantly associated with CIN2+ after accounting for multiple testing [130]. Some studies have contradictory results because most were quite small and heterogeneous and did not always include (1) comparable sets of specimens (cancer, high-grade CIN, cell lines), (2) exactly the same CG sites, or (3) the same methodology [12]. Overall, as cervical cancer prevention moves to DNA testing methods, DNA-based biomarkers, such as HPV methylation could serve as a reflex strategy to identify women at high risk for cervical cancer [131], but the region with the best predictive value must be established.In addition to the already reported genomic alterations in cervical cancer development by hrHPV, many studies underline the involvement of viral or cellular miRNAs, mainly based on the fact that some RNA micromolecules target transcriptional factors that modulate both cellular and viral gene expression [132, 133].In HPV infection, E6 decreases miR-34a [132, 134], which is a target of p53, thus the effect of E6 on miRNA-34a is mediated by decreased p53 [132,134]. On the other hand, one of the targets of miR-34a is p18Ink4c [135], an inhibitor of CDK4/6 that promotes the cell cycle. E7 decreases miR-203 during keratinocyte differentiation, which is a tumor suppressor and thus increases carcinogenesis [136] through an increase of cell survival targeting antiapoptotic protein bcl-w [137], induction of G1 cell cycle arrest targeting survivin [138], inhibition of migration and invasion targeting LIM and SH3 protein [139]. E7 upregulates miR-15a, miR-15b, and miR-15b through E2F1 and E2F3 [140, 141] and in turn, these miR decrease cyclin E1, leading to cell cycle arrest [142]. A lot of other miRs are upregulated or decreased by virus oncogenes inducing changes in cellular signaling pathways, some of these have not yet been elucidated [143].

cancer, the progesterone receptor is lost but it has been demonstrated that reestablishing progesterone signaling in these cells can inhibit endometrial cancer cell proliferation and invasion and increase sensitivity to apoptotic stimuli [100]. The epigenetic restoration of progesterone receptor expression could result in resensitization of endometrial tumors to progestin therapy. The functional role of epigenetic factors in endometrial cancer development began to be evaluated. A study by Jones and collaborators (2013) emphasizes the role of HAND2 hypermethylation, which is a key step in endometrial carcinogenesis [101]. HAND2 is a basic helix-loop-helix transcription factor and developmental regulator [102], expressed in the normal endometrial stroma. The physiological function of HAND2 is to suppress the production of fibroblast growth factors that mediate the paracrine mitogenic effects of estrogen on the endometrial epithelium [103]. HAND2 is under progesterone regulation [104, 105], entering in the progesterone-mediated suppression of estrogen-induced pathways. Conse‐ quently, the methylation of HAND2 is able to predict the response to progesterone [101]. HAND2 methylation is the most common molecular alteration in endometrial cancer and, on the other hand, is an early event in endometrial carcinogenesis that makes it a sensitive test to correctly identify endometrial cancer patients amongst those women who present with

22 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

Histologically, *the cervix* shows mucosa, muscle wall, and the peritoneal serosa. The mucosa of the cervix has an exocervical portion (covered by squamous epithelium, nonkeratinized) and one endocervical (covered by a single-layered cylindrical epithelium, mucus secreting, which contains a small number of ciliated cells, basal stem cells and racemic, tubular, or branched type glands). Cancer of the uterine cervix is the major cause of death from gyneco‐ logical cancers and in over 90% of cases is associated with high-risk human papilloma virus (hrHPV). Etiological factors include cigarette smoking, impairment of cell-mediated immun‐ ity, and long-term estrogen–progestin use [106, 107, 108]. But the main etiological factor of squamous cell carcinoma (that accounts for about 80% of the cases) as well as adenocarcinoma are human papilloma virus infections [109]. The role of other sexually transmitted infections (*Chlamydia trachomatis* and herpes simplex virus) is still unclear [108, 110].In cervical cancer, tumorigenesis of both squamous cell carcinoma and adenocarcinoma is HPV-related [109]. The transforming potential of E6 and E7 viral oncoproteins is based on their numerous actions on cellular proteins, mainly on p53 and pRB tumor suppressors, which are degraded and inactivated, respectively. In addition to the already reported genomic alterations in cervical cancer development by hrHPV, many studies underline the involvement of epigenetic alteration in host cell genes or at the levels of RNA. In order to find some diagnostic and prognostic biomarkers, the methylation of host cell genes and methylation of viral genes were evaluated [12]. The CpG hypermethylation of promoters of tumor suppressor genes, an early and frequent alteration in carcinogenesis, affects all important pathways: cell adhesion (cell adhesion molecule 1 (CADM1)) [13], E-cadherin [111, 112], apoptosis (DAPK, a proapoptotic serine/threonine kinase [113, 114]), cell cycle (cyclin A1 methylation [114, 115]), fragile histidine triad (FHIT) [116], cell signaling pathways (retinoic acid receptor [117], Ras association domain family 1 isoform A (RASSF1) [118]), Wnt/β catenin pathway (adenomatous polyposis coli (APC) [119] and PTEN [120]), p53 signaling pathway (p73 [121]), and DNA repair (O6 methylguanine DNA methyltransferase (MGMT) [113, 122]).For cervical scrapings, some

postmenopausal bleeding [101].

*Ovaries*, paired organs, constitute the female sexual gland with endocrine lunette and also produce ova. The ovary is covered by germinal epithelium (formed from cuboid or cylindrical cells) and subjacent is a thin layer of dense connective tissue. The ovary presents a cortical area (comprised of follicles, corpus luteum, and stroma) and a medulla. Starting from puberty till menopause, there is a growth and maturation of one ovarian follicle during each menstrual cycle and the formation of one corpus luteum after rupture of the follicle and oocyte removal. If the egg is not fertilized, the corpus luteum regresses, undergoes progressive sclerosis forming a hyaline. If the egg is fertilized, the corpus luteum becomes more voluminous and luteal cells increase, constituting the corpus luteum of pregnancy. Ovarian stroma is formed from fibroblastic and mesenchymal cells. Stromal cells present both characters of connective cells and steroid activity (secreting androgens and estrogens). Ovarian medulla consists of lax connective tissue containing blood and lymph vessels, nerves, and embryonic elements. The growth and development of the follicle during the ovarian cycle are driven by two gonado‐ trophic hormones, secreted by the anterior pituitary: follicle-stimulating hormone (FSH) and luteinising hormone (LH). Both FSH and LH are under the control of gonadotrophin-releasing hormone (GnRH) secreted by the hypothalamus through negative feedback carried out by estrogens that are secreted by thecal cells of the follicle.

Ovarian cancer ranks second after cervical cancer worldwide. On the other hand, ovarian cancer is in seventh place in terms of incidence among malignant tumors in women and eighth with respect to death due to malignant tumors in women worldwide [144]. If approximately 90% of ovarian cancers arise from epithelial cells, 3% are from germ cells and 7% from granulosa-theca cells. Ovarian cancer comprises different types of tumors with widely differing clinicopathologic features and behaviors. Based on clinicopathologic and molecular genetic studies, two histologic types of epithelial ovarian serous carcinomas were established: low-grade serous carcinomas (LGSCs) and high-grade serous carcinomas (HGSCs) [145]. Although they are developed independently along different molecular pathways, both types develop from fallopian tube epithelium and involve the ovary secondarily. Type I tumors (LGSCs) are comprised of low-grade serous, low-grade endometrioid, mucinous, and clear cell carcinomas; typically present as large cystic masses confined to one ovary; have a relatively indolent course; and are relatively genetically stable being associated with mutations in KRAS, BRAF, PTEN, PIK3CA, CTNNB1, ARID1A, and PPP2R1A [146, 147] that perturb signaling pathways. Type II tumors (HGSCs) are composed of high-grade serous, high-grade endome‐ trioid, undifferentiated carcinomas and malignant-mixed mesodermal tumors; clinically aggressive and typically present at an advanced stage, which contributes to their high fatality [148]; at the time of diagnosis, they demonstrate marked chromosomal aberrations but over the course of the disease these changes remain relatively stable [149]; approximately 60% of HGSC have the fallopian tube as the origin of serous tumors [150], because the expression profiles of ovarian HGSCs more closely resemble fallopian tube epithelium than the ovarian surface epithelium [151]; they harbor TP53 mutations in over 95% of cases [152, 153], but rarely harbor the mutations detected in the low-grade serous tumors; another possible origin of HGSC is from inclusion cysts through a process of implantation of tubal (müllerian-type) tissue rather than by a process of metaplasia from ovarian surface epithelium (mesothelial). Hyper‐ methylation has been found to be associated with the inactivation of almost every pathway involved in ovarian cancer development, including DNA repair, cell cycle regulation, apop‐ tosis, cell adherence, and detoxification pathways [154]. Complete or partial inactivation of the BRCA1 gene through hypermethylation of its promoter has been reported in 15% of sporadic ovarian tumors [155, 156], 31% of carcinomas but not in the benign or borderline tumors [157], or in the hereditary type of the disease, nor in samples from women with a germ line BRCA1 mutation [158, 159]. On the other hand, hypermethylation of BRCA1 was detected at a significantly higher frequency in serous carcinomas than in tumors of the other histological types [160]. The homeobox genes (HOX), a family of transcription factors that function during embryonic development and control pattern formation, differentiation, and proliferation [161] was associated with ovarian cancers [162]. In addition, based on the high percentage of methylation of the HOXA9 gene observed in 95% of patients with high-grade serous ovarian

carcinoma [163, 164], it has been suggested that the methylation status of HOXA9 and HOXAD11 genes may serve as potential diagnostic and prognostic biomarkers [163,164]. Some other genes found hypomethylated were associated with progression towards cancer: LINE-1 elements [165], SNGG (synucelin-γ), encoding an activator of the MAPK and Elk-1 signaling cascades [166, 167], etc. Overall, DNA hypomethylation may promote tumorigenesis by transcriptional activation of proto-oncogenes and on the other hand loss of imprinting or genomic instability. DNA hypermethylation predisposes to gene mutation because the methylated cytosines are often deaminated and converted to thymine leading to inactivation of tumor suppressor genes. However, these phenomena deregulate the main functions of gynecological cancer cells (Figure 1 and Table 1).

connective tissue containing blood and lymph vessels, nerves, and embryonic elements. The growth and development of the follicle during the ovarian cycle are driven by two gonado‐ trophic hormones, secreted by the anterior pituitary: follicle-stimulating hormone (FSH) and luteinising hormone (LH). Both FSH and LH are under the control of gonadotrophin-releasing hormone (GnRH) secreted by the hypothalamus through negative feedback carried out by

Ovarian cancer ranks second after cervical cancer worldwide. On the other hand, ovarian cancer is in seventh place in terms of incidence among malignant tumors in women and eighth with respect to death due to malignant tumors in women worldwide [144]. If approximately 90% of ovarian cancers arise from epithelial cells, 3% are from germ cells and 7% from granulosa-theca cells. Ovarian cancer comprises different types of tumors with widely differing clinicopathologic features and behaviors. Based on clinicopathologic and molecular genetic studies, two histologic types of epithelial ovarian serous carcinomas were established: low-grade serous carcinomas (LGSCs) and high-grade serous carcinomas (HGSCs) [145]. Although they are developed independently along different molecular pathways, both types develop from fallopian tube epithelium and involve the ovary secondarily. Type I tumors (LGSCs) are comprised of low-grade serous, low-grade endometrioid, mucinous, and clear cell carcinomas; typically present as large cystic masses confined to one ovary; have a relatively indolent course; and are relatively genetically stable being associated with mutations in KRAS, BRAF, PTEN, PIK3CA, CTNNB1, ARID1A, and PPP2R1A [146, 147] that perturb signaling pathways. Type II tumors (HGSCs) are composed of high-grade serous, high-grade endome‐ trioid, undifferentiated carcinomas and malignant-mixed mesodermal tumors; clinically aggressive and typically present at an advanced stage, which contributes to their high fatality [148]; at the time of diagnosis, they demonstrate marked chromosomal aberrations but over the course of the disease these changes remain relatively stable [149]; approximately 60% of HGSC have the fallopian tube as the origin of serous tumors [150], because the expression profiles of ovarian HGSCs more closely resemble fallopian tube epithelium than the ovarian surface epithelium [151]; they harbor TP53 mutations in over 95% of cases [152, 153], but rarely harbor the mutations detected in the low-grade serous tumors; another possible origin of HGSC is from inclusion cysts through a process of implantation of tubal (müllerian-type) tissue rather than by a process of metaplasia from ovarian surface epithelium (mesothelial). Hyper‐ methylation has been found to be associated with the inactivation of almost every pathway involved in ovarian cancer development, including DNA repair, cell cycle regulation, apop‐ tosis, cell adherence, and detoxification pathways [154]. Complete or partial inactivation of the BRCA1 gene through hypermethylation of its promoter has been reported in 15% of sporadic ovarian tumors [155, 156], 31% of carcinomas but not in the benign or borderline tumors [157], or in the hereditary type of the disease, nor in samples from women with a germ line BRCA1 mutation [158, 159]. On the other hand, hypermethylation of BRCA1 was detected at a significantly higher frequency in serous carcinomas than in tumors of the other histological types [160]. The homeobox genes (HOX), a family of transcription factors that function during embryonic development and control pattern formation, differentiation, and proliferation [161] was associated with ovarian cancers [162]. In addition, based on the high percentage of methylation of the HOXA9 gene observed in 95% of patients with high-grade serous ovarian

estrogens that are secreted by thecal cells of the follicle.

24 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

**Figure 1.** Biological functions influenced by alterations of DNA methylation in gynecological cancers.




**Genes Functions Expression change Epigenetic regulation References**

Overexpression

Overexpression

DNA

Overexpression Hypomethylation 176

Overexpression miR-9 downregulation177

DNA

DNA

miR-125a

miR-125b downregulation

Overexpression Hypermethylation 182

Overexpression Hypermethylation 159,182, 183

hypomethylation

miR-15a and miR-16 down regulation

hypomethylation, chromatin acetylation

downregulation via EGFR signaling

hypomethylation/ hypermethylation 164, 173, 174,

175

167

178

179

180

181

*HOXA10 HOXA11*

*MAL*

*NFKB1*

*ARID3B*

*BRCA1*

*RASSF1A*

Fertility, embryo viability, regulation of hematopoietic lineage commitment; regulation of uterine development and is required for

26 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

Formation, stabilization and maintenance of glycosphingolipidenriched membrane microdomains

Cell proliferation; Inflammation, immunity, differentiation, cell growth, tumorigenesis, and

*SNCG* Cell proliferation Overexpression

*BMI1* Cell proliferation Overexpression

*TUBB3* Taxane drug resistance Overexpression

*BCL3* Cell proliferation, tumorigenesis Overexpression

DNA repair, cell cycle checkpoint control, and maintenance of

PTEN, *p14ARF* Cell cycle regulation Overexpression Hypermethylation 182 *DAPK* Regulator of programmed cell death Overexpression Hypermethylation 182

*p16INK4A* Cell cycle regulation Overexpression Hypermethylation 183

the WNT. Overexpression Hypermethylation 159, 183

Epithelial-to-mesenchymal transition; Embryonic patterning, cell lineage gene regulation, cell cycle control, transcriptional regulation and possibly in chromatin

structure modification

genomic stability

Negative regulator of cell proliferation through inhibition of

G1/S-phase progression

*APC* Tumor suppression by antagonizing

female fertility

apoptosis


**Table 1.** Altered DNA methylation in gynecological cancer

miRNA as key players in cell fate decisions are strongly linked to gynecological cancer. But, although the methods to discover miRNA were improved, research is still in progress. Some of these miRNA that have been associated with gynecologic cancers are shown in Figure 2 and Table 2.

**Genes Functions Expression change Epigenetic regulation References**

maintenance; cancer development Hypermethylation <sup>204</sup>

and proliferation Hypermethylation <sup>206</sup>

regulators of cytokine signaling Hypermethylation <sup>206</sup>

and tumorigenesis Hypermethylation <sup>210</sup>

Hypermethylation 210

177

177

214

215

216

miR-214 downregulation

miR-214 downregulation

miR-101 downregulation

miR-29a downregulation

miR-203

DNA

downregulation by

hypermethylation

*HOPX* Tumorigenesis Hypermethylation 205

*HSPA2, MLH1* Regulation of cell growth Hypermethylation 206

*PAX2* Transcriptional factor Hypomethylation 207

*TERT* Cellular senescence Hypermethylation 209

*TFPI2* Tumor suppressor gene Hypermethylation 209 *TP73, FHIT* Cell cycle regulation; apoptosis Hypermethylation 211 *TSLC-1* Hypermethylation 212

*CAGE* RNA processing Overexpression Hypomethylation 213

*CDKN2A* Cell cycle regulation Hypermethylation 208, 209 *MGMT* Potential prognostic cancer Hypermethylation 210 *RASSF2A* Tumor suppressor gene Hypermethylation 210

*PER1* Cells circadian rhythms

*CDH13* Regulation of cell growth, survival

28 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

Negative regulator of cell proliferation through inhibition of

G1/S-phase progression

*MAP2K3* Cell proliferation Overexpression

*MAPK8* Cell proliferation Overexpression

*SERPINH1* Metastasis Overexpression

*VEGFA* Tumor growth, angiogenesis Overexpression

invasion Overexpression

*TSP1* Platelet aggregation, angiogenesis,

*PTGS2* Cell proliferation, migration,

**Table 1.** Altered DNA methylation in gynecological cancer

*SOCS2* Cytokine-inducible negative

Vulvar cancer

Cervical cancer

*RASSF1A*

Figure 2. Venn diagram showing dysregulated miRNAs in gynecological cancers. (A) miRNAs downregulated, (B) miRNAs upregulated. Common miRNAs dysregulated signature between ovarian and other cancers are shown in red. **Figure 2.** Venn diagram showing dysregulated miRNAs in gynecological cancers. (A) miRNAs downregulated, (B) miRNAs upregulated. Common miRNAs dysregulated signature between ovarian and other cancers are shown in red.

Specific miRNAs have effects on various molecular pathways, and specific miRNA expression signatures in gynecological cancers can be associated with diagnosis, prognosis, and therapy response. miRNAs can regulate a large

Table 2. Dysregulated miRNAs in gynecological cancer.

Let‐7a,b, c, d, e, f, g Down c‐Myc, KRAS, HMGA2, IL‐6, LIN28B, HIC2 217

Let‐7i Down HMGA2, LIN28Bm TRIM71,IGF2BP1 <sup>219</sup> 1 Down FOXP1, HDAC4 c‐Met, Pim1, HAND2 220 9 Down NF‐kB, Bcl2, Bcl6, FGF, b‐Raf 220;221 15a, 16 Down BMI1 178 21 Down PTEN 222 30b,d Down Unknown 223

34a,b,c Down SIRT1, MYC, NOTCH, BCL2, CCND1,WNT3 222, 223; <sup>225</sup>

**Estimated target(s) References**

; 218

;224

number of target genes and Table 2 lists the estimated targets.

**miRNA(s) Expression**

Ovarian

cancer

**(Up/downre gulated)**

Specific miRNAs have effects on various molecular pathways, and specific miRNA expression signatures in gynecological cancers can be associated with diagnosis, prognosis, and therapy response. miRNAs can regulate a large number of target genes and Table 2 lists the estimated targets.



Specific miRNAs have effects on various molecular pathways, and specific miRNA expression signatures in gynecological cancers can be associated with diagnosis, prognosis, and therapy response. miRNAs can regulate a large number of target genes and Table 2 lists the estimated

Let-7a,b, c, d, e, f, g Down c-Myc, KRAS, HMGA2, IL-6, LIN28B, HIC2 217, 218 Let-7i Down HMGA2, LIN28Bm TRIM71,IGF2BP1 219 1 Down FOXP1, HDAC4 c-Met, Pim1, HAND2 220 9 Down NF-kB, Bcl2, Bcl6, FGF, b-Raf 220, 221 15a, 16 Down BMI1 178 21 Down PTEN 222 30b,d Down Unknown 223, 224 34a,b,c Down SIRT1, MYC, NOTCH, BCL2, CCND1,WNT3 222, 223, 225

95 Down AIB1, GNAI2 226 98 Down HMGA2, LIN28B, HIC2 223

TNFa, BMPR1B

 Down SPRED1, PIK3R2, RGS4, RGS5, PI3K 229 Down CDK6, MITF, KLF12, PDLIM3 2 Down c-SRK, MMP13, FGF2 220,230 Down MAP3K3, MAP4K4, SOX2, OCT4, KLF4, c-myc 220, 230, 231

150 Down c-Myb, MAK9, Akt3, MAP2K4 230 184 Down TTK69, K10, Sax(A) 230

<sup>335</sup> Down P18SRP, HLF, CALU, MAX, HOXD8, SOX4, JAG1, TNC, c-Met, TNC

519a, d,e Down FLJ31818, TGFBR2, HuR, EIF2C1, ARID4B,

<sup>210</sup> Down E2F3, EFNA3, HoxA1, HoxA9 226, 234, 235,

377 Down REST, SOD1 230, 237 517a, b Down CREAP-1, MAPKAPK5, NFKBIE, PTK2B 238

ARID3B, LIN28b, Akt3, ETS1ARID3B, RBB2, ERBB3,

ZEB1, ZEB2, FN1, PPM1E, EXOC5, GATA4, GATA6,

TUBB3, TNC, TGF-b 219 ; 232, 233

GATA2BD, SUV39H1 223,238, 239

**Estimated target(s) References**

223, 227, 228

236

223, 228

**Expression (Up/ downregulat ed)**

30 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

targets.

Ovarian cancer

**miRNA(s)**

125a, b Down

200a,b,c Down



**miRNA(s)**

Endometrial cancer

**Expression (Up/ downregulat ed)**

32 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

1 Down c-Met, TIMP-3, TRIM2, ITGB3, ZNF264 257, 258 Let-7 Down KRAS, c-Myc, HMG2A, IL-6, HIC2 229 26 Down SMAD1, SOX2, Bcl6, SMAD4, BCL2,KLF4 229 29b Down IGF1, Mcl-1 257 30c Down MYH11, GPRASP2, DDR2, CKS2,C5 250 34b,c Down NOTCH, BCL2, CCND1, WNT3, MYC, SIRT1 257, 259 101 Down COX2, EZH2 257 125 Down LIN28, ERBB2, ERBB3, Akt3 and ETS1 229 129-2 Down SOX4 192 133a,b Down PKM2, Mcl-1,Bcl2l2 257 136 Down Rtl1 257 152 Down ENPP2, SNCAIP, LTBP4, MLH1,Bcl2l11 259, 260 193a,b Down KIT, RAMP1, TSPYL5, ERBB4, ROBO4, UPA 250, 261 204 Down Ezrin, ESR1, CHD5, CAMTA1 261 221 Down LMOD, p27Kip1, p57Kip2, c-Kit 260 376a,c Down PRPS1, BMPR2, KLF15,GRIK2 257, 262 377 Down ETS1, XIAP, RNF38 257 379 Down FOXP2, MTMR2, HLCS,CCNB1 257 411 Down MAP3K1, SP2, CDH2, FOXO1, SMAD4,SET 257 424 Down CCNE1, CCND1,NFI-A 257 455-5p Down PP1R12A, KDR, SUZ12, FOXN3,PTPRJ 257, 263 518c Down ID-1, HOXA3,HOXC8,RAP1B,ABCG2,HLA-G 245,257 542-3p,5p Down COX-2, HSPG2, ZNF618, CREB5 257, 264 654-3p Down KLF12, SORBS1, WDR26, RNF145, AP1S3 229, 265 765 Down KLK4, POU2F2, TIMP3, ADAM19, BCL6B 257 873 Down FOXK2, TBL1X, TMOD2, BMPR2, SFRS1 257 1226 Down MARCH9, PPFIBP1 257

10a Up USF2, HOXA1, HOXD10, HOXB1, HOXB3, RB1CC1

31 Up FOXCP2, FOXP3 261 96 Up CHES1, FOXO1, FOXO3A 261, 266

and ribosomal proteins

**Estimated target(s) References**

250


**Table 2.** Dysregulated miRNAs in gynecological cancer.

Specific biological functions affected by histone modifications in gynecological cancers are presented in Table 3.


**miRNA(s)**

Vulvar cancer

1291

19b-1-5p; 22-5p; 26b-3p; 29c-5p; 106b-3p; 142-3p; 144-5p; 151a-5p; 193a-5p; 342-3p; 365a-3p; 519b-3p;

16-5p; 21-5p; 29c-5p; 142-3p;

**Table 2.** Dysregulated miRNAs in gynecological cancer.

**Expression (Up/ downregulat ed)**

34 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

218 Down LAMB3 280 372 Down CDK2, Cyclin A1 281 513 Down IGF-1, BCL2L2, VEGFA CDK6 274 519a Down HuR 282 9 Up Unknown 283 10a Up (HOX) genes 274 21 Up PTEN,TPM1, PDCD4 271, 284 27a Up Unknown 285 100 Up PLK1 286 126, 127 Up Unknown 278, 287 132 Up (HOX) genes 274 133a Up Unknown 278 133b Up MST2,CDC42, RHOA,MAPK1,AKT1 288 146a Up Unknown 285 148a Up PTEN, P53INP1 and TP53INP2 274 155 Up Unknown 272, 278 182, 199b Up Unknown 278, 280

200a Up MYH10, ZEB1, DCP2, YWHAG, KIDINS220, ZEB2,

203 Up p63 136 205, 221 Up Unknown 272, 285 302b, 522 Up Unknown 274 886-5p Up BAX 289

186-5p; 454-3p; 708-5p; 1267 Up Unknown <sup>72</sup>

TGFB2, RANBP5, EXOC5

Down Unknown 72

**Estimated target(s) References**

283


#### **3. The roles of microenvironment-mediated epigenetic perturbations in the development of gynecological neoplasia**

The complexity that governs the tumor phenotype cannot be explainedonly atthe genetic level, as genetic abnormalities occur with low frequency. Therefore, major attention was focused on the study of the role of tumor microenvironment (TME) not only in tumor initiation but also in progression and metastasis. The hypothesis of cancer cell development and proliferation only in a conducive environment has been made by Paget since 1889 [308]. While Paget suggested that the microenvironment facilitates or inhibits metastasis through growth-promoting/ inhibiting factors,recentresearch sustains thatthe tumoris directed into one or several possible molecular evolution pathways by signals originating in native and/or modified microenviron‐ mental factors [309]. The tumor microenvironment consists of epithelial cells, vascular endothelial cells, fibroblasts and myofibroblasts, macrophages, leukocytes, and the extracellu‐ lar matrix (ECM). Together with the ECM, these nonmalignant cell types constitute the stromal tissue of the tumor that secretes ECM components, cytokines, and growth factors involved in tumor growth and invasion. All these components are dynamically interconnected around the tumor. In the tumorigenesis process, studies have shown the critical role of chronic inflamma‐ tion by hyperexpression of the inflammatory mediators in the microenvironment. The inflammatory microenvironment is both the result of genetic alterations in cancer cells and of the tumor-infiltrating cells that produce inflammatory mediators [310].

While normal fibroblasts prevent tumor progression, cancer-associated fibroblasts (CAFs) that display a different secretory pattern generate an environment that favors tumor growth and invasiveness. Tumor formation is characterized by changes in cell behavior, like accelerated growth with loss of tissue architecture and epithelial dysfunction, angiogenesis, stromal activation, and migratory and invasive features. Therefore, dysfunction in the tumor micro‐ environment, in addition to epithelial dysfunction, is crucial for carcinogenesis as altering its components leads to impaired immune response. TME promotes tumorigenesis through new blood vessel formation. Although studies have suggested that some cells in TME contained mutations, recent data pointed, first, to the presence of mutations only in tumorigenic cells and second, to the contribution of these mutations to epigenetic changes in both nontumori‐ genic cells and TME. In turn, the cells in the microenvironment produce epigenetic changes in tumor cells reflected in their pattern of differentiation [311] and animal models demonstrate that the tumor microenvironment can induce epigenetic alterations and changes in gene expression in tumors [312].

**Genes Functions Expression Up/**

chromatin remodeling Up <sup>304</sup>

activation Up <sup>305</sup>

bacteria, regulates histone methylation, macrophage

Histone deacetylase 1; a transcriptional regulator that mediates histone deacetylation, antiapoptosis, synapse

differentiation, and protein localization EP300 Histone acetyltransferase and regulates transcription via

36 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

pCAF (KAT2B) Histone acetyltransferase (HAT) to promote transcriptional

maturation, and hippocampus development

the tumor-infiltrating cells that produce inflammatory mediators [310].

Histone deacetylase 2; a histone deacetylase and a transcriptional corepressor that acts in chromatin remodeling, inflammatory response, and regulation of

**3. The roles of microenvironment-mediated epigenetic perturbations in the**

The complexity that governs the tumor phenotype cannot be explainedonly atthe genetic level, as genetic abnormalities occur with low frequency. Therefore, major attention was focused on the study of the role of tumor microenvironment (TME) not only in tumor initiation but also in progression and metastasis. The hypothesis of cancer cell development and proliferation only in a conducive environment has been made by Paget since 1889 [308]. While Paget suggested that the microenvironment facilitates or inhibits metastasis through growth-promoting/ inhibiting factors,recentresearch sustains thatthe tumoris directed into one or several possible molecular evolution pathways by signals originating in native and/or modified microenviron‐ mental factors [309]. The tumor microenvironment consists of epithelial cells, vascular endothelial cells, fibroblasts and myofibroblasts, macrophages, leukocytes, and the extracellu‐ lar matrix (ECM). Together with the ECM, these nonmalignant cell types constitute the stromal tissue of the tumor that secretes ECM components, cytokines, and growth factors involved in tumor growth and invasion. All these components are dynamically interconnected around the tumor. In the tumorigenesis process, studies have shown the critical role of chronic inflamma‐ tion by hyperexpression of the inflammatory mediators in the microenvironment. The inflammatory microenvironment is both the result of genetic alterations in cancer cells and of

While normal fibroblasts prevent tumor progression, cancer-associated fibroblasts (CAFs) that display a different secretory pattern generate an environment that favors tumor growth and

HDAC1

HDAC2

translation

**development of gynecological neoplasia**

**Table 3.** Histone modifications in gynecological cancer.

**downregulate**

Up 306, 307

Up 307

**References**

It was suggested that the epigenome serves as the interface between the genome and the environment [313, 314]. The epigenetic role of TME in growth induction seems to be linked with transforming growth factor (TGF)-β and its receptor, whose expressions are regulated through chromatin remodeling [315], although no research on stromal fibroblasts was performed. TGFβ pathways are involved in the oncogenesis process, acting either as tumor suppressor or as tumor promotor, depending on TME crosstalk in the tumor microenviron‐ ment [316]. In malignant progression, epigenetic changes in the expression of 12 genes responsive to the TME stress suggest that coordinated transcriptional response of eukaryotic cells to microenvironment might be correlated with chemotherapy resistance of solid tumors [317]. Since tumor development is lead by physiological responses to an aberrant stromal environment, the interaction between the tumor and stromal cells determines tumoral progression [318]. In the chemokine network, epigenetic silencing of CXCR4 in SDF-1α/CXCR4 signaling of tumor microenvironment of cervical cancer cell lines and primary biopsy samples limited the cell response to the paracrine source of SDF-1α, which lead to loss of cell adhesion and disease progression [319]. Other authors reported miRNA's contribution to cancer progression and metastasis. While extracellular miRNAs are involved in cell–cell communi‐ cation and stromal remodeling [320], specific intracellular ones lead to cell proliferation through cancer-associated fibroblast activation [321].

The acquisition of invasive properties in tumor cells seems to be partially linked to epithelialmesenchymal transition by abrogation of homotypic cell–cell adhesion due to the absence of E-cadherin expression. Starting from the important role of transient E-cadherin expression in neoplasia, DesRoches and collaborators investigated its regulation by the microenvironment. Using 3D human tissue constructs, the authors suggested the role of epigenetic changes (DNA methylation, chromatin remodeling, and specific miRNA regulation) in the plasticity of Ecadherin-mediated adhesion in different tissue microenvironments during tumor cell invasion and metastasis [322]. The entry of the epithelial cells into the stroma is promoted through the E-cadherin intercellular junction disruption by MMP-3 and break down of the ECM collagen fibers by MMP-2 and MMP-9 [323]. MicroRNA suppression also influences the changes involved in epithelial–mesenchymal transition [324]. Reexpression of E-cadherin might reestablish cell–cell adhesion and may result in a mesenchymal–epithelial transition that might lead to proliferative growth of metastases.

Metastasis, as a multistage process (tumor cell migration from primary tumor, invasion of the surrounding tissues, intravasation into the circulation or the lymphatic system metastasis) involves communication with surrounding nonneoplastic cells [325] that can be epigenetically modulated to lead to ECM remodeling. Also, the epigenetic changes in the microenvironment have a significant impact on distant metastasis. In order to create a favorable local environment for cell proliferation in the metastatic sites, carcinoma cells induce epigenetic changes in both the stromal cells and bone marrow–derived cells [326]. The bone marrow cells are mobilized by the primary tumors to the metastatic sites before the actual metastasis creating a suitable microenvironment for metastasis [315, 327].

Due to their reversal character, epigenetic changes of TME might be targeted for controlling diseases and for therapeutic approach as drug resistance seems to also depend on TME. But, chemotherapeutic drug resistance depends at least partly on the TME rather than the tumor itself [328] and the combined treatment of both the tumor and the TME may be more efficient in the fight with cancer [315].

#### **4. Molecular and epigenetic factors involved in drug resistance**

Chemotherapy success is challenged by a multitude of intrinsic or acquired, molecular, genetic and epigenetic factors involved in drug transport, detoxification, signal transduction, gene expression, DNA repair, and programmed cell death. Drug resistance is a major challenge that chemotherapy should overcome. Even if the drug itself is efficient in destroying cancer cells, it is much more complicated to avoid triggering resistance than might appear at different levels of interaction between the drug and its cellular components.

The efflux mechanism is considered to be mainly responsible for the multiple drug resistance phenotypes in gynecologic cancers as well as in all types of cancers [329]. The process may be managed by cancer cells at the genetic and/or epigenetic level. While the genetic modifications of MDR1 and related multidrug resistance proteins were intensely explored over the past few decades, the contribution of epigenetic modification to the expression of MDR1 remains insufficiently explored in human gynecological cancers. It was observed that MDR1 was hypermethylated in 100% of ovarian cancer cell lines, and in 5 out of 13 (38%) primary ovarian cancers associated with loss of MDR1 mRNA expression in ovarian cancer cell lines, sustaining the importance role of epigenetic regulation in the expression of MDR1 and clinical treatment outcomes in human ovarian cancer [330]. However, in six ovarian cancer cell lines—W1MR, W1CR, W1DR, W1VR, W1TR, and W1PR that are respectively resistant to methotrexate, cisplatin, doxorubicin, vincristine, topotecan, and paclitaxel, P-gp is responsible for chemore‐ sistance and, in the case of methotrexate, was found to have a relation between the MRP2 transcript level and drug resistance [331]. Among inhibitors of Pgp MDR, valspodar, an analog of cyclosporine A, showed no clinical benefit in a phase III trial with paclitaxel and carboplatin [332], because while these agents can block drug efflux at the cellular level, the effects are not tumor specific, requiring a reduction in dosage for minimizing the side effects but also the therapeutic advantage. On the other hand, miRNA was involved in resistance through the regulation of MDR proteins at a posttranscriptional level. The interaction of miRNAs with the targeted mRNA can downmodulate MDR proteins improving the response to anticancer drugs. It was described [329] that miR-223 can downregulate ABCB1 and mRNA levels. miR-124a and miR-506 significantly decreased the protein level of MRP4 (ABCC4), which is another efflux membrane transporter; however, these miRNAs did not change the gene transcription levels [333]. In addition, although there are many modalities acting on efflux proteins in order to circumvent drug resistance, their effective action can be compromised due to the diversity of signal transduction pathways involved in transporter-mediated MDR, such as MAPK, JNK, PI3K, among others; as well as some transcription factors, like NF-κB, TNFα, and PTEN that could influence the levels of carrier proteins in different conditions [334].

Metastasis, as a multistage process (tumor cell migration from primary tumor, invasion of the surrounding tissues, intravasation into the circulation or the lymphatic system metastasis) involves communication with surrounding nonneoplastic cells [325] that can be epigenetically modulated to lead to ECM remodeling. Also, the epigenetic changes in the microenvironment have a significant impact on distant metastasis. In order to create a favorable local environment for cell proliferation in the metastatic sites, carcinoma cells induce epigenetic changes in both the stromal cells and bone marrow–derived cells [326]. The bone marrow cells are mobilized by the primary tumors to the metastatic sites before the actual metastasis creating a suitable

Due to their reversal character, epigenetic changes of TME might be targeted for controlling diseases and for therapeutic approach as drug resistance seems to also depend on TME. But, chemotherapeutic drug resistance depends at least partly on the TME rather than the tumor itself [328] and the combined treatment of both the tumor and the TME may be more efficient

Chemotherapy success is challenged by a multitude of intrinsic or acquired, molecular, genetic and epigenetic factors involved in drug transport, detoxification, signal transduction, gene expression, DNA repair, and programmed cell death. Drug resistance is a major challenge that chemotherapy should overcome. Even if the drug itself is efficient in destroying cancer cells, it is much more complicated to avoid triggering resistance than might appear at different levels

The efflux mechanism is considered to be mainly responsible for the multiple drug resistance phenotypes in gynecologic cancers as well as in all types of cancers [329]. The process may be managed by cancer cells at the genetic and/or epigenetic level. While the genetic modifications of MDR1 and related multidrug resistance proteins were intensely explored over the past few decades, the contribution of epigenetic modification to the expression of MDR1 remains insufficiently explored in human gynecological cancers. It was observed that MDR1 was hypermethylated in 100% of ovarian cancer cell lines, and in 5 out of 13 (38%) primary ovarian cancers associated with loss of MDR1 mRNA expression in ovarian cancer cell lines, sustaining the importance role of epigenetic regulation in the expression of MDR1 and clinical treatment outcomes in human ovarian cancer [330]. However, in six ovarian cancer cell lines—W1MR, W1CR, W1DR, W1VR, W1TR, and W1PR that are respectively resistant to methotrexate, cisplatin, doxorubicin, vincristine, topotecan, and paclitaxel, P-gp is responsible for chemore‐ sistance and, in the case of methotrexate, was found to have a relation between the MRP2 transcript level and drug resistance [331]. Among inhibitors of Pgp MDR, valspodar, an analog of cyclosporine A, showed no clinical benefit in a phase III trial with paclitaxel and carboplatin [332], because while these agents can block drug efflux at the cellular level, the effects are not tumor specific, requiring a reduction in dosage for minimizing the side effects but also the therapeutic advantage. On the other hand, miRNA was involved in resistance through the

**4. Molecular and epigenetic factors involved in drug resistance**

of interaction between the drug and its cellular components.

microenvironment for metastasis [315, 327].

38 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

in the fight with cancer [315].

Also, the signal transduction pathways can be involved in drug resistance. The Wnt signaling pathway, which is regulated by a multiprotein complex consisting of, among others, members of β-catenin, adenomatous polyposis coli APC, Axin, and GSK-3β [335], are involved in calcium-dependent cell adhesion due to the interaction between β-catenin and cadherin [336]. Different mutations in APC, promotes β-catenin proteolysis and reduces its transcriptional activity. PTEN, a lipid and protein phosphatase that is a negative regulator of phosphatidyli‐ nositol 3 (PI-3) kinase-dependent signaling interacts with the WNT pathway by impeding activation of integrin-linked kinase (ILK), which inhibits GSK-3β and thus causes accumula‐ tion of β-catenin [337]. The WNT signaling pathway is the most frequently altered pathway in the majority of cancers; therefore, individual components of the pathway are interesting targets for epigenetic inactivation. PI3K/Akt is another signaling pathway that is involved in acquired resistance of many cancers including gynecological ones. All of its isoforms (Akt1, Akt2, and Akt3) are activated (phosphorylated) by phosphatidylinositol 3-kinase (PI3-K) in response to growth factors and promote cell survival. It was demonstrated that the Akt pathway is directly related to the resistance of cancers against different drugs like sorafenib, trastuzumab, and erlotinib [329]. The epigenetic control of Akt and NF-κB is important for the establishment of drug resistance. RUNX3 suppresses Akt1 transcription by directly binding to the Akt1 promoter, and methylation of RUNX3 induces activation of the Akt signaling pathway [329].

Acquired resistance may develop additionally as blockage of apoptotic pathways or defective apoptotic signaling, often associated with loss of tumor suppressor protein p53, but also independent of p53, alteration of the control points of the cell cycle, increased ability to repair DNA, increased DNA damage tolerance, oncogene induction, and downmodulation of tumor suppressor genes. Eluding the normal process of programmed cell death is already known as a crucial strategy for cancer development and progression, but even more importantly, its participation in the intrinsic or acquired resistance of cancer cells to chemotherapy and radiation. Identification of the points of therapeutic intervention could potentially open up more efficient treatment opportunities. Epigenetic strategies might also be a feasible strategy to reactivate apoptosis or on the contrary to inactivate apoptosis-related genes that inhibit the process. However, it has now been demonstrated that inhibitors of DNA methylation and histone deacetylases can reactivate expression of tumor suppressor genes and induce histone hyperacetylation in the tumors of patients with cervical cancer after treatment with these agents. Preclinical studies have suggested a multitude of strategies to prevent or overcome resistance, but these approaches have not successfully translated to clinical practice yet [338].

#### **5. Conclusions**

This chapter underlined the importance of epigenetic events in gynecological cancer. Deci‐ phering the relevant epigenetic changes associated with each step of tumor development might improve molecular diagnostic and cancer risk assessment. Advances in elucidating epigenetic regulation in cancer disease, as well as in the development of technology, lead to the identifi‐ cation of potential biomarkers for diagnostic screening. As epigenetic changes occur early in neoplastic process, epigenetic biomarkers seem to be more sensitive and specific in cancer detection and some have already been tested for several types of cancer, alone or in combina‐ tion with traditional biomarkers. Unlike genetic changes, epigenetic alterations are essentially reversible and allow plasticity. These features are exploited and new therapeutic agents targeting epigenetic processes have been developed. The epigenetic changes of the trans‐ formed cells or TME can be modified by chemotherapeutic drugs and this epigenetic reversal therapy has potential in the future. In addition, miRNAs should be heavily explored as they might represent future alternatives for combined therapy of cancer. Many epigenetic targets are druggable and in order to overcome drug resistance, epigenetic therapy might also be a feasible strategy for induced cell death. Moreover, epigenetic patterns might be useful tools for therapy response prediction.

#### **Author details**

Coralia Bleotu1\*, Demetra Socolov2 , Mariana Anton3 , Anca Botezatu1 , Adriana Plesa1 , Iulia Virginia Iancu1 , Lorelei Irina Brasoveanu1 , Gabriela Anton1 and Carmen Cristina Diaconu1


#### **References**


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**5. Conclusions**

40 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

for therapy response prediction.

Coralia Bleotu1\*, Demetra Socolov2

Pol. 2006;53(2):245–256.

6–21.DOI: 10.1101/gad.947102.

**Author details**

Iulia Virginia Iancu1

**References**

Carmen Cristina Diaconu1

This chapter underlined the importance of epigenetic events in gynecological cancer. Deci‐ phering the relevant epigenetic changes associated with each step of tumor development might improve molecular diagnostic and cancer risk assessment. Advances in elucidating epigenetic regulation in cancer disease, as well as in the development of technology, lead to the identifi‐ cation of potential biomarkers for diagnostic screening. As epigenetic changes occur early in neoplastic process, epigenetic biomarkers seem to be more sensitive and specific in cancer detection and some have already been tested for several types of cancer, alone or in combina‐ tion with traditional biomarkers. Unlike genetic changes, epigenetic alterations are essentially reversible and allow plasticity. These features are exploited and new therapeutic agents targeting epigenetic processes have been developed. The epigenetic changes of the trans‐ formed cells or TME can be modified by chemotherapeutic drugs and this epigenetic reversal therapy has potential in the future. In addition, miRNAs should be heavily explored as they might represent future alternatives for combined therapy of cancer. Many epigenetic targets are druggable and in order to overcome drug resistance, epigenetic therapy might also be a feasible strategy for induced cell death. Moreover, epigenetic patterns might be useful tools

, Mariana Anton3

[1] Siedlecki P, Zielenkiewicz P. Mammalian DNA methyltransferases. Acta Biochim

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, Lorelei Irina Brasoveanu1

2 University of Medicine and Pharmacy "Gr. T. Popa", Iassy, Romania

3 Carol Davila University of Medicine and Pharmacy, Bucharest, Romania

\*Address all correspondence to: cbleotu@yahoo.com

1 Stefan S Nicolau Institute of Virology, Bucharest, Romania

, Anca Botezatu1

and

, Gabriela Anton1

, Adriana Plesa1

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## **Antiangiogenic Therapy in Epithelial Ovarian Cancer**

M.A. Alonso Bermejo, L. Rey Iglesias , M.E. Pérez López, A. Fernández Montes and J. García Mata

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60822

#### **Abstract**

Approximately 75% of women with ovarian cancer are diagnosed at advanced stages (FIGO stage III/IV), with 15-23 months median global survival and 20% 5 year survival. Angiogenesis plays an important role in tumour development and proliferation. Increased angiogenesis is associated with worse clinical outcome in ovarian cancer. Here we review the play of bevacizumab in the treatment of ovarian cancer and also other antiangiogenic drugs. In total, to date there are no promising results for most of the reviewed antiangiogenic agents, except those already known for bevacizumab, trebananib, pazopanib, cediranib and nintedanib. Ongoing re‐ search will shed more light on this fascinating tumour process and its control.

**Keywords:** angiogenesis, ovarian cancer

#### **1. Introduction**

Approximately 75% of women with ovarian cancer are diagnosed at advanced stages (FIGO stage III/IV), with 15–23 months median global survival and 20% 5-year survival [1].

Although approximately 80% of patients respond to first-line chemotherapy, more than 70% relapse and develop resistance to chemotherapy [2]. This requires the development of more effective treatments to improve survival in advanced disease. This was not achieved by adding a third cytotoxic agent to the standard treatment [3–7], and so the latest research is focused on new molecular targets.

© 2016 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.

Angiogenesis plays an important role in tumor development and proliferation. Increased angiogenesis is associated with worse clinical outcome in ovarian cancer.

The vascular endothelial growth factor (VEGF) family comprises VEGF-A (known as VEGF), VEGF-B, VEGF-C, VEGF-D, placental growth factor (PGF), VEGF-E, and VEGF-F (Figure 1).

**Figure 1.** VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and PGF bind differentially to receptors VEGFR-1, VEGFR-2, and VEGFR-3.

The relationship between VEGF overexpression, increased angiogenesis, and ovarian cancer development is well established, as well as in peritoneal dissemination and malignant ascites development [8]. Ovarian tumors overexpress several proangiogenic factors such as vascular endothelial growth factor (VEGF), angiopoietin, fibroblast growth factors, platelet-derived growth factors (PDGFs), and proangiogenic cytokines [9].

The most investigated is VEGF, which promotes endothelial cell proliferation and migra‐ tion for the formation of new blood vessels and increases the permeability of existing blood vessels [10].

**Figure 2.** Monoclonal antibodies (MoAb) inhibit VEGF ligand, and TKI inhibits the VEGFR.

The angiogenesis pathway can be inhibited by two strategies (Figure 2): inhibition of the VEGF ligand with antibodies or soluble receptors and inhibition of the VEGF receptor with tyrosine kinase inhibitors (TKI). Several antiangiogenic drugs have been studied in ovarian cancer in different settings, and we will review them in this chapter.

#### **2. Bevacizumab**

Angiogenesis plays an important role in tumor development and proliferation. Increased

The vascular endothelial growth factor (VEGF) family comprises VEGF-A (known as VEGF), VEGF-B, VEGF-C, VEGF-D, placental growth factor (PGF), VEGF-E, and VEGF-F (Figure 1).

**Figure 1.** VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and PGF bind differentially to receptors VEGFR-1,

The relationship between VEGF overexpression, increased angiogenesis, and ovarian cancer development is well established, as well as in peritoneal dissemination and malignant ascites development [8]. Ovarian tumors overexpress several proangiogenic factors such as vascular endothelial growth factor (VEGF), angiopoietin, fibroblast growth factors, platelet-derived

The most investigated is VEGF, which promotes endothelial cell proliferation and migra‐ tion for the formation of new blood vessels and increases the permeability of existing blood

growth factors (PDGFs), and proangiogenic cytokines [9].

**Figure 2.** Monoclonal antibodies (MoAb) inhibit VEGF ligand, and TKI inhibits the VEGFR.

VEGFR-2, and VEGFR-3.

vessels [10].

angiogenesis is associated with worse clinical outcome in ovarian cancer.

70 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

Bevacizumab is a recombinant humanized monoclonal antibody that binds to all VEGF isoforms [11]. It has shown its efficacy in different neoplasms, such as colorectal cancer, breast cancer, or kidney cancer.

To date, bevacizumab is the antiangiogenic agent that has shown the best results in the treatment of ovarian cancer.

The first-line efficacy data come from two phase III clinical trials: ICON7 and GOG 218.

The ICON7 clinical trial [12] randomized 1528 women to receive carboplatin (AUC 5 or 6) and paclitaxel (175 mg/m2 ) every 3 weeks for 6 cycles, or the same regimen plus bevacizumab (7.5 mg/kg) every 3 weeks during chemotherapy, followed by 12 cycles or until unacceptable toxicity or disease progression. The study included patients with initial (FIGO I/IIA), high-risk (G3 or clear cells), or advanced (FIGO IIB–IV) cancer stage. The primary objective was progression-free survival (PFS) measured by RECIST criteria, and the secondary objectives included overall survival (OS), response to treatment, toxicity, and quality of life. Most of the patients (94%) had good performance status (ECOG Performance Status 0–1). A total of 70% patients were diagnosed at FIGO stage IIIC/IV.

With a median follow-up of 19.4 months, PFS was greater in the group with bevacizumab, and the difference was statistically significant (19.0 months vs. 17.3 months; HR = 0.81 (95% CI = 0.70–0.94); *p* = 0.0041). This difference in PFS was greater in the high-risk-of-progression group (stage IIIC with suboptimal surgery and IV), 15.9 vs. 10.5 months; HR = 0.68 (95% CI = 0.55– 0.85); *p* < 0.001).

In the final analysis, with a median follow-up of 49 months, the increase in PFS is maintained in the high-risk-of-progression group, with an increase of 5.5 months (16.0 vs. 10.5 months; HR = 0.73 (95% CI = 0.61–0.88); *p* = 0.001), and there is an increase of 9.4 months in OS in the high-risk-of-progression group (39.7 vs. 30.3 months; HR = 0.78 (95% CI = 0.63–0.97); *p* = 0.03).

The treatment with bevacizumab was associated with an increase in bleeding (especially grade 1 mucocutaneous bleeding), grade 2 (G2) or greater acute hypertension (18% vs. 2%), grade 3 (G3) or higher thromboembolic events (7% vs. 3%), and gastrointestinal perforation (10 cases vs. 3 cases). The quality of life scores measured by the EORTC QLQ-C30 and QLQ-OV28 questionnaires show that continuation of treatment with bevacizumab appears to be associated with a small but clinically significant decline in quality of life compared to standard chemo‐ therapy, so PFS and quality of life over the period of time in question have to be considered when treatment decisions are made [13].

The GOG 218 clinical trial [14] randomized 1873 women with stage III (incompletely resected) or stage IV epithelial ovarian cancer after cytoreduction surgery in three groups. In the three groups, the patients received carboplatin AUC 6 and paclitaxel 175 mg/m2 every 3 weeks for 6 cycles and the study treatment. In the first arm (placebo arm), patients received placebo every 3 weeks from cycle 2 to cycle 22; in the second arm (bevacizumab initiation), patients received bevacizumab 15 mg/kg every 21 days from cycle 2 to cycle 6 followed by placebo from cycle 7 to cycle 22; in the third arm (bevacizumab throughout), patients received bevacizumab at the same dose from cycle 2 to cycle 22. The primary objective was PFS according to RECIST, CA-125, or clinical criteria. The secondary objectives included OS, safety, and quality of life. Once again, most of the patients (93%) maintained an ECOG PS 0-1. The group had a relatively poor prognosis, as 40% had stage III disease with residual disease greater than 1 cm and 26% had stage IV disease.

With a median follow-up of 17.4 months, the arm with bevacizumab (bevacizumab through‐ out) compared with the standard chemotherapy arm (placebo arm) showed a statistically significant increase in PFS (14.1 vs. 10.3 months; HR = 0.717 (95% CI = 0.625–0.824); *p* = 0.0001). In the bevacizumab initiation group, there was no increase in PFS (11.2 vs. 10.3 months; HR = 0.908 (95% CI = 0.759–1.040); *p* = 0.080). OS was similar in the three groups: 39.3, 38.7, and 39.7 months for the placebo arm, bevacizumab initiation group, and bevacizumab-throughout group, respectively, with no statistically significant differences.

G2 or higher hypertension was the only toxicity that was more common in a statistically significant manner in the bevacizumab groups than in the placebo arm (22.9% in bevacizumab throughout vs. 7.2% in the placebo arm). There were no differences in other toxicities such as gastrointestinal perforation or fistula, G3 or higher proteinuria, G4 neutropenia, febrile neutropenia, or venous or arterial thrombosis.

The main open questions left by these two trials are dosage and duration of treatment with bevacizumab. The dosage recommendation is to use the 15 mg/kg dose, which is in the summary of product characteristics, although benefit with bevacizumab is shown in a 7.5–15 mg/kg dose range [14]. As for treatment duration, both studies show that the widest separation of the PFS curves is found at 12 months in ICON7 and 15 months in GOG 218, which is at the termination of the bevacizumab. Thus, it is advisable to maintain bevacizumab treatment up to 15 months and to consider extending this period.

Two phase II studies showed the activity of bevacizumab in patients pretreated with chemo‐ therapy. Those studies included platinum-sensitive and platinum-resistant patients.

A first study conducted by the GOG, GOG 170D, evaluated the efficacy of bevacizumab at 15 mg/kg every 3 weeks in 62 patients with advanced ovarian cancer after having received one or more treatment lines for advanced disease and found a 21% response rate and 10.3 months median response duration, with no intestinal perforation [15].

A second study in 70 patients evaluated the addition of cyclophosphamide (50 mg orally per day) to bevacizumab (10 mg/kg every 2 weeks) in patients who had received one to three previous chemotherapy lines and found a 24% response rate, 56% of patients relapse-free at 6 months, and 5.7% suffering intestinal perforations [16].

Subsequently, the OCEANS trial was initiated as a phase II study; after a safety review focused on intestinal perforations, it was converted to a phase III trial. Then 484 patients with platinumsensitive recurrent ovarian cancer were randomized to receive carboplatin/gemcitabine for 6– 10 cycles with bevacizumab (15 mg/m² every 3 weeks) or placebo until disease progression or unacceptable toxicity. Progression was documented by radiological (RECIST 1.0) or clinical criteria but not by CA-12.5 elevations. The primary objective was PFS determined by the investigators. The secondary objectives included response rate and OS. It also included an analysis of the primary objective by an independent committee.

The GOG 218 clinical trial [14] randomized 1873 women with stage III (incompletely resected) or stage IV epithelial ovarian cancer after cytoreduction surgery in three groups. In the three groups, the patients received carboplatin AUC 6 and paclitaxel 175 mg/m2 every 3 weeks for 6 cycles and the study treatment. In the first arm (placebo arm), patients received placebo every 3 weeks from cycle 2 to cycle 22; in the second arm (bevacizumab initiation), patients received bevacizumab 15 mg/kg every 21 days from cycle 2 to cycle 6 followed by placebo from cycle 7 to cycle 22; in the third arm (bevacizumab throughout), patients received bevacizumab at the same dose from cycle 2 to cycle 22. The primary objective was PFS according to RECIST, CA-125, or clinical criteria. The secondary objectives included OS, safety, and quality of life. Once again, most of the patients (93%) maintained an ECOG PS 0-1. The group had a relatively poor prognosis, as 40% had stage III disease with residual disease greater than 1 cm and 26%

With a median follow-up of 17.4 months, the arm with bevacizumab (bevacizumab through‐ out) compared with the standard chemotherapy arm (placebo arm) showed a statistically significant increase in PFS (14.1 vs. 10.3 months; HR = 0.717 (95% CI = 0.625–0.824); *p* = 0.0001). In the bevacizumab initiation group, there was no increase in PFS (11.2 vs. 10.3 months; HR = 0.908 (95% CI = 0.759–1.040); *p* = 0.080). OS was similar in the three groups: 39.3, 38.7, and 39.7 months for the placebo arm, bevacizumab initiation group, and bevacizumab-throughout

G2 or higher hypertension was the only toxicity that was more common in a statistically significant manner in the bevacizumab groups than in the placebo arm (22.9% in bevacizumab throughout vs. 7.2% in the placebo arm). There were no differences in other toxicities such as gastrointestinal perforation or fistula, G3 or higher proteinuria, G4 neutropenia, febrile

The main open questions left by these two trials are dosage and duration of treatment with bevacizumab. The dosage recommendation is to use the 15 mg/kg dose, which is in the summary of product characteristics, although benefit with bevacizumab is shown in a 7.5–15 mg/kg dose range [14]. As for treatment duration, both studies show that the widest separation of the PFS curves is found at 12 months in ICON7 and 15 months in GOG 218, which is at the termination of the bevacizumab. Thus, it is advisable to maintain bevacizumab treatment up

Two phase II studies showed the activity of bevacizumab in patients pretreated with chemo‐

A first study conducted by the GOG, GOG 170D, evaluated the efficacy of bevacizumab at 15 mg/kg every 3 weeks in 62 patients with advanced ovarian cancer after having received one or more treatment lines for advanced disease and found a 21% response rate and 10.3 months

A second study in 70 patients evaluated the addition of cyclophosphamide (50 mg orally per day) to bevacizumab (10 mg/kg every 2 weeks) in patients who had received one to three previous chemotherapy lines and found a 24% response rate, 56% of patients relapse-free at 6

therapy. Those studies included platinum-sensitive and platinum-resistant patients.

group, respectively, with no statistically significant differences.

72 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

neutropenia, or venous or arterial thrombosis.

to 15 months and to consider extending this period.

median response duration, with no intestinal perforation [15].

months, and 5.7% suffering intestinal perforations [16].

had stage IV disease.

The addition of bevacizumab to carboplatin/gemcitabine showed a median increase in PFS of 12.4 months versus 8.4 months (HR = 0.484, 95% CI = 0.388–0.605). These figures were repeated when evaluated by an independent committee (12.3 months vs. 8.6 months, *p* = 0.0001). This increase benefited all subgroups irrespective of age, ECOG, presence of cytoreduction surgery, time since last recurrence, and CA-12.5 levels. The secondary objectives also showed a 21.1% increase in response rate in the bevacizumab arm (response rate 78.5% vs. 57.4%, *p* = 0.0001). At cutoff date with a small number of events, there were not statistically significant differences in median survival, 35.2 months in the placebo arm and 33.3 months in the bevacizumab arm.

The most relevant G3 toxicities in the bevacizumab arm were proteinuria (0.9% vs. 8.5%) and hypertension (0.4% vs. 17.4%). No gastrointestinal perforation was documented during the study [17].

This study is the first randomized trial to describe the role of bevacizumab in platinumsensitive disease. In this context, there is an ongoing study, GOG 213, which is evaluating the addition of bevacizumab to carboplatin and paclitaxel in platinum-sensitive relapse, with OS as its primary objective [18].

Two more studies are also evaluating bevacizumab in this context. The MITO-16/Mango OV-2BBP study is evaluating the addition of bevacizumab versus placebo to a carboplatin regimen with gemcitabine or pegylated liposomal adriamycin or paclitaxel in platinumsensitive disease. Its primary objective is PFS, and OS is one of its secondary objectives [19]. The AGO/OVAR 2.21 study aims to show superiority in PFS for the carboplatin regimen with pegylated liposomal adriamycin and bevacizumab versus carboplatin with gemcitabine and bevacizumab [20].

There is a single phase II study in platinum-resistant disease; 44 patients who had received two or three previous treatment lines that included topotecan or liposomal anthracyclines were treated with bevacizumab, finding a 15.9% response rate and 27.8% of patients disease-free at 6 months. Perforations were found in a large percentage of patients, 11.4%, leading to its premature closure [21].

The AURELIA trial tested the addition of an antiangiogenic drug to chemotherapy in plati‐ num-resistant disease. In this trial, 361 women with platinum-resistant ovarian cancer were randomized to receive single-agent chemotherapy (paclitaxel, pegylated liposomal doxorubi‐ cin, or topotecan) or the same chemotherapy with bevacizumab (10 mg/kg every 2 weeks or 15 mg/kg every 3 weeks in the regimen that included topotecan every 3 weeks). The therapeutic regimen was to be decided by the investigators, permitting any of those mentioned above. On the basis of the high rate of gastrointestinal perforations found in the aforementioned phase II study, the inclusion of both platinum-resistant patients who had received at least two lines of chemotherapy and platinum-refractory patients (progression while being treated with platinum) was ruled out, as well as patients with a history of intestinal obstruction (including subocclusive cases), intestinal perforation, abdominal fistula, intra-abdominal abscess, rectum or sigmoid colon affected by the disease, intestinal affectation by CT, or radiotherapy on the abdomen or pelvis. Its primary objective was PFS evaluated by the investigator. The secondary objectives included radiological response rate according to RECIST 1.0 criteria and CA-12.5, OS, safety, tolerability, and quality of life.

PFS was greater in the bevacizumab arm, with 6.7 months versus 3.4 months (HR = 0.42, *p* < 0.001), showing its efficacy in all the subgroups analyzed. There was also a greater response rate (27.3% in the bevacizumab arm vs. 11.8% in the placebo arm, *p* = 0.001) according to radiologic criteria and also a greater CA-12.5 serologic response rate, 11.6% in the placebo arm and 31.8% in the bevacizumab arm. There were no differences in OS (HR = 0.85; 95% CI = 0.66– 1.08; *p* = 0.174). This finding can possibly be explained by the crossover in 40% of the patients assigned to the chemotherapy arm.

Toxicity in the bevacizumab arm included greater proteinuria and G3 hypertension. There was also a 2.2% incidence of gastrointestinal perforations. Overall, this is the first study to evaluate that the addition of an antiangiogenic to chemotherapy has an impact on PFS [22].

Another question to be investigated is the combination of bevacizumab with other strategies that have shown good results in the first-line treatment of ovarian cancer, such as intense doses of chemotherapy or intraperitoneal chemotherapy.

The combination of bevacizumab with intense-dose chemotherapy was studied in the OCTA‐ VIA phase II clinical trial [23]. The primary objective was PFS according to RECIST criteria; the secondary objectives included the overall response rate, response duration in responder patients, OS, progression defined by CA-125, safety, and tolerability. The study included 189 patients diagnosed with stage I/IIA (grade 3/clear cells) or stages IIB–IV (any grade) ovarian cancer who underwent surgery. The patients received 6–8 cycles of bevacizumab (7.5 mg/kg, day 1 every 3 weeks) with weekly paclitaxel (80 mg/m2 , days 1, 8, and 15 every 3 weeks) and carboplatin (AUC 6, day 1 every 3 weeks); bevacizumab continued at the same dose as a single agent every 3 weeks up to a total of 17 cycles (1 year). A PFS of more than 18 months was considered to be clinically significant. Most of the patients were stage IIIC/IV (74%). With a median follow-up of 26.3 months, PFS was 23.7 months (95% CI = 19.8–26.4). PFS in the patients with stage III disease and >1.0 cm of residual disease after debulking surgery, or with stage IV disease, was 18.1 months. The response rate by RECIST in the 91 patients with measurable disease was 84.6% (95%CI = 75.5–91.3%), with 30.8% (95% CI = 21.5–41.3%) of complete responses. OS at 1 and 2 years was 97.8% and 92.1%, respectively. The most common grade ≥ 3 undesirable effects related to bevacizumab were hypertension (4.2%) and thromboembolic events (6.3%). In the study update [24], the limited number of events (17% of patients) for the planned final study analysis means that the OS results would be premature.

The combination of bevacizumab and chemotherapy at intense doses was also studied in the phase III GOG 262 clinical trial, which compared the standard chemotherapy regimen with carboplatin and paclitaxel every 3 weeks versus carboplatin every 3 weeks and paclitaxel weekly. The patients could also receive bevacizumab at the investigator's discretion [25].

The combination of bevacizumab and intraperitoneal chemotherapy is being studied in the GOG 252 phase III clinical trial [26].

#### **3. Trebananib**

the basis of the high rate of gastrointestinal perforations found in the aforementioned phase II study, the inclusion of both platinum-resistant patients who had received at least two lines of chemotherapy and platinum-refractory patients (progression while being treated with platinum) was ruled out, as well as patients with a history of intestinal obstruction (including subocclusive cases), intestinal perforation, abdominal fistula, intra-abdominal abscess, rectum or sigmoid colon affected by the disease, intestinal affectation by CT, or radiotherapy on the abdomen or pelvis. Its primary objective was PFS evaluated by the investigator. The secondary objectives included radiological response rate according to RECIST 1.0 criteria and CA-12.5,

PFS was greater in the bevacizumab arm, with 6.7 months versus 3.4 months (HR = 0.42, *p* < 0.001), showing its efficacy in all the subgroups analyzed. There was also a greater response rate (27.3% in the bevacizumab arm vs. 11.8% in the placebo arm, *p* = 0.001) according to radiologic criteria and also a greater CA-12.5 serologic response rate, 11.6% in the placebo arm and 31.8% in the bevacizumab arm. There were no differences in OS (HR = 0.85; 95% CI = 0.66– 1.08; *p* = 0.174). This finding can possibly be explained by the crossover in 40% of the patients

Toxicity in the bevacizumab arm included greater proteinuria and G3 hypertension. There was also a 2.2% incidence of gastrointestinal perforations. Overall, this is the first study to evaluate

Another question to be investigated is the combination of bevacizumab with other strategies that have shown good results in the first-line treatment of ovarian cancer, such as intense doses

The combination of bevacizumab with intense-dose chemotherapy was studied in the OCTA‐ VIA phase II clinical trial [23]. The primary objective was PFS according to RECIST criteria; the secondary objectives included the overall response rate, response duration in responder patients, OS, progression defined by CA-125, safety, and tolerability. The study included 189 patients diagnosed with stage I/IIA (grade 3/clear cells) or stages IIB–IV (any grade) ovarian cancer who underwent surgery. The patients received 6–8 cycles of bevacizumab (7.5 mg/kg,

carboplatin (AUC 6, day 1 every 3 weeks); bevacizumab continued at the same dose as a single agent every 3 weeks up to a total of 17 cycles (1 year). A PFS of more than 18 months was considered to be clinically significant. Most of the patients were stage IIIC/IV (74%). With a median follow-up of 26.3 months, PFS was 23.7 months (95% CI = 19.8–26.4). PFS in the patients with stage III disease and >1.0 cm of residual disease after debulking surgery, or with stage IV disease, was 18.1 months. The response rate by RECIST in the 91 patients with measurable disease was 84.6% (95%CI = 75.5–91.3%), with 30.8% (95% CI = 21.5–41.3%) of complete responses. OS at 1 and 2 years was 97.8% and 92.1%, respectively. The most common grade ≥ 3 undesirable effects related to bevacizumab were hypertension (4.2%) and thromboembolic events (6.3%). In the study update [24], the limited number of events (17% of patients) for the

planned final study analysis means that the OS results would be premature.

, days 1, 8, and 15 every 3 weeks) and

that the addition of an antiangiogenic to chemotherapy has an impact on PFS [22].

OS, safety, tolerability, and quality of life.

74 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

assigned to the chemotherapy arm.

of chemotherapy or intraperitoneal chemotherapy.

day 1 every 3 weeks) with weekly paclitaxel (80 mg/m2

Angiopoietins 1 and 2 (Ang1 and Ang2) are ligands of the Tie2 receptor, which is expressed in endothelial and some hematopoietic and lymph cells, mediating in vascular remodeling; it has a different signaling pathway from VEGF.

Trebananib (AMG 386) is a peptibody that inhibits angiopoietin 1 and 2, preventing interaction with the Tie2 receptor; it shows antiangiogenic effects in preclinical ovarian cancer models.

Having shown its safety and efficacy when administered intravenously in monotherapy at a maximum dose of 30 mg/kg weekly in phase I studies [27], and increasing PFS in phase II studies that used a dose of 10 mg/kg [28], it can be inferred that this benefit will be even greater using higher doses (such as 15 mg/kg), as doses of up to 30 mg/kg are tolerated without an increased toxicity.

The phase III study (TRINOVA-1) [29] examines the addition of trebananib to weekly paclitaxel versus weekly paclitaxel, showing a significant increase in PFS. It is a randomized, doubleblind, placebo-controlled study. The inclusion criteria are as follows: woman over 18 years of age with histological diagnosis of epithelial ovarian cancer; primary peritoneal cancer or Fallopian tube cancer; having previously received chemotherapy based on a platinum regimen and progressed to 2 treatment lines; performance status 0–1; correct hematological, hepatic, and renal function; correct blood pressure figures (accepting appropriate control by taking antihypertensive treatment); and life expectancy of 3 months or more. Platinum-sensitive patients (platinum-free interval of more than 12 months) and platinum-refractory patients (disease recurrence or progression in the first 6 months or less after starting first-line platinumbased chemotherapy) were excluded and were also excluded in other histologies such as borderline, mucous and clear-cell tumors, patients who had presented a thromboembolic or hemorrhagic event in the last 12 months, unhealed wound, ulcer, fracture or infection, metastasis in the central nervous system, presence of grade 1 or higher neuropathy, presence of hepatitis B or C virus, and HIV infection. A total of 912 patients were randomized to receive in a 1:1 proportion of placebo and paclitaxel weekly or trebananib and paclitaxel weekly. The patients were stratified by platinum-free interval (0–6 months, or more than 6 months but less than 12), geographic region, and presence of radiologically measurable disease or not.

Patients received 80 mg/m² of paclitaxel IV (3 weeks on and 1 week off) and placebo or 15 mg/ kg i.v. trebananib weekly, until progression according to RECIST 1.1 criteria, toxicity, or withdrawal of consent. A reduction in the dose of paclitaxel was allowed, but not of the placebo or trebananib. If necessary due to toxicity (e.g., edema), the drug was suspended until the toxicity was resolved, and it was definitively suspended if the delay due to toxic effects lasted more than 28 days. The patients were reassessed every 8 weeks by computerized tomography (CT) of the chest, abdomen, and pelvis. The tumor marker (specific cancer antigen (CA-125)) did not contribute to the assessment of disease response and progression. The primary objective was PFS, and the secondary objectives are survival and response rate.

The groups comprised 458 patients in the control arm (paclitaxel–placebo) and 461 in the experimental arm (paclitaxel–trebananib); median patient follow-up was 10.1 months.

Median PFS was greater in the group that received trebananib (5.4 vs. 7.2 months, HR = 0.6, 95% CI = 0.57–0.77), *p* < 0.0001, with all patient subgroups benefiting. The response rate by RECIST was greater in the experimental group (30% vs. 38%), and this difference was greater according to CA-125 levels, with a significant reduction of this (49% vs. 56%, *p* = 0.03). The interim overall OS analysis showed no differences between the groups (17.3 months vs. 19 months, HR = 0.86, 95% CI = 0.69–1.08, *p* = 0.19). Grade 3 or more side effects were described in 28% of the control subjects (paclitaxel–placebo) and 34% of the experimental group (pacli‐ taxel–trebananib). The most common adverse reaction to the study drug was edema, which even became a cause of suspension of the treatment. Cases of hypertension, bleeding, pulmo‐ nary and arterial thromboembolism, proteinuria, and gastrointestinal perforations were also described. Trebananib added to paclitaxel for the treatment of recurring ovarian cancer significantly increases PFS versus placebo.

There are 2 phase III studies in which trebananib is added to pegylated liposomal doxorubicin (TRINOVA-2) [30] and trebananib in the first line associated to carboplatin–paclitaxel (TRI‐ NOVA-3) [31], although no conclusions have yet been reached.

#### **4. Pazopanib**

Pazopanib is an orally administered multikinase inhibitor of vascular endothelial growth factor receptor (VEGFR)-1/-2/-3 and of platelet-derived growth factor receptor (PDGFR)-α/-β and of c-Kit.

This drug has been approved for the treatment of metastatic renal cancer and soft tissue sarcomas.

Its role in several combinations has been analyzed, initially in a phase I/II study in combination with carboplatin and paclitaxel, after surgery, in order to increase the disease-free interval. This open-label phase I/II study was conducted to evaluate the safety and efficacy of paclitaxel 175 mg/m² plus carboplatin (AUC 5 (group A) or AUC 6 (group B)) once every 3 weeks for up to six cycles, with either 800 or 400 mg per day of pazopanib. Dose-limiting toxicities (DLT) were detected in two of the first six patients included in the pazopanib 800 mg plus paclitaxel 175 mg/m² plus carboplatin AUC 5 arm. There was also DLT in 2 of these first 6 patients at the lowest dosage level (pazopanib 400 mg plus paclitaxel 175 mg/m² plus carboplatin AUC 5). Two of the 4 DLTs were gastrointestinal perforations, and severe myelotoxicity was reported in 6 of the 12 patients, leading to suspension of the study [32].

Subsequently, its use in monotherapy was investigated in a multicenter, nonrandomized, phase II study (VEG104450; NCT00281632) in patients with recurrence of epithelial ovarian or Fallopian tube cancer or primary peritoneal carcinoma who had presented complete response of CA-125 levels with platinum-based chemotherapy regimens. At relapse, patients with CA-12 levels reaching ≥42 U/mL (>2 × ULN) were treated with pazopanib 800 mg/day until progression or toxicity. Inclusion criteria were ECOG 0–1 and good hepatic and renal function. The primary objective was the response rate (determined by normalization of CA-125 levels or not), and the secondary objectives were overall response (measured as biochemical, radiological, and physical response) and PFS. Eleven out of 36 patients (31%) presented CA-125 response with a mean duration of 113 days; the overall response rate in patients with meas‐ urable disease was 18%. The most common adverse events leading to suspension of the treatment were transaminase elevation, with only 1 case of grade 4 edema [33].

This led to the study of the role of pazopanib as maintenance therapy in ovarian cancer patients who had not progressed during first-line chemotherapy. A total of 940 patients were included, with epithelial ovarian, Fallopian tube, or primary peritoneal cancer; FIGO stages II–IV, with no evidence of progression after surgery; and 6 cycles of platinum plus taxane chemotherapy. They were randomized 1:1 to receive pazopanib 800 mg once daily or placebo for 24 months. The primary objective was PFS by RECIST 1.0 criteria. It was shown that maintenance therapy with pazopanib increased PFS compared with placebo: 17.9 vs. 12.3 months, HR = 0.77; 95% CI = 0.64–0.91; *p* = 0.0021. Grades 3 and 4 adverse events were hypertension (30.8%), neutro‐ penia (9.9%), transaminase elevation (9.4%), diarrhea (8.2%), fatigue (2.7%), thrombocytopenia (2.5%), and palmoplantar erythrodysesthesia (1.9%) in the pazopanib arm. Suspension of the treatment was significantly greater in the pazopanib arm (33.3%) versus placebo (5.6%) [34].

Maintenance therapy with pazopanib leads to an improvement in the median PFS of 5.6 months (HR = 0.77), with a 23% risk reduction for 2 years in women with FIGO stages II–IV who had not progressed to the first line of treatment. An increase in OS has not yet been shown, so the use of pazopanib is not currently recommended for this clinical situation.

#### **5. Nintedanib**

or trebananib. If necessary due to toxicity (e.g., edema), the drug was suspended until the toxicity was resolved, and it was definitively suspended if the delay due to toxic effects lasted more than 28 days. The patients were reassessed every 8 weeks by computerized tomography (CT) of the chest, abdomen, and pelvis. The tumor marker (specific cancer antigen (CA-125)) did not contribute to the assessment of disease response and progression. The primary

The groups comprised 458 patients in the control arm (paclitaxel–placebo) and 461 in the experimental arm (paclitaxel–trebananib); median patient follow-up was 10.1 months.

Median PFS was greater in the group that received trebananib (5.4 vs. 7.2 months, HR = 0.6, 95% CI = 0.57–0.77), *p* < 0.0001, with all patient subgroups benefiting. The response rate by RECIST was greater in the experimental group (30% vs. 38%), and this difference was greater according to CA-125 levels, with a significant reduction of this (49% vs. 56%, *p* = 0.03). The interim overall OS analysis showed no differences between the groups (17.3 months vs. 19 months, HR = 0.86, 95% CI = 0.69–1.08, *p* = 0.19). Grade 3 or more side effects were described in 28% of the control subjects (paclitaxel–placebo) and 34% of the experimental group (pacli‐ taxel–trebananib). The most common adverse reaction to the study drug was edema, which even became a cause of suspension of the treatment. Cases of hypertension, bleeding, pulmo‐ nary and arterial thromboembolism, proteinuria, and gastrointestinal perforations were also described. Trebananib added to paclitaxel for the treatment of recurring ovarian cancer

There are 2 phase III studies in which trebananib is added to pegylated liposomal doxorubicin (TRINOVA-2) [30] and trebananib in the first line associated to carboplatin–paclitaxel (TRI‐

Pazopanib is an orally administered multikinase inhibitor of vascular endothelial growth factor receptor (VEGFR)-1/-2/-3 and of platelet-derived growth factor receptor (PDGFR)-α/-β

This drug has been approved for the treatment of metastatic renal cancer and soft tissue

Its role in several combinations has been analyzed, initially in a phase I/II study in combination with carboplatin and paclitaxel, after surgery, in order to increase the disease-free interval. This open-label phase I/II study was conducted to evaluate the safety and efficacy of paclitaxel 175 mg/m² plus carboplatin (AUC 5 (group A) or AUC 6 (group B)) once every 3 weeks for up to six cycles, with either 800 or 400 mg per day of pazopanib. Dose-limiting toxicities (DLT) were detected in two of the first six patients included in the pazopanib 800 mg plus paclitaxel 175 mg/m² plus carboplatin AUC 5 arm. There was also DLT in 2 of these first 6 patients at the lowest dosage level (pazopanib 400 mg plus paclitaxel 175 mg/m² plus carboplatin AUC 5).

objective was PFS, and the secondary objectives are survival and response rate.

76 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

significantly increases PFS versus placebo.

**4. Pazopanib**

and of c-Kit.

sarcomas.

NOVA-3) [31], although no conclusions have yet been reached.

Nintedanib (BIBF 1120) is a powerful triple angiokinase inhibitor. It inhibits VEGFR-1, VEGFR-2, and VEGFR-3; PDGFR α and β; and FGFR-1, FGFR-2, and FGFR-3. The first data, obtained in a phase I study, in combination with carboplatin and paclitaxel administered twice a day, with doses of 100–250 mg, in 22 patients with locally advanced or recurring metastatic ovarian cancer, indicated that the maximum tolerated dose was 200 mg/12 h. Higher doses were related to higher significant gastrointestinal toxicity in the form of diarrhea, alanine aminotrnasferase (ALT) and aspartate aminotransferase (AST) elevation, and hematological toxicity, basically in white blood cells [35].

Nintedanib 250 mg/12 h was studied in a phase II randomized trial versus placebo in 83 women with recurring (in second or subsequent lines) ovarian cancer who had responded to chemo‐ therapy but presented high risk of relapse. The primary objectives were PFS, OS, and toxicity. The patients received at least 9 cycles of the treatment or placebo, continuing until disease progression (unless the patient withdrew from the study). PFS at 36 weeks was 16.3% versus 5%, with HR = 0.65, 95% CI = 0.42–1.02, and *p* = 0.06. The reported G3/4 adverse events were similar in both arms (34.9% vs. 27.5%, respectively, *p* = 0.49), but in the nintedanib group, there was greater gastrointestinal toxicity in the form of diarrhea, nausea, and vomiting (*p* < 0.001) versus placebo. Likewise, there was greater G3/4 hepatotoxicity with nintedanib versus placebo (51.2% vs. 7.5%, *p* < 0.001 [36]).

There is currently an ongoing phase III, randomized, double-blind trial (AGO-OVAR 12/ LUME) in first-line treatment of locally advanced or metastatic ovarian cancer (stages IIB–IV) that compares the combination of carboplatin, paclitaxel, and nintedanib 200 mg/12 h and maintenance with nintedanib for 120 weeks versus the same chemotherapy regimen with placebo and maintenance with placebo, also for 120 weeks. A total of 1366 patients have been included, and to date, it has reported an advantage in PFS in favor of the arm with the combination of nintedanib with chemotherapy and subsequent maintenance (17.3 months vs. 16.6 months), HR = 0.84 and 95% CI (0.72–0.98), *p* = 0.0239, versus placebo [37].

Another phase II trial is being conducted with this drug in recurrent or persistent disease or disease already treated with bevacizumab [38].

#### **6. Cediranib**

Cediranib (AZD 2171) is a tyrosine kinase agent with antiangiogenic activity, blocking the VEGF receptor (VEGFR 1, VEGFR2 and VEGFR3) and c-Kit.

Although women were included in the phase I study of the drug, they did not have ovarian cancer. It was found that the dose was tolerable up to 45 mg/24 h [39]. The phase II studies used both this and lower doses.

At the 2008 ASCO meeting, Hirte et al. presented data from 60 patients with relapse of ovarian or Fallopian tube carcinoma or peritoneal carcinomatosis treated with cediranib, showing that the 30 mg/24 h dose was well tolerated and active in this patient group [40].

In 2009, the results of a phase II study were published, including 46 patients with platinumsensitive and platinum-resistant relapse of ovarian carcinoma, Fallopian tube carcinoma, or peritoneal carcinomatosis. The patients received cediranib 45 mg/24 h until progression, intolerable toxicity, or withdrawal of consent. After the toxicity seen in 11 patients, the dose was reduced to 30 mg/24 h. More than 20% of the patients presented G3 adverse events, the most common being hypertension (46%), fatigue (24%), and diarrhea (13%); 8.7% of them presented G4 adverse events [41].

The randomized, double-blind ICON 6 study compares a platinum-based chemotherapy arm with cediranib (concurrent), another similar arm (concurrent) plus continuation (for 18 months or until progression) of maintenance cediranib, and the same chemotherapy regimen with placebo in relapse of platinum-sensitive disease. The preliminary data (60 women enrolled) show benefit with the combination of cediranib and its maintenance versus the arm in which placebo was added, both in PFS (11.4 vs. 9.4 months, HR = 0.68, *p* = 0.0022) and in OS (20.3 vs. 17.6 months, HR = 0.70, *p* = 0.049). The dose of cediranib had to be reduced during the study to 20 mg/24 h due to toxicity and reduced adherence to treatment [42]. The results update at the 2013 ESMO congress show greater benefit in PFS (12.5 vs. 9.4 months, HR = 0.57, *p* = 0.00001) and a benefit of 2.7 months in OS in the cediranib maintenance group. It is the first oral antiangiogenic agent to date to show benefit in terms of OS. The most common adverse events were diarrhea, nausea, and fatigue [43].

Cediranib was combined with olaparib in a randomized phase II study versus olaparib in monotherapy in women presenting platinum-sensitive relapse of ovarian cancer associated with BRCA mutation. The patients received olaparib 200 mg/24 h and cediranib 30 mg/24h in the combination arm and olaparib 400 mg/24 h in the monotherapy arm. PFS was 17.7 months with the combination and 9 months with olaparib in monotherapy, HR = 2.9 and 95% CI (1.5– 5.6), *p* = 0.001. There were 2 complete and 21 partial responses, 56% objective responses with the monotherapy, and 3 complete, 33 partial, and 84% objective responses in the experimental group. The incidence of G3/4 adverse events was 70% with the combination and only 7% with olaparib; the most common were fatigue, diarrhea, and hypertension [44].

#### **7. Aflibercept**

Nintedanib 250 mg/12 h was studied in a phase II randomized trial versus placebo in 83 women with recurring (in second or subsequent lines) ovarian cancer who had responded to chemo‐ therapy but presented high risk of relapse. The primary objectives were PFS, OS, and toxicity. The patients received at least 9 cycles of the treatment or placebo, continuing until disease progression (unless the patient withdrew from the study). PFS at 36 weeks was 16.3% versus 5%, with HR = 0.65, 95% CI = 0.42–1.02, and *p* = 0.06. The reported G3/4 adverse events were similar in both arms (34.9% vs. 27.5%, respectively, *p* = 0.49), but in the nintedanib group, there was greater gastrointestinal toxicity in the form of diarrhea, nausea, and vomiting (*p* < 0.001) versus placebo. Likewise, there was greater G3/4 hepatotoxicity with nintedanib versus

There is currently an ongoing phase III, randomized, double-blind trial (AGO-OVAR 12/ LUME) in first-line treatment of locally advanced or metastatic ovarian cancer (stages IIB–IV) that compares the combination of carboplatin, paclitaxel, and nintedanib 200 mg/12 h and maintenance with nintedanib for 120 weeks versus the same chemotherapy regimen with placebo and maintenance with placebo, also for 120 weeks. A total of 1366 patients have been included, and to date, it has reported an advantage in PFS in favor of the arm with the combination of nintedanib with chemotherapy and subsequent maintenance (17.3 months vs.

Another phase II trial is being conducted with this drug in recurrent or persistent disease or

Cediranib (AZD 2171) is a tyrosine kinase agent with antiangiogenic activity, blocking the

Although women were included in the phase I study of the drug, they did not have ovarian cancer. It was found that the dose was tolerable up to 45 mg/24 h [39]. The phase II studies

At the 2008 ASCO meeting, Hirte et al. presented data from 60 patients with relapse of ovarian or Fallopian tube carcinoma or peritoneal carcinomatosis treated with cediranib, showing that

In 2009, the results of a phase II study were published, including 46 patients with platinumsensitive and platinum-resistant relapse of ovarian carcinoma, Fallopian tube carcinoma, or peritoneal carcinomatosis. The patients received cediranib 45 mg/24 h until progression, intolerable toxicity, or withdrawal of consent. After the toxicity seen in 11 patients, the dose was reduced to 30 mg/24 h. More than 20% of the patients presented G3 adverse events, the most common being hypertension (46%), fatigue (24%), and diarrhea (13%); 8.7% of them

The randomized, double-blind ICON 6 study compares a platinum-based chemotherapy arm with cediranib (concurrent), another similar arm (concurrent) plus continuation (for 18 months

the 30 mg/24 h dose was well tolerated and active in this patient group [40].

16.6 months), HR = 0.84 and 95% CI (0.72–0.98), *p* = 0.0239, versus placebo [37].

placebo (51.2% vs. 7.5%, *p* < 0.001 [36]).

78 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

disease already treated with bevacizumab [38].

VEGF receptor (VEGFR 1, VEGFR2 and VEGFR3) and c-Kit.

**6. Cediranib**

used both this and lower doses.

presented G4 adverse events [41].

Aflibercept is a recombinant fusion protein, also called VEFG-Trap, that binds to and neutral‐ izes all forms of VEGF-A and VEGF-B and inhibits placental growth factor (PGF) activation. In several preclinical models, it was seen to inhibit tumor growth and metastasis formation, and another study showed evident reduction in ascites and tumor size in murine models that developed human ovarian tumors [45].

Various phase II studies have tested the activity of this drug in ovarian cancer. In the first of these, aflibercept is combined with docetaxel in 49 patients with platinum-resistant relapse with a maximum of two previous chemotherapy regimens. It was administered at a dose of 6 mg/kg until progression or intolerable toxicity. There was a 54% response rate; 10 cases presented complete response, and relapse was not detected in 4 of them at 1-year posttreatment (range 5–22 months). Median PFS was 6.2 months, and OS was 24.3 months [46].

Gotlieb et al. published a multicenter, randomized, double-blind phase II study that included 55 patients with relapse of platinum-resistant ovarian cancer, who had received a median of at least 4 previous lines (range 2–12) that compared aflibercept 4 mg/kg every 14 days versus placebo. The patients were stratified according to the need for paracentesis in 2 periods, ≤2 weeks and >2 weeks. The primary objective was time to new paracentesis. Time to paracentesis from randomization was significantly greater in the aflibercept arm (55.1 vs. 23.3 days), and in two patients, new paracentesis was not required until 6 months later. There was more toxicity in the form of dyspnea (20% vs. 8%) in the aflibercept arm and also intestinal perfo‐ ration (3 patients vs. 1). However, there was more fatigue or asthenia (13% vs. 44%) and more dehydration (10% vs. 12%) with placebo [47].

Similar to the previous study, and with the same doses, another multicenter group tried to answer the same question in another phase II study: the utility of aflibercept in control of gynecologic tumor ascites. They included 16 platinum-resistant and very pretreated patients, with similar results obtained [48].

These last two studies show the activity and effect of the drug in reducing the need for paracentesis, although a small risk of intestinal perforation in peritoneal carcinomatosis cannot be ruled out. It should only be used after thoroughly evaluating the risk/benefit ratio in each specific case.

#### **8. Sorafenib**

Sorafenib is a multikinase inhibitor with activity on different tyrosine kinase receptors, including VEGFR-2 and VEGFR-3, PDGFR-β, c-Kit, and Flt-3 receptor and the v-raf oncogene.

Limited activity of this drug has been shown in 71 women who presented ovarian tumor relapse within 12 months of completing platinum treatment (after one or two previous regimens). The primary objectives were PFS at 6 months and safety; the secondary objectives were percentage response and duration of PFS and OS. The dose used was 400 mg/12 h. Efficacy was evaluated only in the 59 patients with measurable disease: 14 women (24%) presented PFS of at least 6 months. Partial response was obtained in 2 women, stabilization in 20, while 30 patients presented progression; response could not be measured in 7 patients. The most common G3/4 adverse events were as expected (rash, hand/foot syndrome, gastrointestinal, and metabolic and, to a lesser extent, cardiovascular and pulmonary toxicity) [49].

A Canadian group from the Princess Margaret Hospital studied sorafenib, at the same doses, in combination with gemcitabine weekly in a phase II trial in 43 pretreated patients with platinum-resistant relapse; 2 of them presented partial response, and the disease remained stable for at least 6 months in 10 of them. However, the proportion of responses was only 4.7%. The most common G3/4 events were hematological (28% lymphocytopenia and 26% neutro‐ penia), leading to significant delays in the administration of the therapeutic regimen [50].

German investigators tested the addition of the drug to the carboplatin–paclitaxel combina‐ tion in the neoadjuvant context in patients with large disease volume and ascites. This phase II trial included only 4 patients, as it was stopped due to severe G3/4 toxicity, largely cardiovas‐ cular [51].

The combination with topotecan in platinum-resistant patients was also evaluated. There was important hematologic toxicity and G3/4 toxicity in the form of transaminase elevation [52].

The possibility of continuation or maintenance treatment was evaluated in women after they completed the first-line treatment with carboplatin and paclitaxel and had presented complete response. Two hundred and forty-six patients were included and randomized to receive Sorafenib 400 mg/12 h or placebo until progression, intolerable toxicity, or withdrawal of consent. The patient was withdrawn from the study if there was more than a 30-day delay in the administration of the treatment or if more than two dose reductions were required. The primary objectives were to evaluate the efficacy and safety of this approach. There were no differences between the two groups in PFS, with a trend toward better results in the placebo arm, and there were clearly more adverse events with sorafenib, with a toxicity profile similar to that found in previous studies. The conclusions of the study were that maintenance therapy with this drug could not be recommended [53].

The combination of paclitaxel, carboplatin, and sorafenib was investigated in first-line treatment for metastatic disease in women with stages III and IV. After two treatment cycles, the patients with stabilization or partial response continued the chemotherapy for six cycles, and sorafenib was maintained for 52 weeks; 85 patients were included. Efficacy was similar in proportion of responses, PFS, and 2-year survival. The addition of sorafenib clearly increased toxicity: EPP, mucositis, and HT, and so its use was not recommended [54].

#### **9. Sunitinib**

ration (3 patients vs. 1). However, there was more fatigue or asthenia (13% vs. 44%) and more

Similar to the previous study, and with the same doses, another multicenter group tried to answer the same question in another phase II study: the utility of aflibercept in control of gynecologic tumor ascites. They included 16 platinum-resistant and very pretreated patients,

These last two studies show the activity and effect of the drug in reducing the need for paracentesis, although a small risk of intestinal perforation in peritoneal carcinomatosis cannot be ruled out. It should only be used after thoroughly evaluating the risk/benefit ratio in each

Sorafenib is a multikinase inhibitor with activity on different tyrosine kinase receptors, including VEGFR-2 and VEGFR-3, PDGFR-β, c-Kit, and Flt-3 receptor and the v-raf oncogene. Limited activity of this drug has been shown in 71 women who presented ovarian tumor relapse within 12 months of completing platinum treatment (after one or two previous regimens). The primary objectives were PFS at 6 months and safety; the secondary objectives were percentage response and duration of PFS and OS. The dose used was 400 mg/12 h. Efficacy was evaluated only in the 59 patients with measurable disease: 14 women (24%) presented PFS of at least 6 months. Partial response was obtained in 2 women, stabilization in 20, while 30 patients presented progression; response could not be measured in 7 patients. The most common G3/4 adverse events were as expected (rash, hand/foot syndrome, gastrointestinal,

and metabolic and, to a lesser extent, cardiovascular and pulmonary toxicity) [49].

A Canadian group from the Princess Margaret Hospital studied sorafenib, at the same doses, in combination with gemcitabine weekly in a phase II trial in 43 pretreated patients with platinum-resistant relapse; 2 of them presented partial response, and the disease remained stable for at least 6 months in 10 of them. However, the proportion of responses was only 4.7%. The most common G3/4 events were hematological (28% lymphocytopenia and 26% neutro‐ penia), leading to significant delays in the administration of the therapeutic regimen [50].

German investigators tested the addition of the drug to the carboplatin–paclitaxel combina‐ tion in the neoadjuvant context in patients with large disease volume and ascites. This phase II trial included only 4 patients, as it was stopped due to severe G3/4 toxicity, largely cardiovas‐

The combination with topotecan in platinum-resistant patients was also evaluated. There was important hematologic toxicity and G3/4 toxicity in the form of transaminase elevation [52]. The possibility of continuation or maintenance treatment was evaluated in women after they completed the first-line treatment with carboplatin and paclitaxel and had presented complete response. Two hundred and forty-six patients were included and randomized to receive

dehydration (10% vs. 12%) with placebo [47].

80 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

with similar results obtained [48].

specific case.

**8. Sorafenib**

cular [51].

Sunitinib is also another multikinase inhibitor that binds to VEGF, PDGF, c-Kit, and Ftl-3. As with sorafenib, the response rates of therapy with this drug are low.

In a phase II trial that included 30 women with platinum-sensitive (73%) and platinumresistant (27%) relapse who had received one or two previous lines, treated with the standard 50 mg/day dose for 4 weeks and 2 weeks of rest, there was one partial response and 16 stabilizations [55].

In another phase II study with 73 platinum-resistant patients who had received three or more previous lines, they were randomized to receive sunitinib at standard dose or 37.5 mg/day continually. There were differences in median PFS in favor of the standard administration (4.8 vs. 2.9 months) but not in OS (13.6 vs. 13.7 months). The pattern and the frequency of adverse events were similar in the two groups and as expected: fatigue, cardiovascular and gastroin‐ testinal toxicity, hematological alterations, and hepatic function disorders [56].

The continuous administration of 37.5 mg/day was also evaluated in another Dana–Farber phase II study in 18 platinum-resistant patients, continuing to find a response (partial and complete) rate of around 8% and median PFS of just 10 weeks. There was also considerable toxicity in the form of hypertension and gastrointestinal events [57].

#### **10. Imatinib mesylate**

Imatinib belongs to the tyrosine kinase inhibitor family; it prevents PDGF from binding to its receptor and prevents the triggering of the AKT intracellular signaling cascade responsible for tumor growth and metastatic dissemination.

Its possible therapeutic effect on platinum-resistant ovarian tumors after progression to other treatments has also been studied. Most studies treated patients at the standard dose of 600 mg/ day after selecting them according to immunohistochemical c-Kit expression. They included a small number of patients, and the drug was tested in monotherapy [58, 59] and in combina‐ tion with docetaxel [60] or paclitaxel [61]. Few responses were obtained (0–2%), primarily obtaining stabilizations, with repeated dose reductions required due to toxicity in the form of edemas, gastrointestinal, or hematological adverse events.

More recently, Anderson's work found that there was no correlation between responses in platinum-refractory patients who had progressed to taxanes and expression of the aforemen‐ tioned biomarkers. The efficacy and toxicity results were similar to those of previous studies, concluding that it was not an active treatment in this group of patients [62].

#### **11. Vandetanib**

Vandetanib (ZD6474) also belongs to the oral tyrosine kinase inhibitor family and inhibits VEGFR-2 and VEGFR-3, EGFR, and RET.

In a phase I/II study in combination with pegylated liposomal doxorubicin (50 mg/m², day 1/28 days), at a dose of 100 mg/24 h in platinum-resistant patients, 14 patients were included and few responses (around 10%) were found, with close to 40% stabilizations, but significant toxicity led to discontinuation of the treatment in nearly 30% of patients [63].

Combined with concomitant docetaxel, the SWOG S0904 study compared it to docetaxel in monotherapy. A total of 131 patients were included and randomized to one of the arms; no benefit was found from the addition of vandetanib in PFS; there was G4 hematological toxicity in nearly 30% of the included women [64].

#### **12. Ramucirumab**

Ramucirumab, or IMC-1121B, is an Ig G1 humanized monoclonal antibody that has affinity for the extracellular domain of VEGFR-2 and prevents VEGF from binding to its ligands, thus inhibiting endothelial cell proliferation and migration and new vessel formation.

A multicenter study with 70 women with platinum-resistant relapse in 75% of them only obtained 5% partial responses, nearly 60% stabilizations, and 25% 6-month PFS (both primary end points); ramucirumab was not found to be particularly active in this context [65].

#### **13. Zibotentan**

Zibotentan or ZD4054 is an oral ET-A receptor antagonist that is involved in activation of endothelin growth, and thus in cell proliferation and tumor invasion and migration.

In a multicenter study that combines the drug (at a dose of 10 mg/24 h) with carboplatin and paclitaxel versus the same chemotherapy and placebo, no benefit was found in PFS, percentage of responses, or reduction of CA-125 in women with platinum-sensitive disease. The toxicities most commonly found with the drug were anemia and neutropenia, alopecia, nausea, and headache in nearly 50% of patients [66].

In total, to date there are no promising results for most of the reviewed antiangiogenic agents, except those already known for bevacizumab, trebananib, pazopanib, cediranib, and ninteda‐ nib. Ongoing research will shed more light on this fascinating tumor process and its control.

#### **Author details**

Its possible therapeutic effect on platinum-resistant ovarian tumors after progression to other treatments has also been studied. Most studies treated patients at the standard dose of 600 mg/ day after selecting them according to immunohistochemical c-Kit expression. They included a small number of patients, and the drug was tested in monotherapy [58, 59] and in combina‐ tion with docetaxel [60] or paclitaxel [61]. Few responses were obtained (0–2%), primarily obtaining stabilizations, with repeated dose reductions required due to toxicity in the form of

More recently, Anderson's work found that there was no correlation between responses in platinum-refractory patients who had progressed to taxanes and expression of the aforemen‐ tioned biomarkers. The efficacy and toxicity results were similar to those of previous studies,

Vandetanib (ZD6474) also belongs to the oral tyrosine kinase inhibitor family and inhibits

In a phase I/II study in combination with pegylated liposomal doxorubicin (50 mg/m², day 1/28 days), at a dose of 100 mg/24 h in platinum-resistant patients, 14 patients were included and few responses (around 10%) were found, with close to 40% stabilizations, but significant

Combined with concomitant docetaxel, the SWOG S0904 study compared it to docetaxel in monotherapy. A total of 131 patients were included and randomized to one of the arms; no benefit was found from the addition of vandetanib in PFS; there was G4 hematological toxicity

Ramucirumab, or IMC-1121B, is an Ig G1 humanized monoclonal antibody that has affinity for the extracellular domain of VEGFR-2 and prevents VEGF from binding to its ligands, thus

A multicenter study with 70 women with platinum-resistant relapse in 75% of them only obtained 5% partial responses, nearly 60% stabilizations, and 25% 6-month PFS (both primary

Zibotentan or ZD4054 is an oral ET-A receptor antagonist that is involved in activation of

inhibiting endothelial cell proliferation and migration and new vessel formation.

end points); ramucirumab was not found to be particularly active in this context [65].

endothelin growth, and thus in cell proliferation and tumor invasion and migration.

concluding that it was not an active treatment in this group of patients [62].

toxicity led to discontinuation of the treatment in nearly 30% of patients [63].

edemas, gastrointestinal, or hematological adverse events.

82 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

**11. Vandetanib**

**12. Ramucirumab**

**13. Zibotentan**

VEGFR-2 and VEGFR-3, EGFR, and RET.

in nearly 30% of the included women [64].

M.A. Alonso Bermejo, L. Rey Iglesias , M.E. Pérez López, A. Fernández Montes\* and J. García Mata

\*Address all correspondence to: afm1003@hotmail.com

Medical Oncology Service at the University Hospital Ourense, Ourense, Spain

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## **Peritonectomy Procedures and HIPEC for Peritoneal Metastasis from Ovarian Cancer**

Angelo Di Giorgio, Daniele Biacchi, Antonio Ciardi, Alessio Impagnatiello, Maurizio Cardi, Simone Sibio, Bianca Sollazzo, Joseph Maher Fouad Atta, Giuseppe Naso, Fabio Accarpio and Paolo Sammartino

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60844

#### **Abstract**

Peritoneal carcinomatosis (PC) is the most impressive and frequent evidence of locoregional spread of epithelial ovarian cancer (EOC). For most of its natural history, PC remains confined to the peritoneal district, thus representing a target for various combinations of surgery and systemic or loco-regional chemotherapy. PC is observed both in primary settings, i.e. in patients first treated for locally advanced EOC, and in recurrent, previously treated, EOC patients at any FIGO stage. Since 2000s, the use of hyperthermic intraperitoneal chemotherapy (HIPEC) combined with maximum cytoreduction (peritonectomy) has gradually spread in the treatment of PC from ovarian cancer, as well as for gastrointestinal carcinomatosis and primary tumours of the peritoneum. Use of combined peritonectomy + HIPEC in the treatment of ovarian carcinomatosis is the most discussed issue among those concerning peritoneal surface malignancy (PSM). The main criticism concerns the use of HIPEC, since the need for maximal cytoreduction is consolidated and does not raise any doubts. Communities of surgeon and oncologic gynaecologists who believes in the role of HIPEC have started controlled clinical trials aimed at clarifying the role of HIPEC associated to peritonectomy, but these studies are difficult to conduct and time-consuming. At present and pending the results of future prospective trials, the role and limits of application of the procedure are drawn from experiences from three basic study groups: collective reviews, multicentre studies, monocentric case studies produced by high-volume HIPEC centers. A comprehensive literature review and an in-depth

© 2016 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.

analysis of our personal experience, based on the largest monocentric case series (130 cases), have helped to provide an assessment on the role of peritonectomy + HIPEC in about 2000 patients treated for initial and recurrent PC from ovarian cancer. Comparison of the overall results drawn from these studies, indicates that peritonec‐ tomy + HIPEC is able to guarantee in these patients better overall survival (OS) and higher progression-free survival (PFS) rates than those derived from traditional treatments, with acceptable morbidity and mortality. Notwithstanding, some specific aspects, including the role of chemoresistance and neoadjuvant and adjuvant treatments, should be clarified by further experience and the results of on-going trials.

**Keywords:** Epithelial Ovarian Cancer, Peritoneal Carcinomatosis, Peritonectomy, HIPEC

#### **1. Introduction**

Peritoneal metastasis is the most common type of diffusion and the most frequent cause of death from EOC.

Intra-abdominal and pelvic parietal and visceral peritoneal metastases, often associated with ascites, resectable hepatic metastasis, deep bowel wall infiltration up to mucosa, identify stage III or IV ovarian cancer with diffuse PC [1,2]. Treatment of these conditions is traditionally based on cytoreductive surgery (CRS) combined with systemic carbotaxol-based chemother‐ apy at first line. Despite high rates of chemosensitivity, relapses are detected in up to 50% of cases in the first two years and in almost 100% in the first 5 years post-treatment. [3]

For most of its natural history, EOC is confined to the abdominal cavity, developing further peritoneal tumour implants and producing pelvic and lumbar lymph node metastases without extra-abdominal diffusion.

For this reason, new integrate therapeutic strategies have emphasized the role of local aggressive treatments, represented by maximal cytoreductive surgery (peritonectomy) combined with loco-regional HIPEC.

Peritonectomy (PRT) associated with HIPEC has been used since the second half of the 90's in the treatment of ovarian PC, as well as in other primary and metastatic peritoneal surface malignancies.

PC is observed both in primary settings, i.e. in patients first treated for locally advanced EOC, and as a recurrence in patients previously treated for ovarian cancer at any stage.

#### **2. Initial and recurrent ovarian carcinomatosis**

About 75% of ovarian cancers are diagnosed and treated in primary settings as FIGO Stage IIIc/IV, meaning that they are confined to the abdominal and pelvic cavity and characterised by diffuse visceral and parietal PC [4]. PC is frequently associated with lymphnode metastases and less commonly with haematogenous hepatic metastases.

Such a high percentage of PC at first presentation is mainly caused by the relevant delay in diagnosing EOC at early stages, due to the lack of symptoms and to the low sensitivity and specificity of diagnostic tools. Only 20% to 30% of EOC in developed countries are diagnosed at FIGO Stage I and II and the diagnosis is usually accidental: either via sonography, compu‐ terised tomography (CT scanning) or during laparoscopic investigations [5,6].

The pathogenesis of late PC in patients already treated for EOC at any stage is more complex.

At FIGO stages I and II it may be related to a number of factors:


analysis of our personal experience, based on the largest monocentric case series (130 cases), have helped to provide an assessment on the role of peritonectomy + HIPEC in about 2000 patients treated for initial and recurrent PC from ovarian cancer. Comparison of the overall results drawn from these studies, indicates that peritonec‐ tomy + HIPEC is able to guarantee in these patients better overall survival (OS) and higher progression-free survival (PFS) rates than those derived from traditional treatments, with acceptable morbidity and mortality. Notwithstanding, some specific aspects, including the role of chemoresistance and neoadjuvant and adjuvant treatments, should be clarified by further experience and the results of on-going trials.

92 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

**Keywords:** Epithelial Ovarian Cancer, Peritoneal Carcinomatosis, Peritonectomy,

Peritoneal metastasis is the most common type of diffusion and the most frequent cause of

Intra-abdominal and pelvic parietal and visceral peritoneal metastases, often associated with ascites, resectable hepatic metastasis, deep bowel wall infiltration up to mucosa, identify stage III or IV ovarian cancer with diffuse PC [1,2]. Treatment of these conditions is traditionally based on cytoreductive surgery (CRS) combined with systemic carbotaxol-based chemother‐ apy at first line. Despite high rates of chemosensitivity, relapses are detected in up to 50% of

For most of its natural history, EOC is confined to the abdominal cavity, developing further peritoneal tumour implants and producing pelvic and lumbar lymph node metastases without

For this reason, new integrate therapeutic strategies have emphasized the role of local aggressive treatments, represented by maximal cytoreductive surgery (peritonectomy)

Peritonectomy (PRT) associated with HIPEC has been used since the second half of the 90's in the treatment of ovarian PC, as well as in other primary and metastatic peritoneal surface

PC is observed both in primary settings, i.e. in patients first treated for locally advanced EOC,

About 75% of ovarian cancers are diagnosed and treated in primary settings as FIGO Stage IIIc/IV, meaning that they are confined to the abdominal and pelvic cavity and characterised

and as a recurrence in patients previously treated for ovarian cancer at any stage.

**2. Initial and recurrent ovarian carcinomatosis**

cases in the first two years and in almost 100% in the first 5 years post-treatment. [3]

HIPEC

**1. Introduction**

death from EOC.

extra-abdominal diffusion.

malignancies.

combined with loco-regional HIPEC.

Point 1 of the above is sometimes dictated by special clinical situations, which require conservative treatment. Young patients with small ovarian tumours can be treated with simple unilateral oophorectomy, in order to preserve their reproduction function. The results of this strategy are not uniform and tend to show an unjustifiable risk of surgical relapse. Rupture of the ovarian tumour during open surgery, or more often during laparoscopic surgery, is one of the most frequent cause of peritoneal recurrence [7].

Omission of appendectomy or total omentectomy is also not a rare cause of peritoneal recurrence or persistence of the disease (Fig. 1).

As to point 2, despite international guidelines advice for infra-colic limited resection of the greater omentum and for not total omentectomy, the presence of histologically-proven tumour implants in the latter tissue is associated to elevated rates of peritoneal and omental recurrence.

**Figure 1.** Residual greater omentum involved in recurrent peritoneal carcinomatosis.

On the other hand, the anatomical structure of the omentum is unitary and limited resection is, therefore, not plausible. It should be reminded that the omentum often harbours EOC deposits by virtue of its peculiar anatomy and function. It contains milky spots, which are responsible for the concentration and reabsorption of intraperitoneal fluid, including malig‐ nant ascites. It is through the milky spots that the tumour cells take root into the omentum. This phenomenon facilitates the formation of carcinomatous nodules of various sizes, in some cases involving the complete replacement of the omental tissue with tumour tissue ("omental cake").

Omission of lymphadenectomy in early stage EOC is frequent and correlates significantly with subsequent loco-regional lymph-node metastases and PC (over 50% in our series of recurrent EOC patients).

Finally, chemoresistance to first-line adjuvant treatments with carbotaxol is detectable in 20% of cases and is a further cause of relapse after treatment for stage I and II EOC [8].

Peritoneal recurrences after treatments for FIGO stages III or IV intraperitoneal EOC, can be mainly attributed to the lack of aggressiveness of the standard treatments. The current standard therapy, i.e. CRS combined with systemic chemotherapy, shows limited efficacy in high stage EOC, and is followed in most cases by abdomino-pelvic loco-regional recurrence.

Most often relapses occur as PC, associated with ascites in 60% of cases.

Further attempts to treat ovarian cancer at stage III — aimed at curbing the incidence of peritoneal recurrence — involve the use of intra-peritoneal normothermic chemotherapy (IP CHT).

Several randomized trials have demonstrated the effectiveness of this method, especially after optimal CRS; nevertheless it is still rarely used mainly due to catheter-related complications which significantly reduce its applicability. [9,10]

In conclusion, PC is the most frequent and characteristic manifestation of EOC, whether identified early at first assessment or later as persistent or recurrent disease following standard treatments. These include surgical debulking and systemic chemotherapy, are characterised by high recurrence rates and cannot guarantee long-term survival and improvement in the quality of life.

#### **3. Epidemiology**

EOC affects over 200.000 women and causes 125.000 deaths annually worldwide, with a deaths/new cases ratio of 62,5 % [11]. These data demonstrate that standard treatments are not able to deal effectively with this disease, and success rates are distant from other common types of cancer, such as colorectal cancer, for which the deaths/new cases ratio was 45.9% over the same period. The low impact of standard treatments is also corroborated by the analysis of the causes of death for EOC patients. Our National Institute of Statistics (ISTAT) data referred to 2013-2014 showed that 80% of deaths in EOC patients is exclusively due to peritoneal recur‐ rence, 10% to peritoneal recurrence associated with extra-peritoneal metastasis, and only 10% exclusively to extra-peritoneal metastases. Therefore, alternative therapeutic strategies are needed, also considering that distant metastases are a late occurrence in EOC patients, mainly due to the little effectiveness of standard treatments.

#### **4. Macroscopy and microscopy**

On the other hand, the anatomical structure of the omentum is unitary and limited resection is, therefore, not plausible. It should be reminded that the omentum often harbours EOC deposits by virtue of its peculiar anatomy and function. It contains milky spots, which are responsible for the concentration and reabsorption of intraperitoneal fluid, including malig‐ nant ascites. It is through the milky spots that the tumour cells take root into the omentum. This phenomenon facilitates the formation of carcinomatous nodules of various sizes, in some cases involving the complete replacement of the omental tissue with tumour tissue ("omental

Omission of lymphadenectomy in early stage EOC is frequent and correlates significantly with subsequent loco-regional lymph-node metastases and PC (over 50% in our series of recurrent

Finally, chemoresistance to first-line adjuvant treatments with carbotaxol is detectable in 20%

Peritoneal recurrences after treatments for FIGO stages III or IV intraperitoneal EOC, can be mainly attributed to the lack of aggressiveness of the standard treatments. The current standard therapy, i.e. CRS combined with systemic chemotherapy, shows limited efficacy in high stage EOC, and is followed in most cases by abdomino-pelvic loco-regional recurrence.

Further attempts to treat ovarian cancer at stage III — aimed at curbing the incidence of peritoneal recurrence — involve the use of intra-peritoneal normothermic chemotherapy (IP

Several randomized trials have demonstrated the effectiveness of this method, especially after optimal CRS; nevertheless it is still rarely used mainly due to catheter-related complications

In conclusion, PC is the most frequent and characteristic manifestation of EOC, whether identified early at first assessment or later as persistent or recurrent disease following standard treatments. These include surgical debulking and systemic chemotherapy, are characterised by high recurrence rates and cannot guarantee long-term survival and improvement in the

EOC affects over 200.000 women and causes 125.000 deaths annually worldwide, with a deaths/new cases ratio of 62,5 % [11]. These data demonstrate that standard treatments are not able to deal effectively with this disease, and success rates are distant from other common types of cancer, such as colorectal cancer, for which the deaths/new cases ratio was 45.9% over the same period. The low impact of standard treatments is also corroborated by the analysis of the causes of death for EOC patients. Our National Institute of Statistics (ISTAT) data referred to 2013-2014 showed that 80% of deaths in EOC patients is exclusively due to peritoneal recur‐

of cases and is a further cause of relapse after treatment for stage I and II EOC [8].

Most often relapses occur as PC, associated with ascites in 60% of cases.

which significantly reduce its applicability. [9,10]

94 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

cake").

CHT).

quality of life.

**3. Epidemiology**

EOC patients).

Traditionally the origin of carcinomas of the ovary is identified in the Ovarian Surface Epithelium (OSE). Growing evidence indicates that the majority of EOC have an extraovarian source. [12]

The new paradigm that increasingly fits with the extraovarian origin of EOC establishes common characteristics for ovarian, tubal and primitive peritoneal tumours that unite these malignancies in a common family, divided into two broad groups: type I and type II ovarian cancers.

Molecular profiling contributes to better distinguish the two types of ovarian cancer (high grade vs. low grade) as well as identifying various subtypes, i.e., serous, mucinous, endome‐ trioid and clear cell cancer.

The application of these new classifications will be invaluable in identifying "ovarian" tumours with different prognosis and targets for specific therapeutic strategies. [10]

Major studies on ovarian PC include ovarian, tubal and primitive peritoneal carcinomas grouped together due to their histological and pathological similarities and the treatment options which are identical for all three forms.

Macroscopically the ovarian carcinomatosis is similar to other forms of PSM. It can be present as nodules varying in size from less than 1 mm to various centimetres, isolated or conglom‐ erated in the form of solid or cystic masses or plaques of varying sizes and thicknesses.

The serous or mucinous content of carcinomatous implants and their degree of invasiveness of the peritoneum and of the underlying structures is extremely variable.

Previous treatments with chemotherapy can influence the appearance of ovarian carcinoma‐ tosis. After neoadjuvant or adjuvant chemotherapies, the peritoneum can show evident signs of carcinomatosis regression on its surface, ranging from significant reduction to complete disappearance; in each case the signs of previous disease are still evident.

In particular, the increase in thickness of the parietal and visceral peritoneal membrane, its opacification, and the presence of blurs and reddish spots indicate the location and extent of previous carcinomatosis.

Histological and immunohistochemical studies of biopsies of these tissues often show the presence of microscopic foci of disease in the context of thick, fibrotic areas.

These macroscopic and microscopic features are potential justifications for relapse after neoadjuvant or adjuvant chemotherapy in patients subjected to an apparently negative

**Figure 2.** Microfocus of neoplastic cells inside fibrous desmoplastic tissue- CA-125 immunohistochemistry.

second-look. Indeed, fibrosis encapsulating foci of neoplastic cells may preserve them from effects of further systemic or locoregional therapies.

Furthermore, total chemical cytoreduction runs the risk of both surgical and chemotherapic undertreatment, especially if obtained after effective neoadjuvant treatments.

PC may involve any anatomic site and bowel segment or parenchyma in high percentages (parietal and visceral peritoneum 90%; omentum 60%; diaphragm 40%; liver and spleen capsule 15%). Lymphatic and haematogenous metastases in the liver may also be detected contemporaneously (respectively 50-60% and 5%). Ascites is present in about 60%.

#### **5. Diagnosis and staging of peritoneal carcinomatosis**

The ovarian carcinomatosis is paucisymptomatic until it assumes a considerable size or is associated with ascites or occlusion. Therefore diagnosis is often delayed and more than 70% of ovarian cancer patients are diagnosed at FIGO stages IIIC/IV.

Clinical examination with vaginal and rectal exploration plays a critical role in assessing the pelvic spread of the disease.

Diagnosis is based on a set of efficient morphological investigations (CT, MRI, PET). CA 125, in association or not to CA 19.9, and currently to HE-4, are the most sensitive tumour markers for specific diagnosis of ovarian cancer. Laparoscopy plays an important role in doubtful cases, allowing the direct visualization and biopsy of suspected lesions.

The intraoperative staging of PC from ovarian cancer, as in other forms of PSM, relies mainly on PCI classification proposed by Sugarbaker [13], although other classifications have been proposed.

The correct staging of PC is important to assess resectability, prognosis and risk of complica‐ tions. For this reason, much effort is made to adopt the PCI classification also prior to surgery by applying it to data from morphological imaging (CT, MR, PET) or laparoscopy investiga‐ tions.

Being able to determine reliably in the preoperative phase the peritoneal spread of the disease and the involvement of sensitive anatomical areas, PCI could avoid unnecessary surgical approaches and improve the overall strategy as well as identify cases to be submitted to neoadjuvant chemotherapy (NACT).

However results are still unsatisfactory both due the complexity of PCI classification and the difficulty in its preoperative application; recently a new and simpler method to stage peritoneal carcinomatosis via laparoscopy has been proposed [14].

If the above described set of diagnostic procedures increases the percentage of successful diagnosis of PC, including the identification of the primary tumour and the eventual presence of extra-peritoneal disease, the reliability of the current standard diagnostic tools used in staging intraperitoneal spread must be considered as unsatisfactory. Many authors emphasize the role of laparoscopy in staging intraperitoneal spread of carcinomatosis, but the obvious limits of feasibility in pervasive forms of recurrence restrict the use and significance of this method [14-17]. Moreover risk of contamination of port site access by tumor cells at laparo‐ scopy should be considered [18-20].

#### **6. Evaluation of residual disease after cytoreductive surgery**

Evaluation of tumor residues after cytoreductive surgery is of relevant importance because of residual disease volume is the major prognostic factor in the treatment of EOC.[21-30]

The degree of cytoreduction can be assessed with various classification systems, the most used of them is the Sugarbaker scoring classification [Completeness of Cytoreduction score (CC)] [31]. This system provides four values from 0 to 3, where 0 indicates complete cytoreduction of peritoneal carcinomatosis with total absence of macroscopic residual disease at the end of the surgical phase. The maximum therapeutic efficacy of the integrated procedure is carried out in cases where an "optimal" cytoreduction (CC0 - CC1) is achieved.

#### **7. Peritonectomy and HlPEC**

second-look. Indeed, fibrosis encapsulating foci of neoplastic cells may preserve them from

**Figure 2.** Microfocus of neoplastic cells inside fibrous desmoplastic tissue- CA-125 immunohistochemistry.

Furthermore, total chemical cytoreduction runs the risk of both surgical and chemotherapic

PC may involve any anatomic site and bowel segment or parenchyma in high percentages (parietal and visceral peritoneum 90%; omentum 60%; diaphragm 40%; liver and spleen capsule 15%). Lymphatic and haematogenous metastases in the liver may also be detected

The ovarian carcinomatosis is paucisymptomatic until it assumes a considerable size or is associated with ascites or occlusion. Therefore diagnosis is often delayed and more than 70%

Clinical examination with vaginal and rectal exploration plays a critical role in assessing the

Diagnosis is based on a set of efficient morphological investigations (CT, MRI, PET). CA 125, in association or not to CA 19.9, and currently to HE-4, are the most sensitive tumour markers for specific diagnosis of ovarian cancer. Laparoscopy plays an important role in doubtful cases,

undertreatment, especially if obtained after effective neoadjuvant treatments.

contemporaneously (respectively 50-60% and 5%). Ascites is present in about 60%.

**5. Diagnosis and staging of peritoneal carcinomatosis**

of ovarian cancer patients are diagnosed at FIGO stages IIIC/IV.

allowing the direct visualization and biopsy of suspected lesions.

pelvic spread of the disease.

effects of further systemic or locoregional therapies.

96 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

The limits of success of standard treatments of PC from ovarian cancer have led to test new therapeutic possibilities, borrowing from the experiences made in other forms of peritoneal carcinomatosis a therapeutic strategy based on the association of maximal cytoreduction (Peritonectomy) with Hyperthermic Intraperitoneal Chemotherapy (HIPEC).

Peritonectomy is aimed to complete removal of macroscopic disease; HIPEC is aimed to treat microscopic or millimetric tumor residues after surgical cytoreductive phase.

#### **7.1. Rationale**

The association between PRT and HIPEC is based on a complex rationale that takes into account the mechanism of intraperitoneal spread of free cancer cells, Gompertzian tumor growth kinetics, Goldie&Coldman mathematical model about drug resistance, pharmacoki‐ netic and pharmacodynamic events related to intraperitoneal chemotherapy associated with hyperthermia. [32]

Maximal cytoreduction reducing drastically tumor volume, induces the remaining cells to enter the fast proliferating phase of the cell cycle becoming more responsive to chemotherapic drugs. Moreover microscopic or millimetric residual tumor volumes include a minor rate of chemoresistant clones and can be totally permeated by drugs delivered by intraperitoneal chemotherapy [33-36].

The association of HIPEC is based on a series of advantages related by a part to the fact that the chemotherapy is carried out at the end of the surgical stage directly into abdominal cavity and by the other part to the fact that drugs used are brought to a constant temperature of 42-43° for the entire treatment period of infusion (usually 60 minutes).

The benefits of loco-regional chemotherapy consist of:


The combination of hyperthermia provides additional benefits:


In particular, hyperthermia favours drug penetration into the tissues to a depth of 5 mm, a value significantly greater than what occurs in isothermal conditions (2 mm). Therefore the more the peritonectomy is effective achieving "optimal" cytoreduction (CC0 - CC1), i.e. up to allow the total removal of the disease or leaving residues of minimum size (up to 2.5mm), the more associated chemo-hyperthermia will be able to successfully attack microscopic or minimum size tumor residues.

#### **8. Peritonectomy**

carcinomatosis a therapeutic strategy based on the association of maximal cytoreduction

Peritonectomy is aimed to complete removal of macroscopic disease; HIPEC is aimed to treat

The association between PRT and HIPEC is based on a complex rationale that takes into account the mechanism of intraperitoneal spread of free cancer cells, Gompertzian tumor growth kinetics, Goldie&Coldman mathematical model about drug resistance, pharmacoki‐ netic and pharmacodynamic events related to intraperitoneal chemotherapy associated with

Maximal cytoreduction reducing drastically tumor volume, induces the remaining cells to enter the fast proliferating phase of the cell cycle becoming more responsive to chemotherapic drugs. Moreover microscopic or millimetric residual tumor volumes include a minor rate of chemoresistant clones and can be totally permeated by drugs delivered by intraperitoneal

The association of HIPEC is based on a series of advantages related by a part to the fact that the chemotherapy is carried out at the end of the surgical stage directly into abdominal cavity and by the other part to the fact that drugs used are brought to a constant temperature of 42-43°

**•** direct exposure of whole anatomical region to chemotherapy being absent adhesions

**•** increases the effectiveness of some chemotherapics (CDDP, MMC, DOX, gemcitabine)

In particular, hyperthermia favours drug penetration into the tissues to a depth of 5 mm, a value significantly greater than what occurs in isothermal conditions (2 mm). Therefore the more the peritonectomy is effective achieving "optimal" cytoreduction (CC0 - CC1), i.e. up to allow the total removal of the disease or leaving residues of minimum size (up to 2.5mm), the more associated chemo-hyperthermia will be able to successfully attack microscopic or

for the entire treatment period of infusion (usually 60 minutes).

**•** possibility of using high concentrations of chemotherapics

The combination of hyperthermia provides additional benefits:

**•** promotes tissue penetration of chemotherapeutic drugs

The benefits of loco-regional chemotherapy consist of:

**•** possibility of allowing a prolonged exposure time

(Peritonectomy) with Hyperthermic Intraperitoneal Chemotherapy (HIPEC).

98 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

microscopic or millimetric tumor residues after surgical cytoreductive phase.

**7.1. Rationale**

hyperthermia. [32]

chemotherapy [33-36].

**•** low systemic toxicity

**•** hyperthermia damages cancer cells

**•** does not involve increased toxicity

minimum size tumor residues.

The term of peritonectomy identifies precisely the meaning of the surgical procedure: removal of parietal and visceral peritoneum affected by the neoplastic pathology.

Peritonectomy procedures comprise:


At parietal level the procedure entails complete or partial removal of the peritoneum lining the abdominal wall, the diaphragms and the pelvis according to disease extension. General consensus is in removing parietal peritoneum limited to involved areas, sparing a unaffected zones.

If healthy areas are limited, large parietal peritonectomies should be performed up to complete parietal peritonectomy.

In principle, the resection of the parietal and pelvic peritoneum below the transverse umbilical line should be performed in all cases of peritoneal carcinomatosis from ovarian cancer.

Parietal peritonectomy includes greater and lesser omentectomy, resection of round and falciform ligaments, stripping of omental bursa peritoneum.

When PC spreads deeply beyond peritoneal membrane trough abdominal wall, full or partial thickness parietal resection is performed.

Laparoscopic trocar sites are removed by full thickness cylindrical parietal resection when involved by carcinomatosis or when suspected to be contaminated by tumor cells. Umbilicus, regardless its previous use as trocar sites, should be removed on principle in recurrent cases being a frequent site of metastasis.

Visceral peritoneum cannot be separated from underlying visceral tissue and removed separately as with the peritoneum lying the abdominal walls and diaphragms. Therefore visceral peritonectomy involves exeresis of endoperitoneal viscera or organs deeply infiltrated by PC. Rarely and only in special anatomical situations is possible the removal of visceral peritoneum only as when PC does not deeply infiltrate the visceral wall or when it concerned the Glisson's capsule.

Bowel resection is the most frequent peritonectomy procedure in treating peritoneal carcino‐ matosis from ovarian cancer.

Contemporaneous involvement of multiple viscera induces to multivisceral resections for what en bloc resection should be preferred (Fig 3-4).

**Figure 3.** Pelvic peritonectomy: the moment of rectal resection as final step to remove en-bloc the surgical specimen.

Among large bowel resections, which may include all types of colon resection, left colorectal exeresis is the most frequent. Widespread pelvic involvement by primary tumor and peritoneal metastases with infiltration of the pouch and colorectal wall, provides colorectal resection. Such **Figure 4.** Pelvic peritonectomy: en bloc resection of uterus, adnexa, rectosigmoid colon, pelvic and iliac fossae perito‐ neum, right colon and greater omentum.

exeresis should include mesorectal resection and section of mesenteric vessel at their origin to achieve the same radicality requested for primary colorectal cancer treatment. Same radicality criteria should be followed resecting other large bowel sectors. This policy allows to remove both a large amount of mesocolon, frequently infiltrated by implants, and loco-regional lymph nodes which are metastasized in over 50% of cases. [37] Small and large bowel resections are the most frequent surgical procedures because of deep parietal involvement by tumor implants. Thickness of the gastric wall is such as to allow prevailingly a conservative cleaning of the tumor implants without the need to perform major gastric resections.

Lymphadenectomy plays a relevant role in strategy of peritonectomy for ovarian carcinomatosis and its prognostic role is highly significant: the only performing the procedure involves a significant

increase in survival regardless metastastic involvement of lymph nodes[38-40].

Among large bowel resections, which may include all types of colon resection, left colorectal exeresis is the most frequent. Widespread pelvic involvement by primary tumor and peritoneal metastases with infiltration of the pouch and colorectal wall, provides colorectal resection. Such exeresis should include mesorectal resection and section of mesenteric vessel at their origin to achieve the same radicality requested for primary colorectal cancer treatment. Same radicality criteria should be followed resecting other large bowel sectors. This policy allows to remove both a large amount of mesocolon, frequently infiltrated by implants, and locoregional lymph nodes which are metastasized in over 50% of cases. [37]

Lymphadenectomy plays a relevant role in strategy of peritonectomy for ovarian carcinoma‐ tosis and its prognostic role is highly significant: the only performing the procedure involves a significant increase in survival regardless metastastic involvement of lymph nodes[38-40].

The incidence of loco-regional lymph node metastasis is high exceeding 50% of cases and should induce a policy of radicalization of surgery in lymph nodes as well as in peritoneum.

Iliac-obturator and lumbar lymphadenectomy must be performed routinely in primary forms. In secondary forms lymphadenectomy should be performed if it was not done in previous surgery, or if it has been made necessary by evident nodal relapse in the seats already treated.

Additional forms of lymphadenectomy, at the level of hepatic pedicle, splenic hilum, mesen‐ tery or lesser omentum should be performed in the presence of lymphadenopathy macro‐ scopically evident.

#### **9. Removal / "in situ" destruction of implants**

**Fig. 3-4:** Pelvic peritonectomy: en bloc resection of uterus, adnexa, rectosigmoid colon, pelvic and

**Figure 3.** Pelvic peritonectomy: the moment of rectal resection as final step to remove en-bloc the surgical specimen.

right colon and ileum

100 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

pelvic and iliac fossae peritoneum

uterus rectum

sigmoid colon

Small and large bowel resections are the most frequent surgical procedures because of deep parietal involvement by tumor implants. Thickness of the gastric wall is such as to allow prevailingly a conservative cleaning of the tumor implants without the need to perform major gastric resections. Among large bowel resections, which may include all types of colon resection, left colorectal exeresis is the most frequent. Widespread pelvic involvement by primary tumor and peritoneal metastases with infiltration of the pouch and colorectal wall, provides colorectal resection. Such exeresis should include mesorectal resection and section of mesenteric vessel at their origin to achieve the same radicality requested for primary colorectal cancer treatment. Same radicality criteria should be followed resecting other large bowel sectors. This policy allows to remove both a large amount of mesocolon, frequently infiltrated by implants, and loco-regional lymph nodes which are metastasized

**Figure 4.** Pelvic peritonectomy: en bloc resection of uterus, adnexa, rectosigmoid colon, pelvic and iliac fossae perito‐

Small and large bowel resections are the most frequent surgical procedures because of deep parietal involvement by tumor implants. Thickness of the gastric wall is such as to allow prevailingly a conservative cleaning of the tumor implants without the need to perform major

Lymphadenectomy plays a relevant role in strategy of peritonectomy for ovarian carcinomatosis and its prognostic role is highly significant: the only performing the procedure involves a significant

increase in survival regardless metastastic involvement of lymph nodes[38-40].

iliac fossae peritoneum, right colon and greater omentum.

in over 50% of cases. [37]

gastric resections.

neum, right colon and greater omentum.

greater omentum

The treatment of peritoneal implants does not absolutely require the exeresis of wide portions of peritoneum or the mandatory sacrifice of wide tracts of gut or other structures involved in the disease. In relation to quality, quantity, and macroscopic and microscopic (histology) characteristics of carcinomatous implants, the exeresis should respond to general criteria of saving structures and avoiding useless tissue and visceral sacrifices, when local removal or in situ destruction with an appropriate technology allow a radical result.

A conservative approach is achievable when implants are superficial, few infiltrating the underlying structures, and when are prevailingly mucinous. In these conditions, it is possible to spare wide visceral resection especially when small or large intestine are involved. Local excision or local destruction can be assured effectively with curved scissors, electric scalpels with various tips, radiofrequency (Tissue Link), argon beam laser.

In patients undergone neoadjuvant treatments an additional contribution to HIPEC efficacy is given by argon or electric scalpels use over peritoneal areas where an apparent response to chemotherapy was achieved.

These areas are identified by the presence of specific morphological changes, including opacification, thickening, fibrosis of serous peritoneal membrane and presence of red spots. Extensive treatment on such areas with argon or ball tip electro-surgery permits diffuse local damage and partial destruction of fibrosis.

The loss of structural continuity will permit a deeper tissue penetration of chemotherapics and a better contact with eventual encapsulated microscopic residuals in post-chemotherapy fibrosis.

#### **10. HIPEC**

HIPEC is performed at the end of surgical phase by using a 2.5-4.5 litres solution of chemo‐ therapy drugs. Chemotherapy drugs, HIPEC techniques and duration are synthesized in Table 1. Drug solution is infused in peritoneal cavity by catheters appropriately positioned (fig.5). Infusion is performed under a constant temperature of 41-43°.

**Figure 5.** Intra-abdominal catheter position for HIPEC

Open and closed techniques are used for HIPEC but no proven advantage is related to a specific method.

Procedure duration varies from 60 to 90 minutes and CDDP is drug prevailingly administered.

No specific prospective studies have been conducted to verify differences in outcome by specific technique or to test the role of different chemotherapy regimens or drugs.


**Table 1.** HIPEC drugs, duration and techniques. nr: not reported.

#### **11. Inclusion and exclusion criteria**

General criteria provide to include in the therapeutic program patients without extrabdominal disease with optimal ASA and Performance Status scores and with surgically cytoreducible peritoneal carcinomatosis. Isolated and easy resectable liver metastases are not contraindica‐ tion to procedure performing when complete cytoreduction can be achieved. High level of PCI is not an absolute contraindication if surgery can obtain optimal cytoreduction although some authors identify levels beyond which the procedure is not advisable[14, 41-42].

Exclusion criteria include:

Extensive treatment on such areas with argon or ball tip electro-surgery permits diffuse local

The loss of structural continuity will permit a deeper tissue penetration of chemotherapics and a better contact with eventual encapsulated microscopic residuals in post-chemotherapy

HIPEC is performed at the end of surgical phase by using a 2.5-4.5 litres solution of chemo‐ therapy drugs. Chemotherapy drugs, HIPEC techniques and duration are synthesized in Table 1. Drug solution is infused in peritoneal cavity by catheters appropriately positioned (fig.5).

Open and closed techniques are used for HIPEC but no proven advantage is related to a specific

Procedure duration varies from 60 to 90 minutes and CDDP is drug prevailingly administered.

No specific prospective studies have been conducted to verify differences in outcome by

specific technique or to test the role of different chemotherapy regimens or drugs.

Infusion is performed under a constant temperature of 41-43°.

damage and partial destruction of fibrosis.

102 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

**Figure 5.** Intra-abdominal catheter position for HIPEC

method.

fibrosis.

**10. HIPEC**


Age and comorbidity are relative exclusion criteria, being ASA and Performance Status scores the most reliable criteria to be considered even in patient in their eighties or suffering of other concomitant diseases.

#### **12. Settings**

Peritonectomy combined with HIPEC can be used as primary cytoreduction or as secondary. Primary cytoreduction can be performed as frontline or after neoadjuvant chemotherapy as interval debulking surgery. Secondary cytoreduction is performed in patients with recurrent or persistent disease after previous cytoreductive surgery combined or not with various forms of chemotherapy. Tertiary and quaternary cytoreduction combined or not with HIPEC can be performed in patient with repeated intraperitoneal relapses. PRT + HIPEC can be used as consolidation in primary setting during a second look in patients optimally treated with neoadjuvant chemotherapy or in secondary setting during a second look after any combination of surgery and locoregional or systemic chemotherapy.

#### **13. Results**

Over the last 15 years the use of peritonectomy combined with HIPEC has progressively widespread as treatment of peritoneal carcinomatosis from ovarian cancer. Phase III trials about the efficacy of such integrated procedure compared to traditional treatments based on CRS and systemic or normothermic intraperitoneal chemotherapy (IP CHT) are not available. Therefore the role and limits of application of PRT+HIPEC are inferable by results of phase I and mainly phase II studies. At present an overall analysis of the literature allows us to manage data from over 1900 treated cases (Table 2). Collective reviews, multicentric and monocentric case studies are the most available bases to verify the role of PRT combined with HIPEC in treating peritoneal carcinomatosis from ovarian cancer.

Among available collective reviews, the study of de Bree and Helm of 2012 is the more recent and complete. This study is based on 1102 cases collected from 22 monocentric studies and includes the three major previous reviews conducted by Bjelic, Chua and de Bree himself [43, 46-47]. The three multicenter study published between 2010 and 2013 are reported; their study designs were retrospective or prospective phase II. As for monocentric studies, results of a clinical phase II prospective study about the use of PRT and HIPEC in treating peritoneal ovarian carcinomatosis performed by the authors of this chapter is reported. This study is based on 130 cases treated between November 2000 and December 2013 in the same center and by the same staff [48]. This is the largest monocentric case study compared to all other reports included in the collective review of de Bree, the major of which consists of 81 cases.[49]

The multicenter study of Deraco includes exclusively cases undergoing primary CRS as front line, while that of Bakrin comprises prevailingly cases treated for recurrence (83,8%). In the other studies the rates of primary and secondary CRS were almost similar.


**Table 2.** PRT + HIPEC for peritoneal carcinomatosis from EOC: literature review.

Age and comorbidity are relative exclusion criteria, being ASA and Performance Status scores the most reliable criteria to be considered even in patient in their eighties or suffering of other

Peritonectomy combined with HIPEC can be used as primary cytoreduction or as secondary. Primary cytoreduction can be performed as frontline or after neoadjuvant chemotherapy as interval debulking surgery. Secondary cytoreduction is performed in patients with recurrent or persistent disease after previous cytoreductive surgery combined or not with various forms of chemotherapy. Tertiary and quaternary cytoreduction combined or not with HIPEC can be performed in patient with repeated intraperitoneal relapses. PRT + HIPEC can be used as consolidation in primary setting during a second look in patients optimally treated with neoadjuvant chemotherapy or in secondary setting during a second look after any combination

Over the last 15 years the use of peritonectomy combined with HIPEC has progressively widespread as treatment of peritoneal carcinomatosis from ovarian cancer. Phase III trials about the efficacy of such integrated procedure compared to traditional treatments based on CRS and systemic or normothermic intraperitoneal chemotherapy (IP CHT) are not available. Therefore the role and limits of application of PRT+HIPEC are inferable by results of phase I and mainly phase II studies. At present an overall analysis of the literature allows us to manage data from over 1900 treated cases (Table 2). Collective reviews, multicentric and monocentric case studies are the most available bases to verify the role of PRT combined with HIPEC in

Among available collective reviews, the study of de Bree and Helm of 2012 is the more recent and complete. This study is based on 1102 cases collected from 22 monocentric studies and includes the three major previous reviews conducted by Bjelic, Chua and de Bree himself [43, 46-47]. The three multicenter study published between 2010 and 2013 are reported; their study designs were retrospective or prospective phase II. As for monocentric studies, results of a clinical phase II prospective study about the use of PRT and HIPEC in treating peritoneal ovarian carcinomatosis performed by the authors of this chapter is reported. This study is based on 130 cases treated between November 2000 and December 2013 in the same center and by the same staff [48]. This is the largest monocentric case study compared to all other reports included in the collective review of de Bree, the major of which consists of 81 cases.[49] The multicenter study of Deraco includes exclusively cases undergoing primary CRS as front line, while that of Bakrin comprises prevailingly cases treated for recurrence (83,8%). In the

other studies the rates of primary and secondary CRS were almost similar.

of surgery and locoregional or systemic chemotherapy.

104 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

treating peritoneal carcinomatosis from ovarian cancer.

concomitant diseases.

**12. Settings**

**13. Results**

PCI mean ranged from 10.6 to 16.3 and in all series the rate of patients classified as FIGO stage III and IV exceeded 90 %(Tab.3).


**Table 3.** Patients characteristics. nr: not reported.

Peritonectomy was able to achieve optimal cytoreduction in most cases and the rates of complete cytoreduction ranged from 57,7 to 74,9, being the better scores related to lower level of PCI mean.

Platinum based drugs were the most used during HIPEC, alone or in combination with other chemotherapics. Adjuvant systemic chemotherapy was administered in post HIPEC phase in the vast majority of cases.

#### **14. Survival**


Results related to survival are synthesized in Tab 4 - 6.

**Table 4.** PRT + HIPEC for peritoneal carcinomatosis from EOC: survival in multicentre studies.


**Table 5.** PRT + HIPEC for peritoneal carcinomatosis from EOC: survival in collective reviews


**Table 6.** PRT + HIPEC for peritoneal carcinomatosis from EOC: survival in author's monocentric study.

**14. Survival**

**Author - Year**

CC0 Primary

Author – year

**Author - Year**

CC 0

CC 0

Survival 5 yr OS

Survival 5 yr OS

Frontline 47

%

Primary - <sup>66</sup>

58.5

%

Survival 5 yr OS %

Frontline 33.3

Results related to survival are synthesized in Tab 4 - 6.

106 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

25.4

**Helm (HYPER-O) 2010 [44]**

months 5 yr PFS %

19.7

Consolidation 42.4 53.7 24.2 29.6 12.5 33.4

13

Interval debulking 50.2 68.6 9.6 16.8 16 36.5 35.4

Recurrence 18 23.5 9.6 13.7 37 45.7

CC0 Recurrence - - 40.2 51.5

5 yr PFS %

Median OS Months

Interval debulking 54 69 10 17 35

Recurrence 33 42.5 11.5 20.5

Recurrence - - - -

**Table 5.** PRT + HIPEC for peritoneal carcinomatosis from EOC: survival in collective reviews

66.5

Deraco 2011 [45]

Median OS months

**Table 4.** PRT + HIPEC for peritoneal carcinomatosis from EOC: survival in multicentre studies.

33

Consolidation 84 64 63 35

Frontline 60.7 not reached 15.2 30

30.3

Median PFS

13.7

33.7

24.8

26.7 37 - - 23.6 41.5

Median PFS months

**De Bree 2012 [43]**

> 5 yr PFS %

> > 36.5

17,5

(only frontline) - -

Median OS

41.7

**Bakrin 2013 [42]**

52.7

Median PFS months

25

months 5 yr OS % Median OS months

17

In all studies except one, patients treated in primary setting tend to survive more than recurrent; only Bakrin reported better 5- year overall survival in secondary setting (Fig 6).

In an half of reports, 5- year overall survival rate was about 50 % after primary CRS and about 40% after secondary CRS. Overall PF survival ranged across the reported studies between 13 to 43.1% at 5 years.

**Fig 6**: 5- yr Overall and Progression Free survival after primary and secondary CRS +HIPEC **Figure 6.** yr Overall and Progression Free survival after primary and secondary CRS +HIPEC

The values of median survival, both overall and progression free, reflected the general trend of 5-year survival: except for Bakrin's study, patients treated in primary setting survived more than patients treated for recurrence (Fig. 6 ). The values of median survival, both overall and progression free, reflected the general trend of 5-year survival: except for Bakrin's study, patients treated in primary setting survived more than patients treated for recurrence (Fig. 7).

Among patients treated in primary setting, patients undergoing PRT and HIPEC as front line tended to survive more than those neoadjuvated. Data from HYPER-O report are not available by admission of their Authors because of the small number of events

**Fig 6**: Median Overall and Progression Free survival after primary and secondary CRS+HIPEC

of their Authors because of the small number of events

cases in all analyzed studies.

Among patients treated in primary setting, patients undergoing PRT and HIPEC as front line tended to survive more than those neoadjuvated. Data from HYPER-O report are not available by admission

 Results about long term prognosis in patients with PRT and HIPEC administered as consolidation during a second look are not useful for an advisable evaluation because of scarce number of treated treated for recurrence (Fig. 7 ).

%

**Fig 6**: 5- yr Overall and Progression Free survival after primary and secondary CRS +HIPEC

Among patients treated in primary setting, patients undergoing PRT and HIPEC as front line tended **Fig 6**: Median Overall and Progression Free survival after primary and secondary CRS+HIPEC **Figure 7.** Median Overall and Progression Free survival after primary and secondary CRS+HIPEC

Results about long term prognosis in patients with PRT and HIPEC administered as consoli‐ dation during a second look are not useful for an advisable evaluation because of scarce number of treated cases in all analyzed studies. of their Authors because of the small number of events Results about long term prognosis in patients with PRT and HIPEC administered as consolidation during a second look are not useful for an advisable evaluation because of scarce number of treated cases in all analyzed studies. Among patients treated in primary setting, patients undergoing PRT and HIPEC as front line tended to survive more than those neoadjuvated. Data from HYPER-O report are not available by admission of their Authors because of the small number of events Results about long term prognosis in patients with PRT and HIPEC administered as consolidation

during a second look are not useful for an advisable evaluation because of scarce number of treated

to survive more than those neoadjuvated. Data from HYPER-O report are not available by admission

**Figure 8.** yr Overall and Progression Free survival in primary setting

**Fig 8**: 5-yr Overall and Progression Free survival in primary setting

#### **14.1. Prognostic factors**

A lot of potential prognostic factors have been analyzed by uni- and multivariate analyses and completeness of surgical cytoreduction (CC) resulted as the most significant prognostic factor in all series. Among the others, PCI was significantly related to survival in 3 of the 4 studies where has been analyzed.

Platinum response, blood loss, level of bowel wall infiltration by tumor implants, lymph node metastases, use of carboplatin and duration of perfusion, correlated significantly with survival at least once across the study by uni- or multivariate analyses.

#### **14.2. The role of completeness of cytoreduction and PCI**

Since 1970s results of treatment of locally advanced epithelial ovarian cancer emphasized the role of surgical debulking aimed not only at palliation of clinical status borned from intraper‐


Results about long term prognosis in patients with PRT and HIPEC administered as consoli‐ dation during a second look are not useful for an advisable evaluation because of scarce

Among patients treated in primary setting, patients undergoing PRT and HIPEC as front line tended to survive more than those neoadjuvated. Data from HYPER-O report are not available by admission

 Results about long term prognosis in patients with PRT and HIPEC administered as consolidation during a second look are not useful for an advisable evaluation because of scarce number of treated

 Results about long term prognosis in patients with PRT and HIPEC administered as consolidation during a second look are not useful for an advisable evaluation because of scarce number of treated

A lot of potential prognostic factors have been analyzed by uni- and multivariate analyses and completeness of surgical cytoreduction (CC) resulted as the most significant prognostic factor in all series. Among the others, PCI was significantly related to survival in 3 of the 4 studies

Platinum response, blood loss, level of bowel wall infiltration by tumor implants, lymph node metastases, use of carboplatin and duration of perfusion, correlated significantly with survival

Since 1970s results of treatment of locally advanced epithelial ovarian cancer emphasized the role of surgical debulking aimed not only at palliation of clinical status borned from intraper‐

Among patients treated in primary setting, patients undergoing PRT and HIPEC as front line tended to survive more than those neoadjuvated. Data from HYPER-O report are not available by admission

**Fig 7**: Median Overall and Progression Free survival after primary and secondary CRS+HIPEC

**Fig 6**: Median Overall and Progression Free survival after primary and secondary CRS+HIPEC

**Figure 7.** Median Overall and Progression Free survival after primary and secondary CRS+HIPEC

**Fig 6**: 5- yr Overall and Progression Free survival after primary and secondary CRS +HIPEC

**Fig 6**: 5- yr Overall and Progression Free survival after primary and secondary CRS +HIPEC

The values of median survival, both overall and progression free, reflected the general trend of 5-year survival: except for Bakrin's study, patients treated in primary setting survived more than patients

The values of median survival, both overall and progression free, reflected the general trend of 5-year survival: except for Bakrin's study, patients treated in primary setting survived more than patients

number of treated cases in all analyzed studies.

**Fig 8**: 5-yr Overall and Progression Free survival in primary setting

at least once across the study by uni- or multivariate analyses.

**14.2. The role of completeness of cytoreduction and PCI**

**Figure 8.** yr Overall and Progression Free survival in primary setting

of their Authors because of the small number of events

108 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

of their Authors because of the small number of events

treated for recurrence (Fig. 7 ).

treated for recurrence (Fig. 7 ).

%

cases in all analyzed studies.

cases in all analyzed studies.

**14.1. Prognostic factors**

where has been analyzed.

%

**Table 7.** PRT + HIPEC for peritoneal carcinomatosis from EOC: prognostic factors by uni or multivariate analyses. [nr: not reported; ns: not significant]

itoneal disease spread but also to improve long term survival. [50]. The concept of optimal cytoreduction correlated to the dimension of tumor residuals among gynecologic oncologist has progressively induced to reduce from 2 cm to 0.5 cm the maximum acceptable limit. Among surgical oncologist according to Sugarbaker classification such limit is up to 2.5 mm. The role of cytoreduction level in primary resection for locally advanced EOC is well highlighted by the most relevant retrospective and prospective studies reported in literature [51-53].

A meta-analysis of 6.855 cases confirmed these data [54]. The most significant gap was observed between patient without any residue and those with residues of any size. Even in patients undergoing cytoreductive surgery for recurrent disease a lot of retrospective studies [21-30] and a meta-analysis including 2.019 patients [55] confirmed the prognostic role of maximal cytoreduction. Maximal or optimal cytoreductive surgery are correlated to evident advantages improving patients quality of live, decreasing drug resistance clones entity and improving chemotherapy efficacy. Complete removal of peritoneal disease proves to be the most relevant prognostic factor in all setting even in all analyzed studies on HIPEC here reported (Tab 6).

Some authors argued about the role of PCI in selecting patients to be treated with peritonec‐ tomy and HIPEC, identifying level of diffusion of peritoneal disease by scores beyond which such combined procedures should be avoided. In particular Bakrin identified in a value of PCI equal to 10 that limit in relation to related poor prognosis, while other authors [14] identify specific laparoscopic scoring of diffusion of peritoneal carcinomatosis to predict the achieve‐ ment of an optimal cytoreduction.

Results of our monocentric study show that in PC from ovarian cancer high degrees of PCI are not an absolute limit to the execution of the procedure, if it is possible to obtain an optimal cytoreduction. We believe that high degree of PCI does not constitute an absolute contraindi‐ cation to cytoreduction, as some claim [41, 56] and that rather one should take greater account of technical feasibility, quality of carcinomatosis of the individual case and possibility of obtaining an optimal cytoreduction. In our series, which had a PCI mean of 16.3, patients with PCI> 16 have nevertheless demonstrated a 5-year overall survival of 24.3%, with no difference between primary and secondary CRS, and a 5 year survival of 50.2 % (median 61.1 months) when in these patients with high PCI a complete cytoreduction (CC0) was obtained.

#### **14.3. The role of NACT**

Diffuse Peritoneal Carcinomatosis in primary setting is ideal target for neoadjuvant chemo‐ therapy with carboplatin and taxol, due to high rate or responsiveness when administered as first line treatment (> 80%).

Nevertheless advantages of such strategy are not clear and results are conflicting, both in patients treated with and without HIPEC.

In patients undergoing NACT and successively treated with standard cytoreductive surgery and systemic chemotherapy, preoperative chemotherapy failed to improve survival. In EORTC 55971 phase III trial, NACT increased the rate of optimal Cytoreduction and decreased post-operative morbidity compared to front line CRS, but did not influence Overall or PF survival [57-58].

Similar results have been observed also in the studies related to the role of NACT in patients treated with PRT + HIPEC[59-60].

A better comprehension of significance of this strategy may drawn from analysis of chemo‐ sensitivity during NACT. In our monocentric series more than 50% of patients treated in primary setting undergo carbo-taxol-based NACT. 26,3 % didn't respond to this regimen and demonstrated a significant worse prognosis (29,4% 5-yr OS) compared to cases treated front line or NACT responders (56,4% 5-yr OS).

Some studies envisage for NACT disadvantages related to increased risk of platinum resist‐ ance during post-CRS adjuvant chemotherapy [61] or post-NACT histological changes occurring in tumor tissue that correlate with a poor prognosis [62]. These data are reflected in our cases: neoadjuvated patients showed a higher percentage of chemoresistance during post-HIPEC treatment with platinum derivatives (41.7%) than those not neoadjuvated (31.8%) and survived less.

In the near future the results of ongoing trials will better highlight the optimal strategy in using NACT. Based on results of studies now available, NACT regimen should be personalized and administered to patients with bulky intraperitoneal disease at risk of incomplete CRS, or to patients with small metastatic pleural effusion or with small isolated liver metastasis easily resectable during CRS.

#### **14.4. The role of platinum chemoresistance**

most relevant prognostic factor in all setting even in all analyzed studies on HIPEC here

Some authors argued about the role of PCI in selecting patients to be treated with peritonec‐ tomy and HIPEC, identifying level of diffusion of peritoneal disease by scores beyond which such combined procedures should be avoided. In particular Bakrin identified in a value of PCI equal to 10 that limit in relation to related poor prognosis, while other authors [14] identify specific laparoscopic scoring of diffusion of peritoneal carcinomatosis to predict the achieve‐

Results of our monocentric study show that in PC from ovarian cancer high degrees of PCI are not an absolute limit to the execution of the procedure, if it is possible to obtain an optimal cytoreduction. We believe that high degree of PCI does not constitute an absolute contraindi‐ cation to cytoreduction, as some claim [41, 56] and that rather one should take greater account of technical feasibility, quality of carcinomatosis of the individual case and possibility of obtaining an optimal cytoreduction. In our series, which had a PCI mean of 16.3, patients with PCI> 16 have nevertheless demonstrated a 5-year overall survival of 24.3%, with no difference between primary and secondary CRS, and a 5 year survival of 50.2 % (median 61.1 months)

when in these patients with high PCI a complete cytoreduction (CC0) was obtained.

Diffuse Peritoneal Carcinomatosis in primary setting is ideal target for neoadjuvant chemo‐ therapy with carboplatin and taxol, due to high rate or responsiveness when administered as

Nevertheless advantages of such strategy are not clear and results are conflicting, both in

In patients undergoing NACT and successively treated with standard cytoreductive surgery and systemic chemotherapy, preoperative chemotherapy failed to improve survival. In EORTC 55971 phase III trial, NACT increased the rate of optimal Cytoreduction and decreased post-operative morbidity compared to front line CRS, but did not influence Overall or PF

Similar results have been observed also in the studies related to the role of NACT in patients

A better comprehension of significance of this strategy may drawn from analysis of chemo‐ sensitivity during NACT. In our monocentric series more than 50% of patients treated in primary setting undergo carbo-taxol-based NACT. 26,3 % didn't respond to this regimen and demonstrated a significant worse prognosis (29,4% 5-yr OS) compared to cases treated front

Some studies envisage for NACT disadvantages related to increased risk of platinum resist‐ ance during post-CRS adjuvant chemotherapy [61] or post-NACT histological changes occurring in tumor tissue that correlate with a poor prognosis [62]. These data are reflected in our cases: neoadjuvated patients showed a higher percentage of chemoresistance during post-HIPEC treatment with platinum derivatives (41.7%) than those not neoadjuvated (31.8%) and

reported (Tab 6).

ment of an optimal cytoreduction.

110 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

**14.3. The role of NACT**

first line treatment (> 80%).

survival [57-58].

survived less.

patients treated with and without HIPEC.

treated with PRT + HIPEC[59-60].

line or NACT responders (56,4% 5-yr OS).

The role of platinum chemoresistance has been analyzed in three studies and in two of them chemoresistance resulted as a negative prognostic factor [44, 48 - Tab 6]

In two studies platinum chemoresistance was analyzed in pre-HIPEC phase in patients treated for recurrence while in our monocentric study we have evaluated the chemoresistance by referring to the recurrence/progression within six months after the end of post-HIPEC adjuvant treatment with platinum-based drugs, both in primary and in recurrent forms.

In the two multicenter studies where chemoresistance was analyzed in pre- HIPEC phase, it didn't influence survival in Bakrin's report while resulted marginally significant in HYPER-O registry.

In our series,Platinum chemoresistance so assessed was related to a worse prognosis only after primary CRS plus HIPEC, with both univariate and multivariate analyses (Table 6). The negative correlation between platinum chemoresistance and prognosis in primary forms can be partly explained by the possibility that NACT determines chemoresistance against the platinum used in systemic form after CRS as described above [62].

In our series, post-HIPEC chemoresistance did not influence significantly survival of recurrent patients, whose rates of platinum chemoresistance and chemosensitivity were similar (47.2% vs 52.8%).

In patients treated for recurrence, PRT combined with HIPEC may induce, especially for cases CC0, a reset of previous oncologic situation and that the chemosensitivity assessment to platinum based drugs chemotherapy post-HIPEC more faithfully represents the new relation‐ ship between patient and such chemotherapics. Moreover, the possibility that the CRS associated with HIPEC may lead to a retrieval of chemoresistance to platinum is theorized by some authors [54].

#### **14.5. The role of bowel wall infiltration**

Among the analyzed studies carcinomatous infiltration of intestinal wall has been analyzed only in our monocentric study. Progressive infiltration of bowel wall influenced negatively long term survival. The impact of the degree of parietal layers infiltration like the T role in TNM staging of gastro-intestinal tumor but in an inverse sense has been analyzed in previous report by us and other authors in relation to only colorectal resection [63-65]

Recently the evaluation of bowel wall infiltration up to the mucosa has been included in new 2014 FIGO stage for ovarian cancer identifying mucosal infiltration as FIGO stage IVb [1,2].

#### **14.6. The role of lymphadenectomy**

The role of lymphadenectomy and significance of lymph node metastatic involved in locally advanced EOC is controversial. Lymphadenectomy is supported from some authors on the basis of its positive influence on survival [66-67], while other authors are skeptical [68]. The high rate of loco-regional lymph node metastases justify systematic lymphadenectomy in primary setting on principle and in secondary setting when not performed during primary cytoreduction.

The significance of lymph node metastasis was analyzed only in our monocentric study, where iliac-obturatory and lumbar lymphadenectomy was performed routinely in primary settings and when not done in previous CRS in patients treated for recurrence. Colorectal resections were routinely performed with radical technique as previously reported. Lymphadenectomy in other districts such as the hepatic pedicle, perigastric or mesenteric stations were performed when necessary.

In our study, overall 52,6% of patients had lymph node metastases without significant differences between primary or recurrent forms, similarly to what reported in the literature [45]. Although lymph node involvement worsened prognosis, related 5-year Overall survival reached 39,6% corroborating the role of lymphadenectomy.

#### **15. The role of HIPEC — Comparison of HIPEC vs no HIPEC**

Overall, the results so far obtained by using of PRT combined with HIPEC in treating peritoneal carcinomatosis from ovarian cancer even available mainly if not exclusively from non random‐ ized prospective studies show progressive improvement of long term survival both in primary or recurrent forms in high volume activity centers [55].

Although general consensus about the role of maximum cytoreduction is at present undis‐ putable, criticism about HIPEC role is diffuse because of its potential high morbidity risk and lack of prospective controlled studies.

At present both in primary and recurrent settings, a series of cases / controls studies has demonstrated the major efficacy of the association between CRS and HIPEC compared to traditional treatments [69-76]. Results of the first phase III prospective study recently published [77] about this topic confirmed a significant improvement in long term survival in patients treated with HIPEC compared with those undergoing traditional treatment with CRS and adjuvant systemic chemotherapy.

#### **16. Morbidity and mortality**

Peritonectomy and HIPEC are integrated in a complex and aggressive procedure whose specific related complications are difficult to distinguish, being the overall morbidity reason‐ ably related to the whole procedure. Therefore if renal and haematological toxicity have to be related specifically to chemotherapy activity, even for most common surgical complications like anastomotic leak, intestinal fistulas or endoperitoneal haemorrhage, HIPEC influence can't be undervalued.

Overall the incidence of major complications (grade 3 and 4) ranged from 14% to 56% whose treatment provided surgical, radiological or endoscopic re-intervention in a percentage ranging from 13% to 19,2%.

Haematological and renal toxicity accounted for a maximum incidence of 11 and 8 % respectively.

Mortality rate was extremely variable ranging from 0 to 10%.

**14.6. The role of lymphadenectomy**

112 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

cytoreduction.

when necessary.

The role of lymphadenectomy and significance of lymph node metastatic involved in locally advanced EOC is controversial. Lymphadenectomy is supported from some authors on the basis of its positive influence on survival [66-67], while other authors are skeptical [68]. The high rate of loco-regional lymph node metastases justify systematic lymphadenectomy in primary setting on principle and in secondary setting when not performed during primary

The significance of lymph node metastasis was analyzed only in our monocentric study, where iliac-obturatory and lumbar lymphadenectomy was performed routinely in primary settings and when not done in previous CRS in patients treated for recurrence. Colorectal resections were routinely performed with radical technique as previously reported. Lymphadenectomy in other districts such as the hepatic pedicle, perigastric or mesenteric stations were performed

In our study, overall 52,6% of patients had lymph node metastases without significant differences between primary or recurrent forms, similarly to what reported in the literature [45]. Although lymph node involvement worsened prognosis, related 5-year Overall survival

Overall, the results so far obtained by using of PRT combined with HIPEC in treating peritoneal carcinomatosis from ovarian cancer even available mainly if not exclusively from non random‐ ized prospective studies show progressive improvement of long term survival both in primary

Although general consensus about the role of maximum cytoreduction is at present undis‐ putable, criticism about HIPEC role is diffuse because of its potential high morbidity risk and

At present both in primary and recurrent settings, a series of cases / controls studies has demonstrated the major efficacy of the association between CRS and HIPEC compared to traditional treatments [69-76]. Results of the first phase III prospective study recently published [77] about this topic confirmed a significant improvement in long term survival in patients treated with HIPEC compared with those undergoing traditional treatment with CRS and

Peritonectomy and HIPEC are integrated in a complex and aggressive procedure whose specific related complications are difficult to distinguish, being the overall morbidity reason‐

reached 39,6% corroborating the role of lymphadenectomy.

or recurrent forms in high volume activity centers [55].

lack of prospective controlled studies.

adjuvant systemic chemotherapy.

**16. Morbidity and mortality**

**15. The role of HIPEC — Comparison of HIPEC vs no HIPEC**

It is difficult to compare various experiences mainly because of different criteria by which complications are defined and of different classifications with which morbidity levels are synthetized. The number of possible complications after PRT + HIPEC is high and the likely to have a complete scenarios of all adverse events is difficult and depends on the accuracy with which databases are prepared and on the prospective or retrospective modalities with which data are updated.

A detailed example of database dedicated to morbidity is described in the book edited in 2013 by Sugarbaker about the treatment of peritoneal carcinomatosis [78] with an indication of 48 adverse events arranged within 9 categories. Each adverse event is graded with a score from I to IV, and 14 prognostic indicators have been used in uni and multivariate analyses with the aim to identify the most significant risk factors for postoperative morbidity and mortality.

It is an interesting try to organize the adverse events but results difficult to reproduce and not yet used in other studies. Its use can be considered particularly important for studies dedicated to this problem. An acceptable compromise to obtain comparable data can be gained by using of more simplified and diffused classifications of complications, such as that of Dindo's or CTCAE, and by performing multivariate analyses to infer the risk factors for various compli‐ cations.

Among the analyzed studies, only Bakrin's multicentre study and the author's monocentric study reported the results of uni or multivariate analyses on risk factors and PCI and CC score resulted as the most significant parameters correlated to an increased occurrence of major complications. Cascales Campos on 91 patients treated with PRT + HIPEC for ovarian carci‐ nomatosis in various settings [76] has confirmed with multivariate analysis the role of PCI as risk factor for major complications, associated to the performing of digestive anastomoses.

These results reliably correlate with operative mortality and re-intervention rates, as reported in Deraco and Di Giorgio's studies that include cases with highest mean of PCI, and with lowest morbidity rate in patients treated as consolidation which are free of disease at second look. An exception is represented by Pomel's prospective study dedicated to cases treated as consoli‐ dation with Oxaliplatin based-HIPEC (CHIPOVAC); the study was stopped for excessive morbidity. (70)

The duration of procedure resulted as risk factor in the monocentric study, as in other reports about using of PRT + HIPEC in both ovarian and extra-ovarian PC [72-74].

Among major complications, the anastomotic dehiscences are the most dangerous for con‐ comitant risk of severe sepsis and postoperative mortality. Risk factors for these events are numerous and correlate with the extension of carcinomatosis, the number of intestinal resections required for cytoreduction, the duration of procedure, the blood loss, the extensive use of in situ destruction of parietal implants, the type of anastomosis, in particular, colorectal, the lack of adequate bowel cleaning in occluded and sub-occluded patients, the previous treatment with bevacizumab.

The containment of the risks lies in reducing the number of anastomoses with appropriate evaluation of the intestinal tracts to be resected and avoiding the simultaneous performing of multiple digestive anastomoses in conjunction with low colorectal anastomosis. In these cases it is strategically correct to perform a colorectal resection according to Hartmann and delay recanalization in a second intervention after the end of adjuvant treatment and after further 6 months follow up [65].

In summary, also in presence of remarkable variability of data from the analyzed studies, the incidences of complication and mortality appear limited and comparable to those related to major abdominal and pelvic surgery. Morbidity rate control is possible in highly active centers with consolidated experience and specialized medical, nursing and logistic organization. Results of trials in progress on the specific role of HIPEC shall furnish also significant data about HIPEC related morbidity, while the use of specific protocols and prospective databases, connected to multi-institutional experiences, can give useful data to limit morbidity in medium period.

#### **17. Future**

The use of PRT combined with HIPEC for treating peritoneal carcinomatosis from ovarian cancer is being widely diffused thanks to promising results in terms of survival but is not without its critics that are primarily focused on the role of HIPEC. To date, the major criticisms about HIPEC involve its potential influence on survival and morbidity and the lack of prospective randomized studies as support of results of this procedure. The differing opinions between oncological surgeons, who are more likely to use HIPEC, and oncologic gynecologists and medical oncologists, who are more likely to use standard treatment with CRS and systemic CHT or, more rarely, isothermic IP-CHT, plays a relevant role in such a scenario. Therefore, it is necessary to verify whether PRT plus HIPEC can guarantee better survival compared with standard treatments and whether the incidence of related morbidity is acceptable in compar‐ ison with other types of treatment. At present many clinical trials are ongoing about the efficacy of PRT and HIPEC, most of them are focused specifically on HIPEC role, both in primary and in recurrent patients (Tab 8).


The duration of procedure resulted as risk factor in the monocentric study, as in other reports

Among major complications, the anastomotic dehiscences are the most dangerous for con‐ comitant risk of severe sepsis and postoperative mortality. Risk factors for these events are numerous and correlate with the extension of carcinomatosis, the number of intestinal resections required for cytoreduction, the duration of procedure, the blood loss, the extensive use of in situ destruction of parietal implants, the type of anastomosis, in particular, colorectal, the lack of adequate bowel cleaning in occluded and sub-occluded patients, the previous

The containment of the risks lies in reducing the number of anastomoses with appropriate evaluation of the intestinal tracts to be resected and avoiding the simultaneous performing of multiple digestive anastomoses in conjunction with low colorectal anastomosis. In these cases it is strategically correct to perform a colorectal resection according to Hartmann and delay recanalization in a second intervention after the end of adjuvant treatment and after further 6

In summary, also in presence of remarkable variability of data from the analyzed studies, the incidences of complication and mortality appear limited and comparable to those related to major abdominal and pelvic surgery. Morbidity rate control is possible in highly active centers with consolidated experience and specialized medical, nursing and logistic organization. Results of trials in progress on the specific role of HIPEC shall furnish also significant data about HIPEC related morbidity, while the use of specific protocols and prospective databases, connected to multi-institutional experiences, can give useful data to limit morbidity in medium

The use of PRT combined with HIPEC for treating peritoneal carcinomatosis from ovarian cancer is being widely diffused thanks to promising results in terms of survival but is not without its critics that are primarily focused on the role of HIPEC. To date, the major criticisms about HIPEC involve its potential influence on survival and morbidity and the lack of prospective randomized studies as support of results of this procedure. The differing opinions between oncological surgeons, who are more likely to use HIPEC, and oncologic gynecologists and medical oncologists, who are more likely to use standard treatment with CRS and systemic CHT or, more rarely, isothermic IP-CHT, plays a relevant role in such a scenario. Therefore, it is necessary to verify whether PRT plus HIPEC can guarantee better survival compared with standard treatments and whether the incidence of related morbidity is acceptable in compar‐ ison with other types of treatment. At present many clinical trials are ongoing about the efficacy of PRT and HIPEC, most of them are focused specifically on HIPEC role, both in primary and

about using of PRT + HIPEC in both ovarian and extra-ovarian PC [72-74].

114 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

treatment with bevacizumab.

months follow up [65].

period.

**17. Future**

in recurrent patients (Tab 8).


**Table 8.** Ongoing clinical trials on HIPEC in EOC. [79-90]

Results of such trials may help to confirm the role of HIPEC in various subsets of patients treated in primary setting and contribute to specify also the prognostic role of NACT and chemoresistance.

An half of ongoing studies are referred to recurrent patients. In the most of such trials, only platinum sensitive recurrences are considered. All of these studies are aimed to evaluate the prognostic role of HIPEC in terms of OS, PFS and DFS, having a variety of secondary outcomes such as the role of different combinations of chemotherapy drugs, the use of IP CHT after HIPEC, the QoL, toxicity and morbidity.

#### **18. Conclusions**

At present, lacking results of prospective randomized phase III studies, the role of PRT and HIPEC in treating peritoneal carcinomatosis from EOC can be reliably evaluated by the studies reported in this research which include over 1900 treated cases. The overall size of these case studies is a solid base to reliably identify the trend of results regardless of the study limitations discussed above.

On the basis of analysed results, following conclusions can be drawn:


**•** Major complications and mortality rates are similar to those related to major abdominal pelvic surgery and are not different after primary or secondary cytoreduction. PCI and CC scores represent the most significant risk factors for major complications.

#### **Author details**

**Studies Time Drug Type of study**

116 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

Primary

Primary

Results of such trials may help to confirm the role of HIPEC in various subsets of patients treated in primary setting and contribute to specify also the prognostic role of NACT and

An half of ongoing studies are referred to recurrent patients. In the most of such trials, only platinum sensitive recurrences are considered. All of these studies are aimed to evaluate the prognostic role of HIPEC in terms of OS, PFS and DFS, having a variety of secondary outcomes such as the role of different combinations of chemotherapy drugs, the use of IP CHT after

At present, lacking results of prospective randomized phase III studies, the role of PRT and HIPEC in treating peritoneal carcinomatosis from EOC can be reliably evaluated by the studies reported in this research which include over 1900 treated cases. The overall size of these case studies is a solid base to reliably identify the trend of results regardless of the study limitations

**•** PRT plus HIPEC guarantee significant percentage of long-term overall and progression free

**•** In all settings, complete cytoreduction represents the most significant prognostic factor.

**•** High PCI levels do not constitute a limitation for this procedure if optimal CRS is technically

**•** The prognostic role of NACT and Platinum-based chemoresistance is uncertain; but NACT and platinum chemoresistance should be better assessed, the first for when to be applied

On the basis of analysed results, following conclusions can be drawn:

and the other for its application even in post-HIPEC setting

*Quality of Life and Survivorship Care in Patients Undergoing Hyperthermic Intraperitoneal Chemotherapy (HIPEC)*

*Surgery and Chemotherapy With or Without Chemotherapy After Surgery in Treating Patients With Ovarian, Fallopian Tube,*

**Table 8.** Ongoing clinical trials on HIPEC in EOC. [79-90]

HIPEC, the QoL, toxicity and morbidity.

survival in primary and recurrent settings.

*Uterine, or Peritoneal Cancer*

chemoresistance.

**18. Conclusions**

discussed above.

feasible.

**Identification number\***

Recurrence - Efficacy NCT01126346

Recurrence Cisplatin Safety NCT01970722

Angelo Di Giorgio1\*, Daniele Biacchi1 , Antonio Ciardi2 , Alessio Impagnatiello1 , Maurizio Cardi1 , Simone Sibio1 , Bianca Sollazzo1 , Joseph Maher Fouad Atta1 , Giuseppe Naso1 , Fabio Accarpio1 and Paolo Sammartino1

\*Address all correspondence to: angelo.digiorgio@uniroma1.it

1 Department of Surgery "Pietro Valdoni", Sapienza University of Rome, Italy

2 Department of Radiology, Oncology and Human Pathology, Sapienza University of Rome, Italy

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**Chapter 5**

## **Individualized Novel Therapies for Patients with Tumor Suppressor Genes** *BRCA1* **and** *BRCA2* **Mutated Epithelial Ovarian Cancer**

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[79] http://www.clinicaltrials.gov/ct2/show/ NCT01387399 [80] http://www.clinicaltrials.gov/ct2/show/NCT01539785 [81] http://www.clinicaltrials.gov/ct2/show/NCT01091636 [82] http://www.clinicaltrials.gov/ct2/show/NCT01628380 [83] http://www.clinicaltrials.gov/ct2/show/NCT00426257 [84] http://www.clinicaltrials.gov/ct2/show/NCT01376752 [85] http://www.clinicaltrials.gov/ct2/show/NCT01709487 [86] http://www.clinicaltrials.gov/ct2/show / NCT01144442 [87] http://www.clinicaltrials.gov/ct2/show/NCT01767675 [88] http://www.clinicaltrials.gov/ct2/show/NCT01659554 [89] http://www.clinicaltrials.gov/ct2/show/NCT01126346 [90] http://www.clinicaltrials.gov/ct2/show/NCT01970722

Sandra García-Nieto, Carmen Guillén-Ponce, Carmen Alonso, María-Carmen Rodríguez-Soriano, María-Luz Pombo, Earl Julie and Samir A. Farghaly

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61938

#### **Abstract**

Ovarian cancer is the leading cause of death in women with gynecological cancer, since a large proportion of patients are diagnosed at later stages of the disease. The incidence of ovarian cancer in the general population is 2%, but patients with germline mutations in the *BRCA* genes have a risk of developing ovarian cancer of up to 2050% with a cumula‐ tive risk of ovarian cancer at 70 years of age of 40% in *BRCA1* and 18% in *BRCA2* muta‐ tion carriers. Although it is a chemosensitive tumor, most of the patients after surgery and chemotherapy based on taxanes and platinum will relapse later in life. Due to the high risk of developing ovarian cancer in patients with *BRCA* germline mutations, new treatments rely increasingly on histological and molecular characteristics of the primary tumor, achieving greater selectivity and lower toxicity compared with standard cytotoxic agents. Poly (ADP-ribose) polymerase (PARPS) inhibitors are the first biologically active agents for patients with ovarian cancer with alterations in the DNA repair pathway, par‐ ticularly in the high-grade serous subtype of ovarian cancer.

The results of clinical trials published so far mean that olaparib has been approved, pend‐ ing the results of the Phase III trials. The European Medicines Agency (EMA) adopted olaparib (lynparza ®) on the December 18, 2014, as a maintenance therapy after response to platinum-based chemotherapy in relapsed platinum-sensitive ovarian cancer patients with a *BRCA* mutation. By contrast, the Food and Drug Administration (FDA) approved olaparib on December 19, 2014, in patients with high-grade ovarian epithelial serous tu‐ mors and a *BRCA* mutation who have progressed during three or more lines of chemo‐ therapy. Olaparib is also used in primary fallopian tube and peritoneal cancers with *BRCA* mutations.

**Keywords:** PARP inhibitors, Olaparib, Mutant epithelial ovarian cancer, Tumor suppres‐ sor Genes *BRCA1* and *BRCA2*, Novel therapies for ovarian cancer

© 2016 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.

#### **1. Introduction**

The usual treatment of advanced disease of ovarian cancer is surgery [1] followed by taxane and platinum-based chemotherapy, although a large proportion of patients will relapse throughout their lives. Therefore, current clinical trials focus on the detection of molecular targets that can act more selectively and efficiently on ovarian cancer [2].

It is known that chemotherapy treatments damage the DNA and there are molecules that are responsible for repair and proper maintenance of the genome such as poly (adenosine diphosphate-ribose) polymerase (PARP), which plays a key role in the repair of DNA singlestrand breaks; so, researchers have focused on the mechanism for the development of new therapies, including the PARP inhibitors.

Olaparib is the first PARP-inhibitor class recently approved for the treatment of ovarian cancer with mutations in *BRCA* (breast cancer), delivered orally and with good tolerance, with myelosuppression and gastrointestinal toxicity as the most frequent adverse effects. Through‐ out the chapter, we describe the features of hereditary ovarian cancer with *BRCA* mutation, and the steps to follow once the mutation is detected in patients at risk of ovarian cancer. The characteristics of PARP inhibitors are discussed, focusing on olaparib, and their use and dosage recommendations after reviewing the main Phase II trials for which it has been approved; also, we comment on Phase III olaparib trials that are currently underway.

#### **2.** *BRCA1* **and** *BRCA2* **genes**

Most breast cancers and hereditary ovarian cancers are associated with mutations in two genes, breast cancer type 1 and 2 susceptibility genes (*BRCA1* and *BRCA2*), whose prevalence varies among different geographical areas and ethnic groups, being known as the founder effect among Ashkenazi Jews, whose descendants have an increased risk with any of the three founder mutations (two *BRCA1* mutations, 187delAG and 5385insC, and one *BRCA2* mutation, 6174delT). Founder mutations come from a single carrier ancestor initially extending from a small town with some degree of inbreeding, highly recurrent alterations, or even characteris‐ tics of an ethnic group [3].

*BRCA1* and *BRCA2* are tumor suppressor genes and are involved in the repair of double-strand breaks of DNA, maintaining genome integrity. Germline mutations in the *BRCA1* and *BRCA2* genes are caused by the loss of one of their wild type alleles, and therefore have a single functioning allele, promoting genomic instability and tumorigenesis [4].

Studies suggest that mutation in p53 favor loss of functionality of the *BRCA 1/2* genes inducing tumorigenesis [5,6]. The function of p53 is to detect DNA damage during the cell cycle, allowing repair; so if p53 is altered, DNA repair is incomplete or inadequate, causing cell death in normal cells [7]. The p53 mutation was detected in almost 90% of patients with high-grade serous carcinoma (HGSC) in patients with *BRCA 1/2* mutation.

The *BRCA1* is located on the long arm of chromosome 17 (17q21) gene. It has a sequence of 5,592 nucleotides, divided into 24 exons. The *BRCA1* protein is localized with *BRCA2*, PALPB2, and RAD51 (essential proteins in homologous recombination) in areas of repair of doublestrand breaks of DNA. *BRCA1* is part of BASC (*BRCA1* Associated genome surveillance) complex multiprotein complex responsible for the detection, removal, and repair of DNA breaks. In conclusion, *BRCA1* interacts with other oncogenes, repressors, and activators of transcription, cell cycle regulators, etc., involved in genomic stability. It has also been linked to the development of other cancers, particularly pancreas, uterus, and prostate cancers [8].

**1. Introduction**

therapies, including the PARP inhibitors.

126 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

**2.** *BRCA1* **and** *BRCA2* **genes**

tics of an ethnic group [3].

The usual treatment of advanced disease of ovarian cancer is surgery [1] followed by taxane and platinum-based chemotherapy, although a large proportion of patients will relapse throughout their lives. Therefore, current clinical trials focus on the detection of molecular

It is known that chemotherapy treatments damage the DNA and there are molecules that are responsible for repair and proper maintenance of the genome such as poly (adenosine diphosphate-ribose) polymerase (PARP), which plays a key role in the repair of DNA singlestrand breaks; so, researchers have focused on the mechanism for the development of new

Olaparib is the first PARP-inhibitor class recently approved for the treatment of ovarian cancer with mutations in *BRCA* (breast cancer), delivered orally and with good tolerance, with myelosuppression and gastrointestinal toxicity as the most frequent adverse effects. Through‐ out the chapter, we describe the features of hereditary ovarian cancer with *BRCA* mutation, and the steps to follow once the mutation is detected in patients at risk of ovarian cancer. The characteristics of PARP inhibitors are discussed, focusing on olaparib, and their use and dosage recommendations after reviewing the main Phase II trials for which it has been approved; also,

Most breast cancers and hereditary ovarian cancers are associated with mutations in two genes, breast cancer type 1 and 2 susceptibility genes (*BRCA1* and *BRCA2*), whose prevalence varies among different geographical areas and ethnic groups, being known as the founder effect among Ashkenazi Jews, whose descendants have an increased risk with any of the three founder mutations (two *BRCA1* mutations, 187delAG and 5385insC, and one *BRCA2* mutation, 6174delT). Founder mutations come from a single carrier ancestor initially extending from a small town with some degree of inbreeding, highly recurrent alterations, or even characteris‐

*BRCA1* and *BRCA2* are tumor suppressor genes and are involved in the repair of double-strand breaks of DNA, maintaining genome integrity. Germline mutations in the *BRCA1* and *BRCA2* genes are caused by the loss of one of their wild type alleles, and therefore have a single

Studies suggest that mutation in p53 favor loss of functionality of the *BRCA 1/2* genes inducing tumorigenesis [5,6]. The function of p53 is to detect DNA damage during the cell cycle, allowing repair; so if p53 is altered, DNA repair is incomplete or inadequate, causing cell death in normal cells [7]. The p53 mutation was detected in almost 90% of patients with high-grade

functioning allele, promoting genomic instability and tumorigenesis [4].

serous carcinoma (HGSC) in patients with *BRCA 1/2* mutation.

targets that can act more selectively and efficiently on ovarian cancer [2].

we comment on Phase III olaparib trials that are currently underway.

The *BRCA2* gene is located on chromosome 13q (13q12), and has a sequence of 11,385 nucleo‐ tides in 27 exones. *BRCA2* plays a key role in the cell cycle, especially in cytokinesis and meiosis, as well as in homologous recombination DNA repair [9]. Mutations in this gene have been linked to other cancers such as cancer of the gallbladder, pancreas, stomach, and malignant melanoma.

Hereditary breast and ovarian cancer (HBOC) syndrome is characterized by an autosomal dominant inheritance with high penetrance, presenting increased susceptibility to breast and ovarian cancer, although it has been shown that *BRCA1* and *BRCA2* genes are expressed in most tissues and cells analyzed, suggesting that the pathological impact of a mutation is tissuespecific and that there must be alternative pathways that compensate for their loss of function in other tissue types [10].

Women with hereditary ovarian cancer may have higher rates of response to chemotherapy and improved survival rates in cases of sporadic cancer.

In 2012, the results of an analysis were published [11] in which data from 26 observational studies on the survival of women with ovarian cancer with germline mutations in *BRCA1* and *BRCA2* mutations.

Data from 1,213 cases with a germline mutation in *BRCA1* (*n* = 909) and *BRCA2* (*n* = 303) and 2,666 no mutation carriers were included. The observed overall survival (OS) for 5 years was 36% in non-carriers of mutation patients versus 44% for patients with a *BRCA1* mutation and 52% for patients with a mutation in *BRCA2*. There was an increased survival in *BRCA* mutation carriers versus non-carriers. *BRCA2* carriers had a better prognosis. There were several significant differences in the clinical characteristics of *BRCA1* and *BRCA2* compared with noncarriers. The *BRCA1* and *BRCA2* tumors were more likely to be serous histology and less likely to be mucinous histology. Patients with *BRCA1* and *BRCA2* mutation were more likely to have a tumor stage III/IV and present greater degree of differentiation compared to non-carriers. *BRCA1* carriers were also younger at diagnosis.

Detection of *BRCA1* and *BRCA2* genes is accomplished by DNA extraction from peripheral blood lymphocytes. Detection techniques must be able to identify everything from small changes to large duplications or deletions of exons.

There are over one thousand different mutations to *BRCA1* and *BRCA2*, most of them being small insertions or deletions causing a change in the reading frame (frameshift) and producing a stop codon. However, amino acid substitutions producing a stop codon (nonsense) or mutations located at sites of exon splicing that alter the splicing of the genes and producing full or partial loss of exons [12] can also be found to occur.

It is known that the mutation in the *BRCA1* gene presents a risk of ovarian cancer throughout life of up to 40%, while the *BRCA2* gene has a 20% risk. Although penetrance may vary in the same family carrying a mutation, suggesting that the risk can be influenced by allelic hetero‐ zygosity, modifier genes, and environmental and hormonal cofactors [13,14].

After the diagnosis of breast cancer in a patient with a *BRCA* mutation, there is a subsequent risk of developing ovarian cancer of up to 12.7% in women for *BRCA1* and 6.8% for *BRCA2* [15].

Diagnosis in elderly or the absence of family history does not exclude the presence of a germline mutation as approximately 35% of the *BRCA* mutation carriers have no family history. Genetic tests are expensive, so you should select the most appropriate individuals for genetic testing, varying recommendations between populations and countries.

The presence of *BRCA* somatic and germline mutations are predictors of response to different chemotherapy treatments because they exhibit greater sensitivity and response to platinumtype drugs or PARP inhibitors, which are involved in the repair of DNA single-strand breaks [16].

It is important that families carrying this mutation are informed about the risks of developing various types of cancer, including education about prenatal diagnosis and assisted reproduc‐ tion. Another option is IVF with previously selected embryos. Although, the decision should finally be made on an individual basis and will depend on the preference of each patient.

#### **3. Patients with ovarian cancer** *BRCA* **genes mutations syndrome**

Ovarian cancer is the principal cause of death in women with gynecological cancer, due to the late onset of symptoms and the absence of a method for early detection. Nulliparity, early menarche, and late menopause are associated with an increased risk of occurrence; however, the strongest risk factor is the history of ovarian cancer in a first-degree relative [17].

Malignant primary ovarian tumors fall into three main groups: epithelial, sex cord / stromal, and germ cell tumors. Epithelial tumors being ovarian carcinomas (CBs), which are the most common group, represent up to 90% of ovarian cancers. Low-grade and high-grade serous carcinoma (LGSC and HGSC), mucinous carcinoma (MC), endometrioid carcinoma (EC), and clear cell carcinoma (CCC) are the five histological subtypes of OCs that are known. It is important to make a proper histological typing to determine the prognosis and response to different treatments, including cisplatin [18].

A large proportion of ovarian tumors are sporadic, and only a minority is due to an inherited cause. *BRCA1* and *BRCA2* mutations have been identified in approximately 15% of all epithelial ovarian cancers and up to 22.6% in HGSC. Somatic mutations in *BRCA1* and *BRCA2* have also been identified in as much as 7% of all ovarian cancers [19,20]. Although up to 50% of patients with HGSC harbor homologous recombination defects including the homologous recombination pathway independent of *BRCA 1/2*, this is known as *BRCA*-like behavior.

mutations located at sites of exon splicing that alter the splicing of the genes and producing

It is known that the mutation in the *BRCA1* gene presents a risk of ovarian cancer throughout life of up to 40%, while the *BRCA2* gene has a 20% risk. Although penetrance may vary in the same family carrying a mutation, suggesting that the risk can be influenced by allelic hetero‐

After the diagnosis of breast cancer in a patient with a *BRCA* mutation, there is a subsequent risk of developing ovarian cancer of up to 12.7% in women for *BRCA1* and 6.8% for *BRCA2* [15].

Diagnosis in elderly or the absence of family history does not exclude the presence of a germline mutation as approximately 35% of the *BRCA* mutation carriers have no family history. Genetic tests are expensive, so you should select the most appropriate individuals for

The presence of *BRCA* somatic and germline mutations are predictors of response to different chemotherapy treatments because they exhibit greater sensitivity and response to platinumtype drugs or PARP inhibitors, which are involved in the repair of DNA single-strand breaks

It is important that families carrying this mutation are informed about the risks of developing various types of cancer, including education about prenatal diagnosis and assisted reproduc‐ tion. Another option is IVF with previously selected embryos. Although, the decision should finally be made on an individual basis and will depend on the preference of each patient.

Ovarian cancer is the principal cause of death in women with gynecological cancer, due to the late onset of symptoms and the absence of a method for early detection. Nulliparity, early menarche, and late menopause are associated with an increased risk of occurrence; however,

Malignant primary ovarian tumors fall into three main groups: epithelial, sex cord / stromal, and germ cell tumors. Epithelial tumors being ovarian carcinomas (CBs), which are the most common group, represent up to 90% of ovarian cancers. Low-grade and high-grade serous carcinoma (LGSC and HGSC), mucinous carcinoma (MC), endometrioid carcinoma (EC), and clear cell carcinoma (CCC) are the five histological subtypes of OCs that are known. It is important to make a proper histological typing to determine the prognosis and response to

A large proportion of ovarian tumors are sporadic, and only a minority is due to an inherited cause. *BRCA1* and *BRCA2* mutations have been identified in approximately 15% of all epithelial ovarian cancers and up to 22.6% in HGSC. Somatic mutations in *BRCA1* and *BRCA2* have also been identified in as much as 7% of all ovarian cancers [19,20]. Although up to 50%

zygosity, modifier genes, and environmental and hormonal cofactors [13,14].

genetic testing, varying recommendations between populations and countries.

**3. Patients with ovarian cancer** *BRCA* **genes mutations syndrome**

the strongest risk factor is the history of ovarian cancer in a first-degree relative [17].

different treatments, including cisplatin [18].

full or partial loss of exons [12] can also be found to occur.

128 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

[16].

*BRCA*-like behavior is similar to when there is a loss of function or mutation of the *BRCA* genes with the same clinical and molecular characteristics. Examples include promoter methylation *BRCA1* (observed up to 35% of patients with epithelial ovarian cancer) and p53 mutation,

c-myc amplification or other proteins are needed for proper homologous recombination [21– 24]. The loss of function of suppressor gene *PTEN* has also been shown to produce *BRCA*-like behavior [25], more common in breast and prostate cancers [26,27].

The HGSC subtype has a greater sensitivity to PARP inhibitors without a *BRCA* mutation, probably due to changes in DNA repair that occur up to 50% of cases as we have mentioned previously [28,18,29].

Studies describe that *BRCA* mutation carriers diagnosed with ovarian cancer have higher survival rates compared with sporadic cases [30–32]. This could be due to increased sensitivity to cisplatin.

In HBOC syndrome, mutations in the *BRCA-1* and *BRCA-2* genes associated with the devel‐ opment of ovarian cancer [29] occur. Other inherited syndromes have also been associated with an increased occurrence of ovarian cancer such as Lynch syndrome (hereditary nonpo‐ lyposis colorectal cancer syndrome), characterized by mutations in the DNA repair genes *MLH1*, *MSH2*, *MSH6*, and *PMS2* genes [33].

#### **3.1. Prevention of ovarian cancer in women who have mutations in** *BRCA1* **and** *BRCA2* **genes**

Primary prevention strategies consist of primarily risk-reducing surgeries, the procedure of choice being prophylactic salpingo-oophorectomy bilateral from 35 to 40 years, or after childbearing. Some experts also recommend prophylactic hysterectomy to dry the small portion of remaining fallopian knotweed, although 92% of fallopian tube neoplasms originate in the middle or distal portion of the tube [34]. Although the final decision will be made by the patient.

Patients with *BRCA1* mutation are at increased risk of developing ovarian cancer from the age of 40; the recommendations as explained above are made from that age. This is not so with patients carrying the *BRCA2* mutation; the increased risk of ovarian cancer starts after age 50, so surgery can be postponed for a few years and secondary effects of surgery reduced [35].

After prophylactic surgery, one of the most important side effects which can deteriorate the quality of life of patients is premature menopause, with increased risk of osteoporosis and cardiovascular diseases such as hypertension, diabetes, and hypercholesterolemia. So that closer monitoring is recommended for cardiovascular risk [36].

Secondary prevention is early detection strategies in women carrying mutations in the *BRCA1* and *BRCA2* genes. Current recommendations include performing transvaginal ultrasound twice a year (preferably day 1–10 of the menstrual cycle), together with detection of serum CA-125 levels (after day 5 of the menstrual cycle for), from age 30 or five to ten years earlier than the earliest age of first diagnosis of ovarian cancer in the family. But these methods have limited sensitivity and specificity, with no observed benefit in women carrying mutation since no mortality was reduced. All women who refuse to perform prophylactic surgeries should undergo screening every six months [37].

Despite the greatly reduced risk of developing ovarian cancer after prophylactic surgery, patients should know that a minority (from 3.9% to 4.3%) of them will develop primary peritoneal carcinoma 20 years after the last oophorectomy in patients with *BRCA*-1 mutations. So before you perform these procedures, patients should be informed of the risks and mor‐ bidities associated with these interventions [38,39].

#### **4. Inhibitors of poly (ADP -ribose) polymerase**

The preservation of the genetic code by DNA repair is essential for proper cell function. Currently, there is a better understanding of the DNA repair pathways, so it has been studied more carefully for potential drug targets [40].

There are at least five ways engaged in DNA repair, two of them involved in the repair of double-strand breaks (error-prone, non–homologous, end–joining, predominantly active in G1 cells, and error-free HRR, which predominates in dividing cells) [41].

The major DNA repair pathways are direct repair, mismatch repair (MMR), the base excision repair (BER), nucleotide excision repair (NER), and double-strand break repair recombination, which includes both non-homologous, end-joining and homologous recombination repair (HRR) [41,42].

There are certain external agents such as ionizing radiation producing damaging DNA strand breaks. Normal cells have the ability to repair this damage by a protection mechanism maintaining its normal function, but the tumor cells' ability to repair DNA is a radio-resistance mechanism. In recent years, studies have identified a number of agents in these pathways such as PARP inhibitors [43].

Poly (ADP-ribose) polymerase (PARP) inhibitors are a new class of targeted agents against ovarian cancer [44–46]. PARP is a nuclear enzyme whose function is to repair single-stranded DNA.

There are three generations of PARP inhibitor. The first generation of inhibitors included nicotinamide analogs. 3-Aminobenzamide was the first PARP inhibitor but was not considered powerful enough compared to the second generation [47]. Currently, clinical trials are aimed at third-generation inhibitors with greater potency and specificity, decreasing side effects, this includes olaparib.

DNA repair is essential for proper cell function. Each cell sustains many thousands of episodes of DNA damage every day, which will be repaired by a wide variety of repair mechanisms [48,49].

The *BRCA 1/2* genes are responsible for DNA repair known as homologous recombination (HR) repair. HR is a form of double-strand break repair that occurs in the G2 phase of the cell cycle where the second double-stranded copy of the DNA is used as a template to form an error-free repair [49].

than the earliest age of first diagnosis of ovarian cancer in the family. But these methods have limited sensitivity and specificity, with no observed benefit in women carrying mutation since no mortality was reduced. All women who refuse to perform prophylactic surgeries should

Despite the greatly reduced risk of developing ovarian cancer after prophylactic surgery, patients should know that a minority (from 3.9% to 4.3%) of them will develop primary peritoneal carcinoma 20 years after the last oophorectomy in patients with *BRCA*-1 mutations. So before you perform these procedures, patients should be informed of the risks and mor‐

The preservation of the genetic code by DNA repair is essential for proper cell function. Currently, there is a better understanding of the DNA repair pathways, so it has been studied

There are at least five ways engaged in DNA repair, two of them involved in the repair of double-strand breaks (error-prone, non–homologous, end–joining, predominantly active in

The major DNA repair pathways are direct repair, mismatch repair (MMR), the base excision repair (BER), nucleotide excision repair (NER), and double-strand break repair recombination, which includes both non-homologous, end-joining and homologous recombination repair

There are certain external agents such as ionizing radiation producing damaging DNA strand breaks. Normal cells have the ability to repair this damage by a protection mechanism maintaining its normal function, but the tumor cells' ability to repair DNA is a radio-resistance mechanism. In recent years, studies have identified a number of agents in these pathways such

Poly (ADP-ribose) polymerase (PARP) inhibitors are a new class of targeted agents against ovarian cancer [44–46]. PARP is a nuclear enzyme whose function is to repair single-stranded

There are three generations of PARP inhibitor. The first generation of inhibitors included nicotinamide analogs. 3-Aminobenzamide was the first PARP inhibitor but was not considered powerful enough compared to the second generation [47]. Currently, clinical trials are aimed at third-generation inhibitors with greater potency and specificity, decreasing side effects, this

DNA repair is essential for proper cell function. Each cell sustains many thousands of episodes of DNA damage every day, which will be repaired by a wide variety of repair mechanisms

G1 cells, and error-free HRR, which predominates in dividing cells) [41].

undergo screening every six months [37].

bidities associated with these interventions [38,39].

130 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

more carefully for potential drug targets [40].

(HRR) [41,42].

DNA.

[48,49].

as PARP inhibitors [43].

includes olaparib.

**4. Inhibitors of poly (ADP -ribose) polymerase**

Other DNA repair pathway, such as the non-homologous, end-joining (NHEJ) pathway, also plays a role in the anti-cancer mechanism of action of PARP inhibitors [50].

PARP inhibitors act by trapping PARP-1 and PARP-2 on the double-strand break and blocks DNA replication, which is more toxic to cells than the accumulation of DNA breaks [51]. Overall, in tumors in where there is an apparent defect in homologous DNA repair (and thus a defect in the repair of double-stranded breaks), they seem to be susceptible to PARP-inhibitor therapy. These include tumors associated with germline or somatic mutations in *BRCA 1/2* [52].

There are at least 17 PARP counterparts, with only three PARP-1, PARP-2, and PARP-3s that play a critical role in DNA repair [53,54]. The best known are PARP-1 and PARP-2 [55, 56], and the most studied PARP-1.

PARP-1 was the first to be reported in 1963 [57]. Durkacz [58] stated that modulating PARP1 could enhance the effect of chemotherapy.

PARP-1 contains three functional domains: the N-terminal DNA-binding domain (DBD), the center self-modification domain (DMA), and the C-terminal catalytic domain (CD). The DBD is involved in recognition of DNA-strand breakage and in the binding of PARP-1 to DNA. AMD can interact with many DNA damage response proteins and the CD includes a PARP signature motif and catalyzes the formation of PAR [59]. PARP-1 is essential for base excision repair (BER).

PARP-1 also contributes to other cellular processes such as gene transcription, and the regulation of the chromatin structure, to restart stalled replication forks due to nucleotide depletion or collisions with bulky lesions [52].

PARP-1 has been used in in vitro studies in combination with chemotherapy, to demonstrate its ability to inhibit the classical mechanisms of DNA repair, showing also increased distribu‐ tion of cytotoxicity to the tumor, increasing their exposure by improving vascular perfusion. This resulted in further studies with PARP-1.

The DNA repair biology has allowed us to identify patients most likely to respond to treatment with PARP inhibitors [60].

PARP inhibitors act by synthetic lethality, which occurs when two independent conditions alone do not cause cell death but in combination are lethal. It occurs when a patient has an alteration in the homologous recombination (HR) such as in carriers of a mutation in *BRCA1* and *BRCA2* genes and the application of PARP inhibitors, causing cell death [61,62]. Up to 5% of cutaneous melanomas and gastric cancers, 1% of prostate cancers, and even 19% of familial pancreatic cancers will carry a germline mutation in *BRCA 1/2*, thus they have an altered HR and therefore they may also respond to PARP inhibitors [63].

PARP-2 cooperates with PARP-1 to synthesize poly (ADP-ribose) [pADPr] after damage in the DNA chain [41]. PARP-3 suppresses error-prone NHEJ [52, 64,65] while associated with PARP-1 for DNA repair. The clinical development of PARP-inhibitors has lead to its use as monotherapy or in combination with chemotherapy agents. Olaparib has been recently approved for the treatment of hereditary breast ovarian cancer syndrome, and other PARPinhibitors such as veliparib, rucaparib, or niraparib are being studied [66].

#### **4.1. Combination therapy of PARP inhibitors and radiotherapy**

The efficacy of radiotherapy in the treatment of cancer have been known for several years either concomitantly with chemotherapy or as adjuvant use in therapy.

New clinical trials not only focus on researching new systemic treatments alone or in combi‐ nation with other chemotherapy agents but also study their association with radiotherapy. These new therapies are the PARP inhibitors that have shown activity in conjunction with radiation therapy in several cancer cell lines. Data suggest that PARP inhibitors may enhance the effects of radiation in various types of tumors, such as lung cancer, colorectal, and cervical among others [67]. However, the mechanism of action is still unknown, one hypothesis is that it is due to mutual damage (of PARP-inhibitors and radiotherapy) of DNA or whether tumor re-oxygenation contributes to this radio sensitization via the vasoactive effects of the PARP inhibitors remains to be fully determined [43].

A recently published Phase I clinical trial [68] combined low-dose abdominal level fractionated radiotherapy with increasing doses of the PARP-inhibitor veliparib in patients with peritoneal carcinomatosis secondary to advanced malignant solid tumors. Patients were treated with veliparib (80–320 mg daily) for a total of 3 cycles.

The dose of radiotherapy consisted of 21.6 Gy in 36 fractions, 0.6 Gy twice daily on days 1 and 5 for weeks 1–3 of each cycle. Twenty-two patients were included. Disease stabilization (≥24 weeks) was observed in 7 patients (33%). Median progression-free survival (MPFS) was 4.47 months and median overall survival (MOS) was 13.04 months. In the trial, there were 8 patients with ovarian and fallopian cancers with an observed MPFS of 6.77 months and an MOS of 17.54 months, combined with a higher quality of life. The toxicity grade 3 and 4 lymphopenia were more frequent (68%), anemia (9%), and thrombocytopenia (14%). With these results, the authors concluded that the combination of radiotherapy and veliparib resulted in a stabiliza‐ tion of the response in patients with solid tumors and peritoneal carcinomatosis, especially in the subgroup of patients with ovarian cancer, besides being a well-tolerated regimen.

#### **5. OLAPARIB**

Because many cytotoxic agents work by damaging the DNA, there has been a great deal of interest in the use of inhibitors of DNA repair such as new treatments against cancer, especially in patients with mutations in the *BRCA* genes with altered function, and will be more likely to develop different types of neoplasms due to increased tumorigenesis [69].

Olaparib is a member of the class of N-acylpiperazines formally obtained by condensation of the carboxyl group of 2-fluoro-5 - [(4-oxo-3,4-dihydrophthalazin-1-yl) methyl] benzoic acid with the free amino group of N- (cyclpropylcarbonyl) piperazine.

#### **5.1. Initial clinical trials with OLAPARIB**

PARP-1 for DNA repair. The clinical development of PARP-inhibitors has lead to its use as monotherapy or in combination with chemotherapy agents. Olaparib has been recently approved for the treatment of hereditary breast ovarian cancer syndrome, and other PARP-

The efficacy of radiotherapy in the treatment of cancer have been known for several years

New clinical trials not only focus on researching new systemic treatments alone or in combi‐ nation with other chemotherapy agents but also study their association with radiotherapy. These new therapies are the PARP inhibitors that have shown activity in conjunction with radiation therapy in several cancer cell lines. Data suggest that PARP inhibitors may enhance the effects of radiation in various types of tumors, such as lung cancer, colorectal, and cervical among others [67]. However, the mechanism of action is still unknown, one hypothesis is that it is due to mutual damage (of PARP-inhibitors and radiotherapy) of DNA or whether tumor re-oxygenation contributes to this radio sensitization via the vasoactive effects of the PARP

A recently published Phase I clinical trial [68] combined low-dose abdominal level fractionated radiotherapy with increasing doses of the PARP-inhibitor veliparib in patients with peritoneal carcinomatosis secondary to advanced malignant solid tumors. Patients were treated with

The dose of radiotherapy consisted of 21.6 Gy in 36 fractions, 0.6 Gy twice daily on days 1 and 5 for weeks 1–3 of each cycle. Twenty-two patients were included. Disease stabilization (≥24 weeks) was observed in 7 patients (33%). Median progression-free survival (MPFS) was 4.47 months and median overall survival (MOS) was 13.04 months. In the trial, there were 8 patients with ovarian and fallopian cancers with an observed MPFS of 6.77 months and an MOS of 17.54 months, combined with a higher quality of life. The toxicity grade 3 and 4 lymphopenia were more frequent (68%), anemia (9%), and thrombocytopenia (14%). With these results, the authors concluded that the combination of radiotherapy and veliparib resulted in a stabiliza‐ tion of the response in patients with solid tumors and peritoneal carcinomatosis, especially in

the subgroup of patients with ovarian cancer, besides being a well-tolerated regimen.

develop different types of neoplasms due to increased tumorigenesis [69].

with the free amino group of N- (cyclpropylcarbonyl) piperazine.

Because many cytotoxic agents work by damaging the DNA, there has been a great deal of interest in the use of inhibitors of DNA repair such as new treatments against cancer, especially in patients with mutations in the *BRCA* genes with altered function, and will be more likely to

Olaparib is a member of the class of N-acylpiperazines formally obtained by condensation of the carboxyl group of 2-fluoro-5 - [(4-oxo-3,4-dihydrophthalazin-1-yl) methyl] benzoic acid

inhibitors such as veliparib, rucaparib, or niraparib are being studied [66].

either concomitantly with chemotherapy or as adjuvant use in therapy.

**4.1. Combination therapy of PARP inhibitors and radiotherapy**

132 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

inhibitors remains to be fully determined [43].

veliparib (80–320 mg daily) for a total of 3 cycles.

**5. OLAPARIB**

In 2008, Rottenberg et al. [70] postulated the hypothesis of the use of olaparib (then called AZD2281, KU-0059436) in cancer triple negative breast, because these tumors harbor defects in DNA repair and mutations in *BRCA1*. To do this, they used PARP inhibitors (AZD2281) in genetically engineered mouse models of breast cancer *BRCA1*, resulting in an inhibition of tumor growth and increased overall survival with no signs of toxicity. Drug resistance developed after long-term treatment due to upregulation of efflux pumps; however, this was overcome by co-administration of the P-glycoprotein inhibitor tariquidar. They observed that the combination of AZD2281 with cisplatin or carboplatin increased progression-free survival, suggesting the effectiveness of AZD2281 as DNA-damaging agents.

Evers et al. [71] studied sensitivity to conventional cytotoxic drugs AZD2281 in cell lines with *BRCA2* mutations. AZD2281 was observed to be the drug that caused greater tumor reduction in the presence of *BRCA2* mutations, alone or in combination with cisplatin.

Fong et al. [60] conducted a Phase I clinical trial with escalating doses of mg to 600mg olaparib, in a population of 60 patients, including 22 mutation carriers in the *BRCA1* and *BRCA2* gene. Dose-limiting toxicity was observed in one of eight patients receiving 400 mg twice daily (grade 3 fatigue and mood alteration) and two of five patients who received 600 mg twice daily (grade 4 thrombocytopenia and drowsiness grade 3). In 63% of patients with cancer and carriers of *BRCA* mutations, a clinical benefit for a period of 4 months or more was observed and 8 patients had ovarian cancer.

In this study, patients resistance to platinum response was observed.

A year later, the same team of Fong et al. confirmed previous results by expanding a cohort of patients with mutations in *BRCA1* and *BRCA2*, including primary peritoneal cancer with ovary, and fallopian tube cancers (13 platinum-sensitive, 24 platinum-resistant, and 13 platinum-refractory) observing a clinical benefit response of up to 46%. The overall clinical benefit rate decreased due to insensitivity to platinum (platinum-sensitive patients: 69%, platinum-resistant: 46% refractory to platinum: 23%). The median response duration was 28 weeks, concluding that patients who were platinum-sensitive present a greater response to olaparib, in addition to showing a benefit in resistant and refractory patients [72].

Seventy five percent of *BRCA1*-mutated breast cancers are classified as triple-negative breast and *BRCA1* or *BRCA2* mutation carriers and have the tendency to develop ovarian cancer. In a Phase II clinical trial [63], the investigators administered 400 mg of olaparib twice daily in patients with ovarian cancer HGSC and triple-negative breast cancer. Patients were stratified according to whether they were carriers of *BRCA1*, *BRCA2*, and *BRCA* wild-type gene. The primary endpoint was objective response rate by Response Evaluation Criteria In Solid Tumors (RECIST). It was observed that 41% of patients with *BRCA* mutation carrier ovarian cancer and 24% of patients with wild-type *BRCA* showed no response to olaparib RECIST criteria. No confirmed objective responses were reported in patients with breast cancer and they concluded that olaparib is an efficient drug for treatment of *BRCA* mutant ovarian cancer.

Kaye et al. published the results of a Phase II trial in 2012. The study included 97 patients with ovarian cancer and *BRCA1* or *BRCA2* germline mutations that had recurred within 12 months of prior platinum therapy. They randomized in a 1:1:1 ratio to receive olaparib 200 mg or 400 mg bd or pegylated liposomal doxorubicin (PLD) 50 mg / m2 intravenously. The median PFS was 6.5 months, 8.8 months, and 7.1 months for the olaparib 200 mg, 400 mg olaparib, and PLD groups, respectively. Objective response rates were 25, 31, and 18% for olaparib 200 mg, 400 mg olaparib, and PLD, respectively. Proving that olaparib 400 mg twice daily is an appropriate dose. A surprising finding was the effectiveness of PLD [74]. These results affirm the data published by Adams et al. [75] which show increased PLD activity in patients carrying a *BRCA* mutation.

Lederman et al. [76] reported on a Phase II trial; they administered olaparib as a maintenance therapy in patients with recurrent ovarian cancer orfallopian tube or primary peritoneal cancer of high-grade, which wasplatinum-sensitive.Patients were randomizedto receive olaparib 400 mg twice daily or placebo within 8 weeks after the last dose of platinum-based chemothera‐ py. The primary endpoint was progression-free survival (PFS). A first analysis performed after progression in 57.7% of patients showed that PFS was significantly longerin the olaparib group than in the placebo group. Median PFS was 8.4 months in the olaparib group versus 4.8 months in the placebo group (*P* < 0.001). Subgroup analysis of progression-free survival showed that, in the olaparib group, patients had a lower risk of progression than those in the placebo group. Having had a complete response to the treatment of platinum-based chemotherapy before entering the study was a significant prognostic factor for longer progression-free survival (hazardratio, 0.46;*P*< 0.001).Time toprogressionaccordingtotheRECISTguidelinesorCA-125 level was significantly longer in the olaparib group than in the placebo group (median, 8.3 months vs. 3.7 months (*P* < 0.001). The response rate was 12% (7 of 57 with measurable disease patients at study entry) in the olaparib group, as compared with 4% (*P* = 0.12).

In the interim analysis of overall survival (OS), 101 patients (38%) had died: 52 in the olaparib group and 49 in the placebo group. No significant difference in overall survival was observed (*P* = 0.75). The median overall survival was similar in the two study groups (29.7 months in the olaparib group and 29.9 months in the placebo group). Although *BRCA* mutation status was known for 37% of all patients who entered the study, a subgroup analysis suggested that olaparib could increase PFS in patients with a *BRCA* mutation.

The incidence of adverse events grade 3 or 4 was higher in the olaparib group (35.3%) compared to the placebo group (20.3%). The most common adverse events leading to discontinuation or dose reduction of olaparib were vomiting, nausea, and fatigue. There were no statistically significant differences in quality of life test performed patients.

In another study, Lederman et al. [77] presented test data 19, a second retrospective analysis of OS, and *BRCA* mutation status of the Phase II trial published by them in 2012. The primary endpoint was PFS, analyzed for the overall population and by *BRCA* status. *BRCA* mutation status was known in 96% of patients in the group of olaparib compared to 95% in the placebo group, of whom 56% versus 50% had a deleterious or suspected tumor or deleterious *BRCA* germline mutation. The median PFS was significantly longer in the olaparib group compared to the placebo group, 11.2 months vs. 4.3 months; in the wild-type *BRCA* patients the findings were similar, 7.4 months vs. 5.5 months. In a second interim analysis (58% maturity), the OS was similar in patients with mutated *BRCA* and wild-type *BRCA*, this may be secondary to the fact that 23% of patients receiving placebo could subsequently receive an inhibitor of PARP. In the results, they observed that some patients responded to olaparib in the absence of a *BRCA* mutation, this could be due to epigenetic silencing of *BRCA* or homologous recombination genes such as RAD51D.

of prior platinum therapy. They randomized in a 1:1:1 ratio to receive olaparib 200 mg or 400 mg bd or pegylated liposomal doxorubicin (PLD) 50 mg / m2 intravenously. The median PFS was 6.5 months, 8.8 months, and 7.1 months for the olaparib 200 mg, 400 mg olaparib, and PLD groups, respectively. Objective response rates were 25, 31, and 18% for olaparib 200 mg, 400 mg olaparib, and PLD, respectively. Proving that olaparib 400 mg twice daily is an appropriate dose. A surprising finding was the effectiveness of PLD [74]. These results affirm the data published by Adams et al. [75] which show increased PLD activity in patients carrying a *BRCA*

134 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

Lederman et al. [76] reported on a Phase II trial; they administered olaparib as a maintenance therapy in patients with recurrent ovarian cancer orfallopian tube or primary peritoneal cancer of high-grade, which wasplatinum-sensitive.Patients were randomizedto receive olaparib 400 mg twice daily or placebo within 8 weeks after the last dose of platinum-based chemothera‐ py. The primary endpoint was progression-free survival (PFS). A first analysis performed after progression in 57.7% of patients showed that PFS was significantly longerin the olaparib group than in the placebo group. Median PFS was 8.4 months in the olaparib group versus 4.8 months in the placebo group (*P* < 0.001). Subgroup analysis of progression-free survival showed that, in the olaparib group, patients had a lower risk of progression than those in the placebo group. Having had a complete response to the treatment of platinum-based chemotherapy before entering the study was a significant prognostic factor for longer progression-free survival (hazardratio, 0.46;*P*< 0.001).Time toprogressionaccordingtotheRECISTguidelinesorCA-125 level was significantly longer in the olaparib group than in the placebo group (median, 8.3 months vs. 3.7 months (*P* < 0.001). The response rate was 12% (7 of 57 with measurable disease

patients at study entry) in the olaparib group, as compared with 4% (*P* = 0.12).

olaparib could increase PFS in patients with a *BRCA* mutation.

significant differences in quality of life test performed patients.

In the interim analysis of overall survival (OS), 101 patients (38%) had died: 52 in the olaparib group and 49 in the placebo group. No significant difference in overall survival was observed (*P* = 0.75). The median overall survival was similar in the two study groups (29.7 months in the olaparib group and 29.9 months in the placebo group). Although *BRCA* mutation status was known for 37% of all patients who entered the study, a subgroup analysis suggested that

The incidence of adverse events grade 3 or 4 was higher in the olaparib group (35.3%) compared to the placebo group (20.3%). The most common adverse events leading to discontinuation or dose reduction of olaparib were vomiting, nausea, and fatigue. There were no statistically

In another study, Lederman et al. [77] presented test data 19, a second retrospective analysis of OS, and *BRCA* mutation status of the Phase II trial published by them in 2012. The primary endpoint was PFS, analyzed for the overall population and by *BRCA* status. *BRCA* mutation status was known in 96% of patients in the group of olaparib compared to 95% in the placebo group, of whom 56% versus 50% had a deleterious or suspected tumor or deleterious *BRCA* germline mutation. The median PFS was significantly longer in the olaparib group compared to the placebo group, 11.2 months vs. 4.3 months; in the wild-type *BRCA* patients the findings were similar, 7.4 months vs. 5.5 months. In a second interim analysis (58% maturity), the OS was similar in patients with mutated *BRCA* and wild-type *BRCA*, this may be secondary to the

mutation.

There were more treatment interruptions and dose reductions in the olaparib group compared to placebo. Adverse effects grade 3 or 4 in the olaparib group were fatigue in 7% vs. 3% in the placebo group, and anemia 5% vs. <1%, respectively. Serious adverse effects were reported in 18% of patients receiving olaparib compared to 9% on placebo. Tolerability was similar regardless of the mutational status.

Although the number of patients treated with somatic *BRCA* mutations was lower, responses were also seen during maintenance therapy.

The test of olaparib superiority over the placebo in platinum-sensitive patients demonstrated that patients relapsed after treatment. In addition, it was shown that *BRCA* mutations, whether somatic or germ line, are the main determinants of response to olaparib.

After the results of test data19 were published, olaparib was approved by EMA as a mainte‐ nance therapy after response to platinum-based chemotherapy in relapsed platinum-sensitive ovarian cancer, fallopian tube, and primary peritoneal cancers patients with a *BRCA* mutation. In contrast, the FDA-approved olaparib in patients with high-grade serous ovarian epithelial tumors, primary fallopian tube and peritoneal cancers with a *BRCA* mutation who had progressed during three or more lines of chemotherapy.

Before starting treatment with olaparib, they confirmed the presence of a *BRCA* germline or somatic mutation, using a valid method of analysis in a specialized laboratory.

It also assessed the efficacy and tolerability of olaparib in combination with chemotherapy followed by maintenance olaparib versus chemotherapy alone in patients with high-grade serous ovarian cancer, including primary peritoneal and fallopian tubes, platinum-sensitive who had received three or more lines of platinum-based chemotherapy. AM Oza et al. [78] published data based on chemotherapy combination of carboplatin (area under the curve [AUC] 4 mg / mL per min) plus paclitaxel (175 mg / m2) every 21 days with olaparib 200 mg twice daily (during days 1–-10 of each cycle of 21 days), 6 total cycles followed olaparib maintenance monotherapy (400 mg twice daily) until disease progression or unacceptable toxicity, compared to chemotherapy alone (carboplatin AUC 6 mg / mL paclitaxel 175 mg / m2) without maintenance. The primary endpoint was PFS, the secondary efficacy endpoints were overall survival; percentage change in tumor size; the proportion of patients with an objective response according to RECIST; cancer antigen 125 (CA-125) response.

The results concluded that the PFS was higher in the olaparib plus chemotherapy group (median 12.2 months) compared with chemotherapy alone (median of 9.6 months). *BRCA* mutation status was known in 107 patients. *BRCA* mutations were observed in 41 (38%) of 107 patients (20 in the chemotherapy group and 21 in more olaparib chemotherapy alone); of the 41 patients with *BRCA* mutation, the PFS at 12 months was 70% for patients in the combination arm vs. 12.5% in the chemotherapy alone arm. There were no statistically significant differences in OS or percentage change in tumor size. The proportion of patients with an objective response by the central review was similar between treatment groups. The two treatment groups had a similar proportion of patients with a CA-125 response and ovarian cancer response. The most frequent grade 3 or 4 adverse effects in the combination arm were neutropenia (43%) vs. (35%) in patients with chemotherapy alone and anemia ([9%] vs. [5%]). The most frequent adverse effects of mild or moderate intensity were alopecia, nausea, diarrhea, headache, and peripheral neuropathy among others, they were all more common in the combination group.

Not only olaparib has been studied in combination with chemotherapy, but their association was also analyzed with cediranib, an anti-angiogenic agent with activity against the VEGF receptor (VEGFR) 1, VEGFR2, and VEGFR3. In Phase I clinical trials, combination responses and manageable toxicities were observed, so that a Phase II clinical trial was developed aimed at demonstrating whether their combination would result in increased PFS versus olaparib monotherapy in women with recurrent platinum-sensitive ovarian cancer. The trial was conducted by Liu [79].

They recruited women with ovarian (HGSC and EC), fallopian tube or primary peritoneal cancer, or patients with a *BRCA* germ line mutation. Patients were randomized to receive olaoparib in 400 mg capsules twice daily or the combination of cediranib 30 mg daily and olaparib 200 mg capsules twice daily. The primary endpoint was PFS. 46 women received 44 olaparib monotherapy and combination therapy women. Median PFS was 17.7 months for the women treated with cediranib plus olaparib compared with 9 months in patients receiving olaparib monotherapy. Grade 3 and 4 adverse events were more common with the combina‐ tion therapy, including fatigue, diarrhea, and hypertension. Thus, the authors concluded that PFS increases considerably with the combination therapy, so Phase III clinical trials are necessary to confirm these results, including assessments of quality of life.

Because of the mechanism of action of olaparib, the use of PARP inhibitor in other tumors with mutations in the *BRCA 1/2* gene could be effective, a Phase II clinical trial with different tumors was performed, all patients had *BRCA 1/2* mutations and recurrent cancer. The study was conducted by Kaufman et al. [52]. It was a prospective, multi-center, randomized trial, and patients were recruited in several centers in Israel, Australia, Germany, Spain, Sweden, and the United States between February 21, 2010, and July 31, 2012. It included patients with ovarian cancer resistant to prior platinum; with three breast cancer chemotherapy regimens for metastatic disease; pancreatic cancer progression during treatment with gemcitabine; with prostate cancer progression or on hormonal and one systemic therapy. Olaparib was admin‐ istered 400 mg twice per day. The primary efficacy end point was tumor response rate according to RECIST. Secondary end points included: objective response rate (in those with measurable disease at baseline), PFS, duration of response, safety, and tolerability.

And 298 patients were evaluated, of whom 193 had ovarian cancer, 62 had breast cancer, 23 had advanced pancreatic cancer, and 8 patients with advanced prostate cancer. The remaining 12 patients had a range of cancers, including cancers of the biliary tract, bladder, colorectum, lung, esophagus, and uterus.

The tumor response rate was 26.2% (78 of 298; 95% CI, 21.3 to 31.6) overall and 31.1% (60 of 193; 95% CI, 24.6 to 38.1) in patients with ovarian cancer, 12.9% (eight of 62; 95% CI, 5.7 to 23.9) in breast, 21.7% (five of 23; 95% CI, 7.5 to 43.7) in pancreatic, and 50.0% (four of eight; 95% CI, 15.7 to 84.3) in prostate cancers. In 42% of all patients, stable disease was observed after 8 weeks of treatment, up to 46.8% having achieved stabilization in cases of breast cancer. Overall median duration of the response was 208 days (ovarian cancer, 225 days; breast cancer, 204 days; pancreatic cancer, 134 days; prostate cancer, 327 days). Median time to onset of response was 56.0 days (ovarian cancer, 56.5 days; breast cancer, 54.5 days; pancreatic cancer, 113.0 days; prostate cancer, 54.5 days). The objective response rate (restricted to those with measurable disease at baseline) was 29.3% (95% CI, 23.9 to 35.2).

A similar response rate for patients with a *BRCA1* mutation (26.3 % ; 95 % CI, 20.3 to 33.0) and those with a *BRCA2* mutation was also noted.

The most common side effects were fatigue, nausea, and vomiting. About 54% of patients experienced grade 3 toxicity, most frequently fatigue and anemia, 40.3 % of patients had to modify (interruption and / or reduction) olparib dose due to adverse effects. Nine patients died as a result of severe adverse effects.

After these results, they concluded that the response to olaparib is independent of the anatomical organ of origin, provided that there is a mutation in *BRCA 1/2*.

Despite the demonstrated efficacy of PARP inhibitors in patients with *BRCA 1/2* mutation, there has been less activity in patients with breast cancer, perhaps due to the biologic hetero‐ geneity and low *BRCA 1/2* mutation rate in somatic triple negative breast cancer [80–83].

#### **5.2. Phase III clinical trials**

in OS or percentage change in tumor size. The proportion of patients with an objective response by the central review was similar between treatment groups. The two treatment groups had a similar proportion of patients with a CA-125 response and ovarian cancer response. The most frequent grade 3 or 4 adverse effects in the combination arm were neutropenia (43%) vs. (35%) in patients with chemotherapy alone and anemia ([9%] vs. [5%]). The most frequent adverse effects of mild or moderate intensity were alopecia, nausea, diarrhea, headache, and peripheral

Not only olaparib has been studied in combination with chemotherapy, but their association was also analyzed with cediranib, an anti-angiogenic agent with activity against the VEGF receptor (VEGFR) 1, VEGFR2, and VEGFR3. In Phase I clinical trials, combination responses and manageable toxicities were observed, so that a Phase II clinical trial was developed aimed at demonstrating whether their combination would result in increased PFS versus olaparib monotherapy in women with recurrent platinum-sensitive ovarian cancer. The trial was

They recruited women with ovarian (HGSC and EC), fallopian tube or primary peritoneal cancer, or patients with a *BRCA* germ line mutation. Patients were randomized to receive olaoparib in 400 mg capsules twice daily or the combination of cediranib 30 mg daily and olaparib 200 mg capsules twice daily. The primary endpoint was PFS. 46 women received 44 olaparib monotherapy and combination therapy women. Median PFS was 17.7 months for the women treated with cediranib plus olaparib compared with 9 months in patients receiving olaparib monotherapy. Grade 3 and 4 adverse events were more common with the combina‐ tion therapy, including fatigue, diarrhea, and hypertension. Thus, the authors concluded that PFS increases considerably with the combination therapy, so Phase III clinical trials are

Because of the mechanism of action of olaparib, the use of PARP inhibitor in other tumors with mutations in the *BRCA 1/2* gene could be effective, a Phase II clinical trial with different tumors was performed, all patients had *BRCA 1/2* mutations and recurrent cancer. The study was conducted by Kaufman et al. [52]. It was a prospective, multi-center, randomized trial, and patients were recruited in several centers in Israel, Australia, Germany, Spain, Sweden, and the United States between February 21, 2010, and July 31, 2012. It included patients with ovarian cancer resistant to prior platinum; with three breast cancer chemotherapy regimens for metastatic disease; pancreatic cancer progression during treatment with gemcitabine; with prostate cancer progression or on hormonal and one systemic therapy. Olaparib was admin‐ istered 400 mg twice per day. The primary efficacy end point was tumor response rate according to RECIST. Secondary end points included: objective response rate (in those with

necessary to confirm these results, including assessments of quality of life.

measurable disease at baseline), PFS, duration of response, safety, and tolerability.

And 298 patients were evaluated, of whom 193 had ovarian cancer, 62 had breast cancer, 23 had advanced pancreatic cancer, and 8 patients with advanced prostate cancer. The remaining 12 patients had a range of cancers, including cancers of the biliary tract, bladder, colorectum,

The tumor response rate was 26.2% (78 of 298; 95% CI, 21.3 to 31.6) overall and 31.1% (60 of 193; 95% CI, 24.6 to 38.1) in patients with ovarian cancer, 12.9% (eight of 62; 95% CI, 5.7 to 23.9)

neuropathy among others, they were all more common in the combination group.

136 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

conducted by Liu [79].

lung, esophagus, and uterus.

Currently, there are two Phase III trials underway with olaparib, both sponsored by Astra Zeneca: SOLO 1 (NCT01844986) and SOLO2 (NCT01874353). Both are multi-center, doubleblind, in which randomly assigned patients (2: 1) receive 300 mg of olaparib daily maintenance in patients diagnosed with high-grade serous or endometrioid ovarian cancer, primary peritoneal including and / or fallopian tube cancer with a *BRCA* mutation, and having partial or complete response after completion of platinum-based chemotherapy. To be included in SOLO1 trial, patients must have been newly diagnosed, with advanced (FIGO stage III-IV) disease, and responding to first-line treatment with platinum. To be included in SOLO2, patients must have completed ≥2 lines of platinum therapy. The main objective for both clinical trials is progression-free survival [84].

Not only they are testing olaparib, but there are also other Phase III trials with PARP inhibitors, such as veliparib, rucaparib, and niraparib, trying to improve the identification of patients who might best respond to PARP inhibitors [80] and reducing associated toxicities.

#### **5.3. Combination therapy of OLAPARIB with other therapeutic agents**

Olaparib has been tested in several clinical trials in combination with other chemotherapy because it was thought that it could increase sensitivity to chemotherapy by inhibiting DNA repair which could be responsible for drug resistance. This has been associated with topotecan [85], dacarbazine [86], paclitaxel [87], and cisplatin and gemcitabine [88]. In several Phase I clinical trials, myelosuppression increased in the combination therapy versus monotherapy, especially with topotecan and cisplatin.

Olaparib has also been studied in combination with bevacizumab. Dean et al. [89] designed a Phase I clinical trial, as they hypothesized that bevacizumab antiangiogenic activity and hypoxia inducing DNA damage may enhance olaparib therapeutic activities. They showed that a dose of olaparib 400 mg twice daily in combination with Avastin 10mg / kg every two weeks was tolerated well. They are considering the combination for future clinical trials.

#### **5.4. Dosage**

The recommended lynparza® (olaparib) dose is 400 mg (eight capsules) twice a day, i.e., a total daily dosage of 800 mg. Treatment should begin before eight weeks of completion of the last cycle of chemotherapy with a regimen containing platinum. Continual treatment is recom‐ mended until disease progression. The recommended dose is reduced to 200 mg orally twice daily (total daily dose 400 mg). Elderly patients require a dose reduction initially, and it can be administered in patients with mild renal impairment (creatinine clearance> 50 mL / min).

#### **5.5. Adverse reactions**

Among the most frequent toxicities in clinical trials are hematologic toxicity with mild to moderate anemia, lymphopenia, neutropenia, and thrombocytopenia at manageable levels. Other frequently observed side effects are headache, fatigue, decreased appetite, abdominal discomfort, nausea, vomiting, diarrhea, and dyspepsia.

Another event that was observed was the development of myelodysplastic / AML syndrome, in only a small number of patients receiving olaparib alone or in combination with other antineoplastic during clinical trials. All had previously received platinum-based chemother‐ apy regimens, radiation, and other DNA-damaging agents.

There have been cases of pneumonitis, some of them being fatal. If patients are treated with Lynparza, respiratory symptoms such as cough, dyspnea, and fever should be closely monitored. If there is any alteration in the chest radiography, treatment must be stopped and the patient is treated appropriately. Paralyzer can cause birth defects if given to pregnant women. A reliable contraception should be recommended during treatment and one month after the last dose [90,63].

#### **6. Mechanisms of resistance to PARP inhibitors**

Targeted therapy based on the patient's mutation status is the future of the treatment of ovarian cancer. *BRCA* deficiency may be reversed by mutational changes in the reading frame, resulting in wild-type *BRCA* protein production. A second mutation (compensatory mutations or crossovers) can cause changes in the reading frame *BRCA* mutation, HR rebuilding, and restoring its functionality, explaining why not all tumors with *BRCA* mutation respond to PARP inhibitors [91]. Some *BRCA1* mutant alleles encode functional proteins but are degraded, stabilizing the activity of the mutated protein and can reset the HR [92]. Another mechanism could be the upregulation of the pump glycoprotein efflux reducing concentrations of the intracellular PARP inhibitor [93,94] or loss of 53BP1, a key protein in the NHEJ pathway.

#### **7. Conclusions**

clinical trials, myelosuppression increased in the combination therapy versus monotherapy,

Olaparib has also been studied in combination with bevacizumab. Dean et al. [89] designed a Phase I clinical trial, as they hypothesized that bevacizumab antiangiogenic activity and hypoxia inducing DNA damage may enhance olaparib therapeutic activities. They showed that a dose of olaparib 400 mg twice daily in combination with Avastin 10mg / kg every two weeks was tolerated well. They are considering the combination for future clinical trials.

The recommended lynparza® (olaparib) dose is 400 mg (eight capsules) twice a day, i.e., a total daily dosage of 800 mg. Treatment should begin before eight weeks of completion of the last cycle of chemotherapy with a regimen containing platinum. Continual treatment is recom‐ mended until disease progression. The recommended dose is reduced to 200 mg orally twice daily (total daily dose 400 mg). Elderly patients require a dose reduction initially, and it can be administered in patients with mild renal impairment (creatinine clearance> 50 mL / min).

Among the most frequent toxicities in clinical trials are hematologic toxicity with mild to moderate anemia, lymphopenia, neutropenia, and thrombocytopenia at manageable levels. Other frequently observed side effects are headache, fatigue, decreased appetite, abdominal

Another event that was observed was the development of myelodysplastic / AML syndrome, in only a small number of patients receiving olaparib alone or in combination with other antineoplastic during clinical trials. All had previously received platinum-based chemother‐

There have been cases of pneumonitis, some of them being fatal. If patients are treated with Lynparza, respiratory symptoms such as cough, dyspnea, and fever should be closely monitored. If there is any alteration in the chest radiography, treatment must be stopped and the patient is treated appropriately. Paralyzer can cause birth defects if given to pregnant women. A reliable contraception should be recommended during treatment and one month

Targeted therapy based on the patient's mutation status is the future of the treatment of ovarian cancer. *BRCA* deficiency may be reversed by mutational changes in the reading frame, resulting in wild-type *BRCA* protein production. A second mutation (compensatory mutations or crossovers) can cause changes in the reading frame *BRCA* mutation, HR rebuilding, and restoring its functionality, explaining why not all tumors with *BRCA* mutation respond to PARP inhibitors [91]. Some *BRCA1* mutant alleles encode functional proteins but are degraded,

especially with topotecan and cisplatin.

138 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

**5.4. Dosage**

**5.5. Adverse reactions**

after the last dose [90,63].

discomfort, nausea, vomiting, diarrhea, and dyspepsia.

apy regimens, radiation, and other DNA-damaging agents.

**6. Mechanisms of resistance to PARP inhibitors**

The context of the *BRCA 1/2*-mutant genotype has a significant impact on disease behavior and outcome. An encouraging data on responses to PARP inhibitors in *BRCA 1/2*-mutant carriers with prostate, pancreatic, and breast cancer have been reported and are likely to be associated with platinum sensitivity. It is apparent that establishing the germ-line and/or tumor *BRCA 1/2* mutation status in patients with cancers known to be associated with *BRCA 1/2* mutations is of notable importance due to the potential therapeutic options. Chemotherapy recommen‐ dation for patients with *BRCA 1/2* mutant epithelial ovarian cancer ought to be based on rechallenging these patients with platinum-based treatment, and prolonging the platinum-free interval in the event of early relapse following platinum-based treatment. The timing and sequence of therapy, and the indications for rechallenging patients with platinum-based chemotherapy, including routes and schedules of administration (IV vs. IP, weekly vs. thrice weekly), olaparib plus paclitaxel and carboplatin followed by laparib maintenance monother‐ apy, significantly improved progression-free survival versus paclitaxel plus carboplatin alone, in patients with *BRCA*-mutated recurrent platinum-sensitive ovarian cancer, with acceptable tolerability profile. It differs as more data emerges from analyses of the mutation status of *BRCA 1/2* genes and other HR-related genes in tumor samples from previously completed studies. In addition, stratification based on HR-deficiency phenotype/genotype may become the standard in future clinical trials involving patients with *BRCA 1/2* mutant epithelial ovarian cancer. It has been established that olaparib plus paclitaxel and carboplatin followed by olaparib maintenance monotherapy significantly improved progression-free survival versus paclitaxel plus carboplatin alone, in patients with *BRCA*-mutated recurrent platinum-sensitive ovarian cancer, with acceptable tolerability profile.

#### **8. Future directions**

Tumors with alterations in DNA repair lead to a defective HR, based on synthetic lethality and being very sensitive to PARP inhibitors. This happens with HGSC *BRCA* mutation carriers demonstrating its response to olaparib. We still need to better identify patients who will respond to PARP inhibitors because a proportion of patients developed resistance to these treatments, so research is needed to understand the mechanisms of action of PARP inhibitors and mechanisms of resistance. The only biomarkers that have been shown to be predictive of response while using PARP inhibitors are the *BRCA 1/2* mutations both somatic and germ line, in the absence of other biomarkers, we are limited to using the PARP inhibitors only in patients with *BRCA* mutation 1/2, although it may be effective in other tumors despite the absence of *BRCA* mutation.

We have studied the operation of Rad5 using antibody detection of Rad5 as new response PARP-inhibitors biomarkers, although initial results suggest that is not sufficient, specific and sensitive for use in clinical application, so we need to keep looking for new biomarkers or methods that help us to identify the appropriate patients for treatment with PARP inhibitors [52]. As not all genes responsible for DNA repair are known, another option that has not yet been implemented is to apply functional tests of DNA-repair capability, this would help us identify the abnormalities and tumors suitable for treatment with PARP inhibitors [95], the molecules involved in *BRCA*-like tumors whose information is essential to broaden the scope of action of PARP-inhibitors, without limiting its use for patients with *BRCA* 1/2 mutations. A current research topic is whether these new drugs work better alone or in combination with standard cytotoxic agents, avoiding toxicities and resistance mechanisms [96]. They have been shown to be well tolerated with manageable toxicity, but the long-term action is unknown. Some experts question whether inhibiting DNA repair can lead to deleterious effects such as an increased risk of developing other types of cancer in the future. Another topic of interest is its effect in combination with radiotherapy or as maintenance therapy. As of now, we do not know what effect PARP inhibitors may have in patients with low tumor burden, and what would be the benefit when these agents are used as maintenance therapy or chemoprevention.

#### **Author details**

Sandra García-Nieto1 , Carmen Guillén-Ponce3\*, Carmen Alonso1 , María-Carmen Rodríguez-Soriano2 , María-Luz Pombo1 , Earl Julie3 and Samir A. Farghaly4


3 Oncology Departmen. Hospital Universitario Ramón y Cajal. Madrid, Spain

4 Joan and Sanford I. Weill Medical College/ The Graduate School of Medical Sciences, and The New York Presbyterian Hospital-Weill Cornell Medical Center, Cornell University, New York, NY, USA

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[1] Chi DS, Eisenhauer EL, Zivanovic O, etal. Improved progression-free and overall survival in advanced ovarian cancer as a result of a change in surgical paradigm. Gy‐ necol Oncol 2009;114:26–31.

[2] Jemal A, Bray F, Center MM, et al. Global cancer statistics.CA Cancer J Clin 2001;61:69–90.

We have studied the operation of Rad5 using antibody detection of Rad5 as new response PARP-inhibitors biomarkers, although initial results suggest that is not sufficient, specific and sensitive for use in clinical application, so we need to keep looking for new biomarkers or methods that help us to identify the appropriate patients for treatment with PARP inhibitors [52]. As not all genes responsible for DNA repair are known, another option that has not yet been implemented is to apply functional tests of DNA-repair capability, this would help us identify the abnormalities and tumors suitable for treatment with PARP inhibitors [95], the molecules involved in *BRCA*-like tumors whose information is essential to broaden the scope of action of PARP-inhibitors, without limiting its use for patients with *BRCA* 1/2 mutations. A current research topic is whether these new drugs work better alone or in combination with standard cytotoxic agents, avoiding toxicities and resistance mechanisms [96]. They have been shown to be well tolerated with manageable toxicity, but the long-term action is unknown. Some experts question whether inhibiting DNA repair can lead to deleterious effects such as an increased risk of developing other types of cancer in the future. Another topic of interest is its effect in combination with radiotherapy or as maintenance therapy. As of now, we do not know what effect PARP inhibitors may have in patients with low tumor burden, and what would be the benefit when these agents are used as maintenance therapy or chemoprevention.

140 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

, Carmen Guillén-Ponce3\*, Carmen Alonso1

1 Oncology Department. Hospital General Universitario de Albacete. Albacete, Spain

3 Oncology Departmen. Hospital Universitario Ramón y Cajal. Madrid, Spain

\*Address all correspondence to: carmenguillenponce@gmail.com

2 Oncology Department. Hospital Virgen de la Luz. Cuenca, Spain

, María-Luz Pombo1

4 Joan and Sanford I. Weill Medical College/ The Graduate School of Medical Sciences, and The New York Presbyterian Hospital-Weill Cornell Medical Center, Cornell University,

[1] Chi DS, Eisenhauer EL, Zivanovic O, etal. Improved progression-free and overall survival in advanced ovarian cancer as a result of a change in surgical paradigm. Gy‐

,

and Samir A. Farghaly4

, Earl Julie3

**Author details**

Sandra García-Nieto1

New York, NY, USA

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[73] Audeh MW, Carmichael J, Penson RT, et al. Oral poly(ADP-ribose) polymerase in‐ hibitor olaparib in patients with *BRCA1* or *BRCA2* mutations and recurrent ovarian

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[75] Adams SF, Marsh EB, Elmasri W, et al. A high response rate to liposomal doxorubi‐ cin is seen among women with *BRCA* mutations treated for recurrent epithelial ovar‐

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146 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives


#### **Chapter 6**

## **Ovarian Cancer Research in the Post Genomic Era — Challenges and Opportunities**

Alicia A. Goyeneche and Carlos M. Telleria

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60600

#### **Abstract**

[94] Rottenberg S, Jaspers JE, Kersbergen A, et al. High sensitivity of *BRCA1*-deficient mammary tumors to the PARP inhibitor AZD2281 alone and in combination with

[95] Pennington KP, Walsh T, Harrell MI, et al. Germline and somatic mutations in ho‐ mologous recombination genes predict platinum response and survival in ovarian,

[96] Liu JF, Konstantinopoulos PA, Matulonis UA. PARP inhibitors in ovarian cancer:

fallopian tube, and peritoneal carcinomas. Clin Cancer Res 2014;20:764–75.

Current status and future promise. Gynecol Oncol 2014;133:362–9.

platinum durgs. PROC Natl Acad Sci USA 2008;105:17079–84.

148 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

The field of ovarian cancer research is undergoing major re-examination. Pathologists are defining the disease in new terms, and—having observed discrepancies in its actual cell(s) and tissue(s) of origin—are asking whether or not ovarian cancer truly represents one disease or a complex group of diseases. Further complexity was unveiled after sequencing a large number of high-grade serous ovarian cancer tumor samples (the most frequent ovarian cancer histotype). The experiments uncovered the existence of at least four different molecular subtypes, which standard pathological assessment cannot determine. These discoveries propelled a need for designing novel model systems to study the disease and to develop therapies tailored to the molecular genetics of the tumor. Though there has been no major breakthrough as regards overall patient survival of ovarian cancer in the last 50 years, this chapter summarizes the many challenges and fascinating opportunities scientists face in altering the fatal course of this disease.

**Keywords:** Epithelial ovarian cancer (EOC), the cancer genome atlas (TCGA), pa‐ tient-derived xenografts (PDXs), peritoneal ovarian carcinomatosis, tumor burden, ; tumor dormancy, minimal residual disease (MRD), high-grade serous ovarian can‐ cer (HGSOC), Genetically engineered mouse models (GEMM), single nucleotide polymorphisms (SNPs), Calculator of Ovarian Carcinoma Subtype Prediction (COSP)

© 2016 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.

#### **1. Introduction**

#### **1.1. Ovarian cancer represents various diseases**

Epithelial ovarian cancer (EOC) is the deadliest gynecological disease. Over 70% of patients are diagnosed at late stages when the disease has disseminated within the abdominopelvic cavity. This is due to a lack of specific symptoms and valid biomarkers to look out for in early screenings, a consequence of the poor understanding of the disease's pathobiology. Upon late diagnosis, the standardized treatment is surgery (to remove all macroscopic disease within the abdominal cavity), followed by 6 cycles of a platinating, DNA-damaging agent combined with a taxane that disrupts microtubule function. Ninety percent of patients with late diagnosis, despite showing a promising initial response to standard of care treatment, ultimately relapse and die of the disease. The 5-year survival rate for EOC has remained below 35% over the past 20 years (rev.in [1-6]).

EOC genetically and biologically represents various diseases with different sites of origin that share common anatomical locations in the abdominal cavity when symptomatic [6]. EOCs are histologically classified as low-grade serous carcinoma, endometrioid carcinoma, clear-cell carcinoma, mucinous carcinoma, and high-grade serous ovarian carcinoma (HGSOC) [4]. Mucinous ovarian tumors are frequently the result of metastatic gastrointestinal cancers. Clear ovarian cancers and endometrioid ovarian cancers likely originate from endometrioid lesions, whereas serous ovarian cancers have 3 likely sites of origin: (1) the secretory cells of the distal fallopian tubes, (2) the ovarian surface epithelium, and (3) a niche of cells found in the hilum region of the ovary in a transitional area among the ovarian surface epithelium, the mesothe‐ lium, and the tubal epithelium [7, 8]. Among serous ovarian cancers, the low-grade serous tumors often carry wild-type p53 gene, are chromosomally stable and frequently unresponsive to platinum therapy, and carry Ras mutations. In contrast, HGSOC are p53 mutant and usually highly responsive to platinum therapy, and carry widespread DNA copy changes and wildtype Ras [9].

HGSOC is the most aggressive subtype of EOC, represents the majority of cases of EOC, and causes almost 70% of all deaths from this disease [9]. The Cancer Genome Atlas (TCGA) network reported the genetic sequencing of 489 tumors histopathologically classified as HGSOC [10]. The study confirmed, in a larger cohort of patients, that the genetic signature of HGSOC involves mutation of tumor suppressor p53 in 96-97% of cases, as previously described in a smaller cohort [11], with almost 50% of the tumors having dysregulation of the homolo‐ gous recombination DNA repair pathway. The study led to revisiting HGSOC in terms of its biology, response to chemotherapy, clinical outcome, and genetic subtypes [6, 9, 12].

#### **2. There are insufficient model systems to study ovarian cancer in vivo**

The stagnation in successfully treating patients with EOC is compounded with an insufficiency of model systems to study the disease when harbored within the abdominopelvic cavity. Four main approaches have been used to study EOC in vivo in mice: (1) genetically engineered mouse models (GEMM) that develop EOC from the epithelium of the ovaries [13-15] or the oviducts (fallopian tubes) [16, 17]; (2) syngeneic models in which mouse EOC cells are orthotopically xenografted into the ovarian bursa [18] or the peritoneal cavity [19] of immu‐ nocompetent mice; (3) patient-derived xenografts (PDX) models in which the tumors of the patients are transplanted into the peritoneal cavity of severe immunodeficient mice (deficient in T-cells, B-cells and NK cells) [20-22]); and (4) xenografts of human EOC cells into the flanks or the abdominal cavity of nude, T-cell deficient mice [23]. Current GEMM of EOC facilitate studying the disease from its inception. Yet, due to a lack of highly specific promoters to target the presumed cells of origin, the GEMM do not develop the same genetic lesions carried by patients, and, hence, do not recapitulate the human phenotype in its entirety (rev.in [24, 25]). The use of mouse EOC cells xenografted in immunocompetent mice is highly relevant since the disease can be assessed in the presence of an intact immune system; however, the number of available models is limited [18, 19]. PDX closely recapitulate the histology of the patient's sample when placed within the peritoneal cavity as a finely minced tumor with some varia‐ bility depending on the host mice. For instance, when human ovarian cancer tissues are xenografted in SCID (C.B-17/IcrHsd-*Prkdc*scid *Lyst*bg) mice, the human stroma accompanying the cancer cells is rapidly replaced with mouse stroma [22]. In contrast, in severe immunode‐ ficient NOD-SCID IL2Rγnull (NSG) mice lacking acquired and innate immunities [26], the tumor associated human stromal cells (e.g. fibroblasts and lymphocytes) remain functional for an extended period of time [20]. Nevertheless, the xenograft of EOC into the peritoneal cavity of immunosuppressed mice recapitulates only a late phase of disease as the cells are directly deposited into the peritoneal cavity of a host. Clearly, each model system for recapitulating EOC in mouse models has shortcomings.

**1. Introduction**

20 years (rev.in [1-6]).

type Ras [9].

**1.1. Ovarian cancer represents various diseases**

150 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

Epithelial ovarian cancer (EOC) is the deadliest gynecological disease. Over 70% of patients are diagnosed at late stages when the disease has disseminated within the abdominopelvic cavity. This is due to a lack of specific symptoms and valid biomarkers to look out for in early screenings, a consequence of the poor understanding of the disease's pathobiology. Upon late diagnosis, the standardized treatment is surgery (to remove all macroscopic disease within the abdominal cavity), followed by 6 cycles of a platinating, DNA-damaging agent combined with a taxane that disrupts microtubule function. Ninety percent of patients with late diagnosis, despite showing a promising initial response to standard of care treatment, ultimately relapse and die of the disease. The 5-year survival rate for EOC has remained below 35% over the past

EOC genetically and biologically represents various diseases with different sites of origin that share common anatomical locations in the abdominal cavity when symptomatic [6]. EOCs are histologically classified as low-grade serous carcinoma, endometrioid carcinoma, clear-cell carcinoma, mucinous carcinoma, and high-grade serous ovarian carcinoma (HGSOC) [4]. Mucinous ovarian tumors are frequently the result of metastatic gastrointestinal cancers. Clear ovarian cancers and endometrioid ovarian cancers likely originate from endometrioid lesions, whereas serous ovarian cancers have 3 likely sites of origin: (1) the secretory cells of the distal fallopian tubes, (2) the ovarian surface epithelium, and (3) a niche of cells found in the hilum region of the ovary in a transitional area among the ovarian surface epithelium, the mesothe‐ lium, and the tubal epithelium [7, 8]. Among serous ovarian cancers, the low-grade serous tumors often carry wild-type p53 gene, are chromosomally stable and frequently unresponsive to platinum therapy, and carry Ras mutations. In contrast, HGSOC are p53 mutant and usually highly responsive to platinum therapy, and carry widespread DNA copy changes and wild-

HGSOC is the most aggressive subtype of EOC, represents the majority of cases of EOC, and causes almost 70% of all deaths from this disease [9]. The Cancer Genome Atlas (TCGA) network reported the genetic sequencing of 489 tumors histopathologically classified as HGSOC [10]. The study confirmed, in a larger cohort of patients, that the genetic signature of HGSOC involves mutation of tumor suppressor p53 in 96-97% of cases, as previously described in a smaller cohort [11], with almost 50% of the tumors having dysregulation of the homolo‐ gous recombination DNA repair pathway. The study led to revisiting HGSOC in terms of its

biology, response to chemotherapy, clinical outcome, and genetic subtypes [6, 9, 12].

**2. There are insufficient model systems to study ovarian cancer in vivo**

The stagnation in successfully treating patients with EOC is compounded with an insufficiency of model systems to study the disease when harbored within the abdominopelvic cavity. Four

#### **3. The progression of epithelial ovarian cancer within the abdominopelvic cavity is not easy to assess**

Studies involving the implantation of EOC cells in the peritoneal cavity (intraperitoneally; i.p.) of host mice are limited when compared to the number of studies done using EOC cells xenografted subcutaneously (s.c.) (rev.in [25, 27]). One main reason for this discrepancy is that the growth of s.c. tumors can be monitored easily using precision calipers; yet, this site fails to represent the environment of the abdominal cavity in which EOC thrive. The struggle to analyze disease progression in the peritoneal cavity is that it requires sophisticated, noninvasive, imaging approaches to follow the development of internal tumor nodules in a context of a lack of well-defined parameters of tumor burden [28]. In most studies done with i.p. xenografts, tumor burden has been assessed by recording overall survival [23], noting volume of ascites accumulated [29], or calculating the total mass of what is considered tumoral tissue after collection from the abdominal cavity at necropsy [30]. More recently, non-invasive imaging methods to evaluate tumor progression in longitudinal studies have been developed, yet their application in evaluating EOC within the peritoneal cavity has been limited [25]. Overall, information as to how EOC develops within the abdominal cavity is scarce. Preferred sites of anatomical distribution of the tumors remain unknown as do their histopathology and molecular genetics.

#### **4. Peritoneal ovarian cancer needs to be studied in different regions of the abdominopelvic cavity**

It is feasible that solid nodules that develop, for instance, in the omentum, have a different genetic profile when compared with sibling nodules found in other sites, such as the dia‐ phragm, the surface of the liver, the bowel, or the lower pelvic cavity. This might be due to tissues (to which each tumor foci must adapt) having different histological and physiological micro-environments, likely impinging on the behavior of the cancer cells. Depending on the nearby tissue microenvironment, cancer cells may hijack otherwise non-malignant cells in a different manner depending on the anatomical location of the foci. As a consequence, this differential tumor adaptation to the environment may explain the apparent heterogeneity observed in tumors found within the peritoneal cavity of patients at the moment of debulking surgery, sometimes leading to difficulties in making the correct histopathological diagnosis of the overall disease. Thus, there is an urgent need to (1) standardize, across a genetically-defined group of available EOC cell lines, a common set of histopathological and genetic biomarkers of disease growing in the abdominal cavity; and (2) determine if such biomarkers, despite being expressed from the same cell types of origin, show heterogeneity according to the site within the abdominal cavity where the tumor develops. For instance, evidence suggests that omental vs. ovarian sites of HGSOC patients show variability in the host stromal responses among the sites [31]. Another study using biopsies from different sites within the peritoneal cavity of patients with HGSOC show heterogeneity or clonal diversity among the tumor sites mani‐ fested by single nucleotide polymorphisms (SNPs) associated with differentially expressed genes [32].

One tool currently available for characterizing the histopathological subtype of ovarian carcinomas is the Calculator of Ovarian Carcinoma Subtype Prediction (COSP), which is an algorithm that encompasses 9 predictive biomarkers and is used to differentiate histotypes of EOCs. The algorithm is freely accessible [33] and permits the scoring by immunohistochem‐ istry, using standardized antibodies and incubation procedures, the abundances of WT1 (Wilms Tumor 1), p16 (cyclin dependent kinase inhibitor 2A; CDKN2A), DKK1 (dickkopf homolog 1), VIM (vimentin), p53 (TP53), PRG (progesterone receptor), TFF3 (trefoil factor 3 [intestinal]), HNF1B (hepatocyte nuclear factor 1β) and MDM2 (mouse double minute 2). The scores for these markers are 0 or 1, except for p53 that has scores of 0 (no expression denoting null p53), 1 (low abundance for wild type p53), or 2 (high abundance for mutant p53). For instance, for the Kuramochi ovarian cancer cell line (see later Table 1), the algorithm predicts a HGSOC histotype with 97% probability, whereas for the popular A2780 ovarian cancer cell line (see later Fig.1), the algorithm predicts an endometrioid histotype with 94% probability [34]. A limitation to the algorithm is its difficulty in clearly differentiating between low-grade and high-grade serous histotypes. However, low-grade serous ovarian carcinomas only account for ~3% of all [35]. Furthermore, a combination of histological assessment and molecular genetic profiles should be able to distinguish between these two serous ovarian cancer subtypes.

sites of anatomical distribution of the tumors remain unknown as do their histopathology and

**4. Peritoneal ovarian cancer needs to be studied in different regions of the**

It is feasible that solid nodules that develop, for instance, in the omentum, have a different genetic profile when compared with sibling nodules found in other sites, such as the dia‐ phragm, the surface of the liver, the bowel, or the lower pelvic cavity. This might be due to tissues (to which each tumor foci must adapt) having different histological and physiological micro-environments, likely impinging on the behavior of the cancer cells. Depending on the nearby tissue microenvironment, cancer cells may hijack otherwise non-malignant cells in a different manner depending on the anatomical location of the foci. As a consequence, this differential tumor adaptation to the environment may explain the apparent heterogeneity observed in tumors found within the peritoneal cavity of patients at the moment of debulking surgery, sometimes leading to difficulties in making the correct histopathological diagnosis of the overall disease. Thus, there is an urgent need to (1) standardize, across a genetically-defined group of available EOC cell lines, a common set of histopathological and genetic biomarkers of disease growing in the abdominal cavity; and (2) determine if such biomarkers, despite being expressed from the same cell types of origin, show heterogeneity according to the site within the abdominal cavity where the tumor develops. For instance, evidence suggests that omental vs. ovarian sites of HGSOC patients show variability in the host stromal responses among the sites [31]. Another study using biopsies from different sites within the peritoneal cavity of patients with HGSOC show heterogeneity or clonal diversity among the tumor sites mani‐ fested by single nucleotide polymorphisms (SNPs) associated with differentially expressed

One tool currently available for characterizing the histopathological subtype of ovarian carcinomas is the Calculator of Ovarian Carcinoma Subtype Prediction (COSP), which is an algorithm that encompasses 9 predictive biomarkers and is used to differentiate histotypes of EOCs. The algorithm is freely accessible [33] and permits the scoring by immunohistochem‐ istry, using standardized antibodies and incubation procedures, the abundances of WT1 (Wilms Tumor 1), p16 (cyclin dependent kinase inhibitor 2A; CDKN2A), DKK1 (dickkopf homolog 1), VIM (vimentin), p53 (TP53), PRG (progesterone receptor), TFF3 (trefoil factor 3 [intestinal]), HNF1B (hepatocyte nuclear factor 1β) and MDM2 (mouse double minute 2). The scores for these markers are 0 or 1, except for p53 that has scores of 0 (no expression denoting null p53), 1 (low abundance for wild type p53), or 2 (high abundance for mutant p53). For instance, for the Kuramochi ovarian cancer cell line (see later Table 1), the algorithm predicts a HGSOC histotype with 97% probability, whereas for the popular A2780 ovarian cancer cell line (see later Fig.1), the algorithm predicts an endometrioid histotype with 94% probability [34]. A limitation to the algorithm is its difficulty in clearly differentiating between low-grade and high-grade serous histotypes. However, low-grade serous ovarian carcinomas only account for ~3% of all [35]. Furthermore, a combination of histological assessment and

molecular genetics.

genes [32].

**abdominopelvic cavity**

152 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

## **5. Most popular epithelial ovarian cancer cell lines used to study peritoneal carcinomatosis in mouse models give rise to disparate intra-abdominal disease phenotypes**

Common EOC cell lines, utilized for years in preclinical studies, were evaluated for their ability to cause i.p. tumors [23]. ES-2, A2780, and HEY cells (all originally diagnosed as undifferen‐ tiated carcinomas), OV2008 (likely originated from an endometrial carcinoma), and SKOV-3 (which depicts a clear-cell adenocarcinoma histotype in xenografts) all develop intra-abdomi‐ nal tumors in less than 3 months. The tumors are described as "dense solid," often accompanied by accumulation of cellular ascites. Our experience with widely used EOC cell lines reveals different times for the establishment of the xenografts and highly diverse anatomical depiction of the solid growths within the abdominal cavity among the different cell lines. IGROV-1 cells generate large solid masses termed omental cakes that expand toward the lower pelvic cavity (Fig.1A). SKOV-3 cells develop small yet multiple nodules in the mesentery and the omentum (Fig.1B), while A2780 cells develop large solid masses, taking the ovaries and the lower pelvic cavity (Fig.1C). The diversity in anatomical growths is likely due to the varied histotypes and genetic profiles represented by these cell lines (see later) [36].

**Figure 1.** Peritoneal disease caused by IGROV-1 (A), SKOV-3 (B), and A2780 (C) EOC cells. The images were taken after 4 weeks of injection for IGROV-1, 10 weeks for SKOV-3, and 6 weeks for A2780 tumors. In A, the growth within the peritoneal cavity is mostly confined to an "omental cake" (white area). In B, dotted black areas show solid tumors adhering to fat in the pelvic region, intestines, and mesentery. In C, right (ROT) and left (LOT) ovarian tumors clearly are connected to the uterine horns (yellow arrows). Ovaries carrying tumors have more blood supply and are larger than the rest of the peritoneal, pale nodules (yellow pattern).

#### **6. The majority of epithelial ovarian cancer cell lines used for preclinical studies do not embody the most frequent histotype of the disease**

Based on the genetic signatures published from over 50 human ovarian cancer cell lines widely available, out of the approximately 100 that have been described in the literature [36-38], it is apparent that the vast majority of the cell lines overwhelmingly used for over 30 years to study the disease have a genotype which does not resemble the most predominant histotype of EOC, HGSOCs. This may be a major contributing factor in the failure to bring new and effective treatments of HGSOC to clinical practice.

The cell lines currently characterized as likely representing HGSOC [36-38] were developed in the 1970s and 1980s and have been poorly described. Oftentimes they lack information on the original histopathological diagnosis, are poorly linked to patient data, and were developed from ascites or solid nodules following an array of protocols not always clearly stated. It is imperative that biomedical researchers worldwide join efforts to develop new, highly stand‐ ardized and annotated ovarian cancer cell lines. These cells lines should be developed under similar isolation and culture protocols as to prevent inter-laboratory variations in their behavior, thus accelerating the creation of new knowledge in the field of preclinical ovarian cancer modelling and therapy. By taking advantage of the progress made in the area of molecular genetics and ovarian cancer biology, it is time to generate new cell lines that genetically and histopathologically can be characterized as pertaining, for instance, to the HGSOC histotype, and, within it, to each one of the molecular subtypes described by Tothill et al. [39] and later on confirmed in a larger cohort of patients [10]. Additionally, there is a timely opportunity to utilize these biological resources with the objective of standardizing mouse models of intra-abdominal disease caused by genetically-identified HGSOC cells.

#### **7. The need for an expanded definition of tumor burden when referring to peritoneal ovarian cancer carcinomatosis**

Limiting preclinical analysis of tumor burden to overall survival, tumor mass, or volume of ascites accumulated is not sufficient if we are to find early metrics of response to new therapies as well as early signs and symptoms of the disease. Re-defining tumor burden in peritoneal ovarian cancer in a comprehensive manner should provide investigators worldwide with multifaceted metrics—anatomical, physiological, and behavioral—to be followed to under‐ stand the biology of the progression of EOC and, most importantly, that of the most frequent HGSOC type. The metrics should also allow inter-laboratory and inter-cell line comparisons of HGSOC as a unique disease, provide standardized benchmarks for testing new preclinical therapies, reveal markers of disease state with clinical implications for earlier diagnosis, and provide a baseline reference for the validation of new HGSOC cell lines established from patients with well-documented medical history and annotated histopathological diagnosis of HGSOC.

Based on recently published genotypes [36-38], it is feasible to begin redefining peritoneal ovarian tumor burden by utilizing the currently available cell lines that have the highest probability of genetically representing HGSOC (some examples are displayed in Table 1). Despite that each cell line was established using different culture conditions, we should standardize all cell lines to grow under the same culture conditions to avoid bias and proceed to authenticate them using DNA microsatellite short tandem repeats (STR) as recently recommended [37]. Only cell lines which match their STR public genetic database 90-100% should be used worldwide [40]. Validated cell lines may be injected i.p. in the lower pelvic region of widely available immunosuppressed female mice lacking T-cell function (Hsd: Athymic Nude-*Foxn1*nu) or in severe immunodeficient NOD/SCID/IL2Rγnull mice lacking acquired and innate immunities [26]. Disease progression can then be followed and the signs and symptoms contrasted against non-cancerous, age-matched controls studied in parallel.

apparent that the vast majority of the cell lines overwhelmingly used for over 30 years to study the disease have a genotype which does not resemble the most predominant histotype of EOC, HGSOCs. This may be a major contributing factor in the failure to bring new and effective

The cell lines currently characterized as likely representing HGSOC [36-38] were developed in the 1970s and 1980s and have been poorly described. Oftentimes they lack information on the original histopathological diagnosis, are poorly linked to patient data, and were developed from ascites or solid nodules following an array of protocols not always clearly stated. It is imperative that biomedical researchers worldwide join efforts to develop new, highly stand‐ ardized and annotated ovarian cancer cell lines. These cells lines should be developed under similar isolation and culture protocols as to prevent inter-laboratory variations in their behavior, thus accelerating the creation of new knowledge in the field of preclinical ovarian cancer modelling and therapy. By taking advantage of the progress made in the area of molecular genetics and ovarian cancer biology, it is time to generate new cell lines that genetically and histopathologically can be characterized as pertaining, for instance, to the HGSOC histotype, and, within it, to each one of the molecular subtypes described by Tothill et al. [39] and later on confirmed in a larger cohort of patients [10]. Additionally, there is a timely opportunity to utilize these biological resources with the objective of standardizing mouse models of intra-abdominal disease caused by genetically-identified HGSOC cells.

**7. The need for an expanded definition of tumor burden when referring to**

Limiting preclinical analysis of tumor burden to overall survival, tumor mass, or volume of ascites accumulated is not sufficient if we are to find early metrics of response to new therapies as well as early signs and symptoms of the disease. Re-defining tumor burden in peritoneal ovarian cancer in a comprehensive manner should provide investigators worldwide with multifaceted metrics—anatomical, physiological, and behavioral—to be followed to under‐ stand the biology of the progression of EOC and, most importantly, that of the most frequent HGSOC type. The metrics should also allow inter-laboratory and inter-cell line comparisons of HGSOC as a unique disease, provide standardized benchmarks for testing new preclinical therapies, reveal markers of disease state with clinical implications for earlier diagnosis, and provide a baseline reference for the validation of new HGSOC cell lines established from patients with well-documented medical history and annotated histopathological diagnosis of

Based on recently published genotypes [36-38], it is feasible to begin redefining peritoneal ovarian tumor burden by utilizing the currently available cell lines that have the highest probability of genetically representing HGSOC (some examples are displayed in Table 1). Despite that each cell line was established using different culture conditions, we should standardize all cell lines to grow under the same culture conditions to avoid bias and proceed to authenticate them using DNA microsatellite short tandem repeats (STR) as recently

treatments of HGSOC to clinical practice.

154 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

**peritoneal ovarian cancer carcinomatosis**

HGSOC.

The animals can be monitored to record body weight, abdominal circumference, body temperature, and food/water intake to build a clinical history of each animal as the disease progresses, using biomarkers of tumor progression [41]. The experimental animals and agematched, non-cancer controls also can be subjected to a battery of behavioral tests to assess visceral pain, motor function, and depression-like behavior (helplessness and social with‐ drawal). In animal models of EOC, depressive-like behaviors may be facilitated by the production of inflammatory cytokines from the cancer cells acting at the level of brain regions like the hippocampus [42, 43] and thus, may be a sensitive marker of disease state. Finally, these parameters can be completed with longitudinal, intra-abdominal anatomy of the tumorcarrying mice using non-invasive imaging approaches (e.g. micro-ultrasound) [44, 45]. The recorded images can then be analyzed longitudinally to identify the formation and progression of intra-abdominal solid masses and accumulation of ascites fluid. On selected masses, it is also possible to study vascularity using 3D power Doppler ultrasound [46, 47].


**Table 1.** Epithelial ovarian cancer cell lines with likely HGSOC genomic classification.

The accumulation of bloody ascites in the abdominopelvic cavity suggests that changes in vascular integrity, with possible effects on blood pressure and oxygen delivered, are taking place. Indeed, declines in oxygenation and blood pressure have been suggested as biomarkers of peritoneal tumor progression [28]. Thus, as part of a comprehensive approach for assessing tumor burden, we suggest determining the level of peripheral blood oxygen saturation [48] and blood pressure [49]. Finally, we propose completing the assessment of tumor burden by taking a blood sample from the animals in order to (1) measure cancer antigen CA-125 used as a biomarker of EOC disease progression [50]; (2) study hematological parameters that can be altered due to tumor burden—e.g. red and white blood cell counts, platelets, hemoglobin concentration, and hematocrit; (3) perform chemical analysis of GOT (glutamic oxaloacetic transaminase) and GPT (glutamic pyruvic transaminase) as surrogate markers of hepatic function; (4) measure serum levels of creatinine and urea as surrogate markers of renal function; and (5) measure serum levels of estradiol and progesterone, since they impact the outcome of the behavioral tests suggested above.

#### **8. Understanding all cellular components of advanced disease**

In ovarian cancer, metastasis through the vasculature is rare and a very late manifestation of the disease. Instead, ovarian cancer cells are prone to spread by direct extension from the ovaries to adjacent tissues, or to detach from the primary ovarian tumor directly into the peritoneal cavity where they seed the mesothelium of the omentum, diaphragm, bowel serosa, and the entire peritoneum [51-53]. Widespread visceral and intestinal wall metastases with formation of adhesions between the loops of the bowel cause intestinal obstruction, prevent normal nutrition, and become a primary cause of death [1].

The high incidence (65%) of peritoneal malignant effusions in ovarian cancer patients at advanced presentation [54, 55], and the development of symptoms due to ascites accumulation at diagnosis as well as recurrence [56], suggest that the "liquid" component is an active pathogenic manifestation of the disease. Ovarian cancer cells isolated from peritoneal ascites of major ovarian cancer histological types were described as organized structures of different sizes and heterogeneous morphology [57]. Furthermore, multicellular structures isolated directly from ascites were shown capable of adhering ex vivo to components of the extracel‐ lular matrix and to monolayers of mesothelial cells, suggesting their participation in the dissemination of ovarian cancer [58]. Cancer cells isolated from ascites and metastatic secon‐ dary sites exhibit a higher percentage of stemness markers when compared to their primary tumors [59-61]. Additionally, cancer-associated proteins and mRNAs are differentially expressed in peritoneal effusions when compared to primary carcinomas or solid metastases. Lastly, there is a differential gene expression among peritoneal effusions when comparing samples at diagnosis (pre-chemotherapy) vs. samples at recurrence (post-chemotherapy) [62]. Altogether, these data suggest that cancer cells within effusions—the "liquid" component of ovarian cancer—represent a biomarker of tumor evolution toward a more aggressive/ advanced disease phenotype of poor prognosis.

Most of our understanding of the biology of ovarian cancer multicellular structures is based on the premise that mono-dispersed ovarian cancer cells, when gathering together either by enforced gravity or prevention of adhesion, mimic the program of assembly followed by ovarian cancer multicellular structures found within malignant effusions [63, 64]. Therefore, it is of importance to define if ovarian cancer multicellular structures found in ascites represent aggregation following shedding from solid tumors or, instead, are active products of disease selection and critical drivers of disease advancement and prognosis (Fig.2).

place. Indeed, declines in oxygenation and blood pressure have been suggested as biomarkers of peritoneal tumor progression [28]. Thus, as part of a comprehensive approach for assessing tumor burden, we suggest determining the level of peripheral blood oxygen saturation [48] and blood pressure [49]. Finally, we propose completing the assessment of tumor burden by taking a blood sample from the animals in order to (1) measure cancer antigen CA-125 used as a biomarker of EOC disease progression [50]; (2) study hematological parameters that can be altered due to tumor burden—e.g. red and white blood cell counts, platelets, hemoglobin concentration, and hematocrit; (3) perform chemical analysis of GOT (glutamic oxaloacetic transaminase) and GPT (glutamic pyruvic transaminase) as surrogate markers of hepatic function; (4) measure serum levels of creatinine and urea as surrogate markers of renal function; and (5) measure serum levels of estradiol and progesterone, since they impact the

**8. Understanding all cellular components of advanced disease**

In ovarian cancer, metastasis through the vasculature is rare and a very late manifestation of the disease. Instead, ovarian cancer cells are prone to spread by direct extension from the ovaries to adjacent tissues, or to detach from the primary ovarian tumor directly into the peritoneal cavity where they seed the mesothelium of the omentum, diaphragm, bowel serosa, and the entire peritoneum [51-53]. Widespread visceral and intestinal wall metastases with formation of adhesions between the loops of the bowel cause intestinal obstruction, prevent

The high incidence (65%) of peritoneal malignant effusions in ovarian cancer patients at advanced presentation [54, 55], and the development of symptoms due to ascites accumulation at diagnosis as well as recurrence [56], suggest that the "liquid" component is an active pathogenic manifestation of the disease. Ovarian cancer cells isolated from peritoneal ascites of major ovarian cancer histological types were described as organized structures of different sizes and heterogeneous morphology [57]. Furthermore, multicellular structures isolated directly from ascites were shown capable of adhering ex vivo to components of the extracel‐ lular matrix and to monolayers of mesothelial cells, suggesting their participation in the dissemination of ovarian cancer [58]. Cancer cells isolated from ascites and metastatic secon‐ dary sites exhibit a higher percentage of stemness markers when compared to their primary tumors [59-61]. Additionally, cancer-associated proteins and mRNAs are differentially expressed in peritoneal effusions when compared to primary carcinomas or solid metastases. Lastly, there is a differential gene expression among peritoneal effusions when comparing samples at diagnosis (pre-chemotherapy) vs. samples at recurrence (post-chemotherapy) [62]. Altogether, these data suggest that cancer cells within effusions—the "liquid" component of ovarian cancer—represent a biomarker of tumor evolution toward a more aggressive/

Most of our understanding of the biology of ovarian cancer multicellular structures is based on the premise that mono-dispersed ovarian cancer cells, when gathering together either by

outcome of the behavioral tests suggested above.

156 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

normal nutrition, and become a primary cause of death [1].

advanced disease phenotype of poor prognosis.

While the presence of multicellular structures in ascites was reported over 25 years ago [57], their biology has been studied using in vitro platforms and multicellular structures that were forced to form from ovarian cancer cell lines by using either gravity or non-adherent condi‐ tions. We should investigate the pathogenic capacity of unforced, spontaneously arranged ovarian cancer multicellular structures in vivo. If a key mechanism for ovarian cancer pro‐ gression takes place within the "liquid" component of the disease, then multicellular structures may represent a druggable target. Developing therapeutic interventions to interrupt formation of multicellular structures free-floating in the peritoneal fluid may be an efficient way of interrupting disease advancement.

**Figure 2.** Proposed model for the role of ovarian cancer multicellular structures (MCS) in peritoneal carcinomatosis. Selected cells from microscopic nodules with distinctive capacity to form MCS, adapt, survive, and grow in the perito‐ neal fluid developing irregular and organized spheroidal MCS that might evade chemotherapy and/or preserve ovari‐ an cancer initiating cells (CIC), leading to a feed-forward, chemo-resistant, and self-renewal recurrent seeding. MCS committed to develop the solid component of the disease will adhere, disaggregate, migrate, and invade the mesothe‐ lial cell layer covering the surface of the peritoneum (maroon), and form foci that neo-vascularize and grow (green). Other MCS might develop non-invasive nodules that amplify the cellularity within the "liquid" compartment causing ascites. Blue: highly differentiated ovarian cancer cells. Pink: less differentiated ovarian cancer cells with self-renewal capacity. Red: new blood vessels. Gray: extracellular matrix. Yellow: fibroblasts.

#### **9. Understanding dormancy after "successful" standard of care (surgery and chemotherapy)**

Although most patients diagnosed with ovarian cancer undergo remission after optimal surgical cytoreduction and platinum-taxane chemotherapy, microscopic foci of cells manage to survive within the peritoneal cavity and recreate the illness. Recurrence develops a more aggressive phenotype for which current therapies almost always fail (rev.in [1-6]). Thus, understanding the biology of minimal residual disease is crucial in developing effective therapies for ovarian cancer.

**Figure 3.** Hypothetical model of ovarian cancer dormancy after debulking surgery and platinum–taxane (PT) therapy, relapse after chemotherapy-associated dormancy, and potential stages of the disease where therapeutic intervention is envisioned. DFS, disease free survival; OS, overall survival. MRD, minimal residual disease.

Within the minimal residual disease, ovarian cancer cells are in a unique, subclinical, biological stage termed dormancy. Long recognized in the clinic, *dormancy* describes a period of time that can last many years between primary therapy and recurrence of metastatic disease (rev.in [65]). In ovarian cancer, dormancy was reported to be represented by small, poorly vascularized fibrotic nodules located on the surface of the peritoneum in patients undergoing second-look surgery after front-line debulking operation and chemotherapy [66]. Dormant cancer cells are usually defined as survivors of primary therapy likely containing drug-resistant, tumorinitiating cells. They are kept either in a status of cell cycle arrest (quiescence) or equilibrium among proliferating and dying cells to preserve constant micro-tumor mass (rev.in [65]). Attempts to eliminate dormant ovarian cancer cells with maintenance therapies have not been efficient: they extend progression-free survival but not overall survival [67, 68].

It is important to investigate the magnitude and location of the disease still present following an objective response to front-line therapy, and characterize the adaptive molecular reprog‐ ramming after chemotherapy leading to the dormant status of the cells comprising the minimal residual disease (Fig.3). Chemotherapy-associated tumor dormancy and awakening from dormancy likely have defined molecular signatures that can be unveiled by combined use of currently available transcriptomic, proteomic, and epigenomic platforms that can be integrat‐ ed utilizing multipronged bioinformatic tools. Knowing the mechanism(s) ovarian cancer cells utilize to achieve dormancy in the peritoneum and awake from it will provide two potential avenues for intervention as follows: (1) perpetuation of the dormant status of the cancer; and/ or (2) interception of the awakening signal that causes disease relapse (Fig.3).

Whereas total elimination of ovarian cancer cells is the ideal goal, the alternative approach of keeping ovarian cancer in a chronic dormant state is highly relevant as this would categorize ovarian cancer patients with an objective response to front-line standard of care as having a chronic manageable disease or "cancer without disease."

#### **10. Conclusions**

**9. Understanding dormancy after "successful" standard of care (surgery**

Although most patients diagnosed with ovarian cancer undergo remission after optimal surgical cytoreduction and platinum-taxane chemotherapy, microscopic foci of cells manage to survive within the peritoneal cavity and recreate the illness. Recurrence develops a more aggressive phenotype for which current therapies almost always fail (rev.in [1-6]). Thus, understanding the biology of minimal residual disease is crucial in developing effective

**Figure 3.** Hypothetical model of ovarian cancer dormancy after debulking surgery and platinum–taxane (PT) therapy, relapse after chemotherapy-associated dormancy, and potential stages of the disease where therapeutic intervention is

Within the minimal residual disease, ovarian cancer cells are in a unique, subclinical, biological stage termed dormancy. Long recognized in the clinic, *dormancy* describes a period of time that can last many years between primary therapy and recurrence of metastatic disease (rev.in [65]). In ovarian cancer, dormancy was reported to be represented by small, poorly vascularized fibrotic nodules located on the surface of the peritoneum in patients undergoing second-look surgery after front-line debulking operation and chemotherapy [66]. Dormant cancer cells are usually defined as survivors of primary therapy likely containing drug-resistant, tumor-

envisioned. DFS, disease free survival; OS, overall survival. MRD, minimal residual disease.

**and chemotherapy)**

158 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

therapies for ovarian cancer.

Progresses made in the field of molecular oncology within the last decade have been remark‐ able. The use of RNA sequencing, micro RNA expression profiles, mutation analysis, shotgun proteomics, reverse-phase protein arrays, and epigenomic platforms, together with novel imaging tools, should be applied in uncovering the hidden secrets of ovarian cancer initiation and progression, and in developing early diagnostic tools. Understanding the location and molecular behavior of the abdominopelvic minimal residual disease after otherwise efficient front-line chemotherapy should lead to the discovery of new molecular targets for disease interception that can be exploited to prevent recurrence. We are at a point in time in which we have a unique opportunity to utilize the vast state-of-the-art technological armamentarium developed in the past decade to revisit the basic biology of peritoneal ovarian cancer and renew hopes for bettering the prognosis of this deadly disease.

#### **Acknowledgements**

This research was supported by award CA164622 from the National Cancer Institute, the National Institutes of Health (NIH), funds from the Division of Basic Biomedical Sciences of the Sanford School of Medicine of the University of South Dakota, and a donation from the SuFuDu Drumline. We thank Nahuel Telleria for proofreading the manuscript.

#### **Author details**

Alicia A. Goyeneche and Carlos M. Telleria\*

\*Address all correspondence to: carlos.telleria@usd.edu

Division of Basic Biomedical Sciences, Sanford School of Medicine, The University of South Dakota, Vermillion, South Dakota, USA

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## **Analysing Molecular Mechanism Related to Therapy-Resistance in** *In-vitro* **Models of Ovarian Cancer**

Jens C. Hahne, Arnd Honig, Jörg B. Engel, Andrea Lampis and Nicola Valeri

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60727

#### **Abstract**

Ovarian cancer is among the most common cause of cancer death and ranks first in the number of deaths each year in the field of gynaecological malignancies. This is due to its late diagnosis and the development of chemoresistance. Platinum derivates, including cisplatinum and carboplatin in combination with paclitaxel, are the first-line chemotherapeutic agents. Platinum derivates irreversibly intercalates into the DNA and creates inter- and intra-strand DNA cross-links. During cell division, platinum-DNA-adducts block the replication machinery, inducing DNA damage and apoptosis. Nearly all patients respond to first-line chemotherapy before it comes later to recurrence of the disease. At time of recurrence, tumours are usually more aggressive, form metastasis in secondary tissues and acquire resistance to conventional chemotherapeutics. Drug resistance is a common problem in tumour therapy not only restricted to ovarian cancer. It is characterized by gene mutations, increased DNA repair, reduced drug efficacy and enhanced drug clearance and detoxification. Up to now the complex molecular mechanism of chemoresistance is not well understood. Increasing evidence points towards AKT over-expression and alteration of the PI3K/AKT/mTOR cascade as a central mechanistic reason for this resistance.

**Keywords:** Ovarian cancer, cisplatinum resistance, AKT, PI3K

© 2016 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.

#### **1. Introduction**

There were 14,1 million new cancer cases, 8,2 million cancer deaths and 32,6 million people living with cancer (within 5 years of diagnosis) in 2012 world-wide [1].

Gynaecological tumours are among the most common cause of cancer death and currently causing more than 100,000 deaths per year [2]. Ovarian cancer is an important public health problem because it has the highest tumour-associated mortality of gynaecological malignan‐ cies and 239,000 women have been diagnosed with ovarian cancer in 2012 [2]. Furthermore there has been no appreciable improvement in survival for woman with advanced ovarian cancer over the past 40 years. The survival of ovarian cancer is poor and more than 70% of cases are diagnosed at late stage.

In ovarian cancer treatment platinum-based chemotherapy plays a pivotal role as first-line chemotherapy option alone or in combination with taxane [3]. Therefore platinum-resistance is the most crucial problem for treating ovarian cancer. Increasing evidence points towards AKT over-expression and alteration of the PI3K/AKT/mTOR cascade as a mechanistic reason for this resistance.

This chapter provides a short overview of the PI3K/AKT/mTOR-signalling network by summarizing *in-vitro* cell culture based studies that confirm the role of AKT as an important mediator of platinum resistance. The rationale for targeting this pathway in cancer will be discussed with a special focus on tumour immunological aspects also based on *in-vitro* studies. Moreover the PI3K/AKT/mTOR-signalling cascade other general mechanisms of resistance will be shortly addressed. Platinum-resistance can be also caused by differential expression of microRNAs as well as by detoxification of bioactive platinum-complexes by sulphur-contain‐ ing peptides or proteins, cellular compartmentation, increased DNA repair and alteration in apoptotic signalling pathways [4]. Furthermore diminished drug accumulation caused by reduced uptake or increased efflux of platinum compounds via heavy metal transporter can result in platinum therapy failure [4].

A better understanding of the molecular mechanisms causing cancer therapy-resistance might result in new therapeutic options for patients suffering from tumours.

#### **2. Phosphatidylinositol-3-Kinase (PI3K)/AKT/mTOR-signal transduction pathway**

One of the most frequently altered signalling pathways involved in cancer as well as in development of resistance especially in ovarian cancer is the PI3K/AKT/mTOR pathway.

PI3K is a member of the lipid-kinase-family that can phosphorylate the 3´-OH-group of inositolphospholipids as phosphatidylinositol-4,5-bisphosphate (PIP2) which is converted into the second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3) [5]. According to different protein structure of the catalytic subunit, PI3Ks are subdivided into three classes. Class I PI3Ks are the most studied class of PI3K and the most interesting with regard to signalling in tumours. Class I PI3Ks are activated by extracellular signal transduction via receptors with tyrosine-kinase activity or via G-protein coupled receptors (GPCR). In tumour cells growth-factors that bind to the specific receptors activate class 1 PI3Ks and this results in inhibition of autophagy [6].

**1. Introduction**

for this resistance.

**pathway**

cases are diagnosed at late stage.

result in platinum therapy failure [4].

There were 14,1 million new cancer cases, 8,2 million cancer deaths and 32,6 million people

Gynaecological tumours are among the most common cause of cancer death and currently causing more than 100,000 deaths per year [2]. Ovarian cancer is an important public health problem because it has the highest tumour-associated mortality of gynaecological malignan‐ cies and 239,000 women have been diagnosed with ovarian cancer in 2012 [2]. Furthermore there has been no appreciable improvement in survival for woman with advanced ovarian cancer over the past 40 years. The survival of ovarian cancer is poor and more than 70% of

In ovarian cancer treatment platinum-based chemotherapy plays a pivotal role as first-line chemotherapy option alone or in combination with taxane [3]. Therefore platinum-resistance is the most crucial problem for treating ovarian cancer. Increasing evidence points towards AKT over-expression and alteration of the PI3K/AKT/mTOR cascade as a mechanistic reason

This chapter provides a short overview of the PI3K/AKT/mTOR-signalling network by summarizing *in-vitro* cell culture based studies that confirm the role of AKT as an important mediator of platinum resistance. The rationale for targeting this pathway in cancer will be discussed with a special focus on tumour immunological aspects also based on *in-vitro* studies. Moreover the PI3K/AKT/mTOR-signalling cascade other general mechanisms of resistance will be shortly addressed. Platinum-resistance can be also caused by differential expression of microRNAs as well as by detoxification of bioactive platinum-complexes by sulphur-contain‐ ing peptides or proteins, cellular compartmentation, increased DNA repair and alteration in apoptotic signalling pathways [4]. Furthermore diminished drug accumulation caused by reduced uptake or increased efflux of platinum compounds via heavy metal transporter can

A better understanding of the molecular mechanisms causing cancer therapy-resistance might

**2. Phosphatidylinositol-3-Kinase (PI3K)/AKT/mTOR-signal transduction**

One of the most frequently altered signalling pathways involved in cancer as well as in development of resistance especially in ovarian cancer is the PI3K/AKT/mTOR pathway.

PI3K is a member of the lipid-kinase-family that can phosphorylate the 3´-OH-group of inositolphospholipids as phosphatidylinositol-4,5-bisphosphate (PIP2) which is converted into the second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3) [5]. According to different protein structure of the catalytic subunit, PI3Ks are subdivided into three classes.

result in new therapeutic options for patients suffering from tumours.

living with cancer (within 5 years of diagnosis) in 2012 world-wide [1].

168 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

PI3K activity is associated with cytoskeletal organization, cell division, inhibition of apoptosis and glucose uptake [7-9]. The second messenger PIP3 in turn activates in the PI3K/AKTpathway different proteins like AKT (protein kinase B), a serine-threonine kinase [5, 10]. PIP3 itself is reconverted in PIP2 via different phosphatases especially PTEN and SHIP [5]. AKT is the key protein in the PI3K/AKT signalling pathway; it binds PIP3 over the plekstrinhomology-domain (PH-domain) and by this AKT translocates to cell membrane where it interacts with various phospholipids [10]. Cell membrane bound AKT is phosphorylated by phosphoinositide-dependent kinase-1 (PDK1) at threonine 308 and by PDK2 at serine 473 [5, 10, 11]. AKT can also be activated by mTOR2 [5, 12]. Phosphorylated AKT is the active form that modulates and regulates a huge range of proteins involved in diverse cellular processes such as cell cycle regulation, cell proliferation and cell viability [13]. Phosphorylation of AKT can be blocked by the carboxy-terminal modulator-protein (CTMP) and by this preventing the AKT activation as well as further signal transduction [5]. Phosphorylated AKT activates another serine-threonine-kinase, the mammalian target of rapamycin (mTOR) an important regulator for translation, cell growth and cell cycle [14, 15]. Furthermore mTOR has an important role in regulation of autophagy [6, 16, 17].

In general the PI3K/AKT-signal transduction pathway is of pivotal importance for mediating and controlling several cellular processes including cell growth, cell proliferation, survival, motility, adhesion, migration, differentiation, metabolic processes and cell cycle progression in cells [18, 19]. Amplifications, mutations, translocations and deregulation result in aberrant activation of this pathway [5, 20-23]. Furthermore the loss-of-function caused by mutation or deletion of phosphatase and tensin homolog (PTEN) protein results in an increased activity of the PI3K/AKT pathway [6]. The PTEN protein acts as a phosphatase and dephosphorylate PIP3, resulting in the biphosphate product PIP2. The dephosphorylation is essential as it triggers the inhibition of the AKT signalling pathway [24, 25].

#### **3. Alteration of the PI3K/AKT/mTOR-signal transduction pathway in tumours**

Recent studies indicate that numerous components of the PI3K/AKT/mTOR-pathway are deregulated by amplification, mutation and translocation more frequently than any other pathway in cancer patients with resultant activation of this pathway [20].

Both genetic and biochemical data suggest that activation of the PI3K/AKT/mTOR survival pathway contributes to ovarian cancer development and tumourigenesis [15]. Such activation is caused by different mechanisms and one mechanism is somatic alterations in PI3KCA gene that have been found in a substantial fraction of ovarian cancers [26]. PIK3CA amplifications are present in 40% of ovarian cancers [19]. Furthermore, activation of PI3K/AKT/mTOR signal transduction pathway is caused by mutations in the gene coding for PIK3CA. Another alteration that results in increased activity of the PI3K/AKT/mTOR pathway is PTEN loss-offunction. PTEN loss is observed in about 7% of all ovarian cancer cases and it seems to be more common in type I ovarian tumours [27-32].

For AKT a point-mutation in the PH-domain has been detected in ovarian cancer [33]. This point-mutation results in conformational change of the PH-domain so that AKT can be activated without the presence of PI3K [33].

Deregulation, mutation or over-expression of cell surface receptors can also result in an increased activity of the PI3K/AKT/mTOR signalling pathway in ovarian cancer [34]. Further‐ more Ras mutations are found in 20% of low-grade ovarian cancers [35]. Since Ras has been shown to activate both the Ras/Raf/MEK/ERK and the PI3K/AKT/mTOR pathways, mutations of Ras should theoretically activate both pathways simultaneously. Nevertheless so far it has not been evaluated in detail if Ras mutations can result in an increased activity of the PI3K/AKT/mTOR-signalling pathway. Although one study demonstrates that some Ras mutations result in deregulated PI3K and downstream AKT activation [36]. Beside Ras mutations also the over-expression of several other proteins e.g. Rab25 [37], Twist2 [38] or MyD88 [39] seems to enhance activation of AKT. The fact that AKT can be activated by a number of different proteins underlines the key role of AKT signalling under physiological and pathophysiological conditions. As evidence, in human specimens of ovarian cancer AKT was found to be activated in 68% of cases [40].

### **4. Effects of altered PI3K/AKT/mTOR-signal transduction pathway in tumours**

As mentioned before, AKT is an important regulator of various cellular pathways that promote cell survival, cell proliferation, angiogenesis and invasion. Furthermore, the epithelialmesenchymal-transformation, an important step for tumour metastasis, has been shown to be related to AKT activation [41]. Deregulation of components of the PI3K/AKT-cascade not only contributes to ovarian cancer development and tumourigenesis but also to chemotherapeutic drug and radiation resistance as it was recently shown [4, 5, 18, 42-56]. The sensitivity of cells to radiation and chemotherapeutic drug-induced apoptosis is determined by the balance between cellular survival and apoptosis [5, 12]. Due to the well-known anti-apoptotic role of AKT, an AKT over-expression in cancer cells might be related to increased resistance to radiation and chemotherapy.

Beside the PI3K/AKT/mTOR signalling cascade other general mechanisms of resistance exist. However in this chapter other possibilities of platinum-resistance will be mentioned only shortly.

In general diminished drug accumulation caused by reduced uptake or increased efflux of platinum compounds via heavy metal transporter can result in platinum therapy failure [4]. Furthermore in some resistant cell lines with increased cisplatinum efflux an increased intracellular pH was detected [57]. Intra-cellularly, cisplatinum´s chlorides are replaced by neutral hydroxyl or highly reactive positively charged aqua groups, with the pKa for the interconversion between chloro-hydroxy and chloro-aqua species being 6.56 [58]. Hence, if intracellular pH is high, a higher proportion of drug may be represented in the uncharged chloro-hydroxy form, with increased passive efflux of this form.

are present in 40% of ovarian cancers [19]. Furthermore, activation of PI3K/AKT/mTOR signal transduction pathway is caused by mutations in the gene coding for PIK3CA. Another alteration that results in increased activity of the PI3K/AKT/mTOR pathway is PTEN loss-offunction. PTEN loss is observed in about 7% of all ovarian cancer cases and it seems to be more

For AKT a point-mutation in the PH-domain has been detected in ovarian cancer [33]. This point-mutation results in conformational change of the PH-domain so that AKT can be

Deregulation, mutation or over-expression of cell surface receptors can also result in an increased activity of the PI3K/AKT/mTOR signalling pathway in ovarian cancer [34]. Further‐ more Ras mutations are found in 20% of low-grade ovarian cancers [35]. Since Ras has been shown to activate both the Ras/Raf/MEK/ERK and the PI3K/AKT/mTOR pathways, mutations of Ras should theoretically activate both pathways simultaneously. Nevertheless so far it has not been evaluated in detail if Ras mutations can result in an increased activity of the PI3K/AKT/mTOR-signalling pathway. Although one study demonstrates that some Ras mutations result in deregulated PI3K and downstream AKT activation [36]. Beside Ras mutations also the over-expression of several other proteins e.g. Rab25 [37], Twist2 [38] or MyD88 [39] seems to enhance activation of AKT. The fact that AKT can be activated by a number of different proteins underlines the key role of AKT signalling under physiological and pathophysiological conditions. As evidence, in human specimens of ovarian cancer AKT

**4. Effects of altered PI3K/AKT/mTOR-signal transduction pathway in**

As mentioned before, AKT is an important regulator of various cellular pathways that promote cell survival, cell proliferation, angiogenesis and invasion. Furthermore, the epithelialmesenchymal-transformation, an important step for tumour metastasis, has been shown to be related to AKT activation [41]. Deregulation of components of the PI3K/AKT-cascade not only contributes to ovarian cancer development and tumourigenesis but also to chemotherapeutic drug and radiation resistance as it was recently shown [4, 5, 18, 42-56]. The sensitivity of cells to radiation and chemotherapeutic drug-induced apoptosis is determined by the balance between cellular survival and apoptosis [5, 12]. Due to the well-known anti-apoptotic role of AKT, an AKT over-expression in cancer cells might be related to increased resistance to

Beside the PI3K/AKT/mTOR signalling cascade other general mechanisms of resistance exist. However in this chapter other possibilities of platinum-resistance will be mentioned only

In general diminished drug accumulation caused by reduced uptake or increased efflux of platinum compounds via heavy metal transporter can result in platinum therapy failure [4].

common in type I ovarian tumours [27-32].

170 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

activated without the presence of PI3K [33].

was found to be activated in 68% of cases [40].

**tumours**

shortly.

radiation and chemotherapy.

Another general resistance mechanism is detoxification of bioactive platinum-complexes by sulphur-containing peptides or proteins. Increased glutathione (GSH) level has been shown to cause resistance by binding and inactivating cisplatinum, enhancing DNA repair and reducing cisplatinum-induced oxidative stress [59-62].

Increased DNA repair and reduced apoptotic response are further possible reasons for platinum resistance [4, 63]. Cisplatinum may induce apoptosis through the Fas/Fas ligand signalling complex (with activation of caspase-8, then caspase-3), or by mitochondrial cyto‐ chrome-c release [64]. In the presence of ATP and cytochrome-c, apoptotic-protease-activatingfactor-1 (Apaf-1) activates caspase-9, with subsequent caspase-3 activation [64]. Cisplatinum may also kill via a caspase-3 independent apoptotic pathway, by a defective apoptotic pathway or by necrosis [64]. Caspase-3, -8 and -9 are important in cisplatinum-induced apoptosis [62]. A cisplatinum-resistant cell showed global down-regulation of caspase and Bax expression, but increased Bcl-2 [65].

Recent reports describe that platinum-resistance can be also caused by differential expression of microRNAs (miRNAs) [66-69]. miRNAs belong to the family of small non-coding RNAs; they are generally 21-25 nucleotides long and play key role in post-transcriptional modulation of gene expression thus representing fine regulators in tumour development and progression as well as response and resistance to anti-tumour agents [70]. miRNA-152 was identified as an autophagy-regulating miRNA down-regulated in cisplatinum-resistant cell lines and also *invivo* in ovarian cancer tissues reduced expression has been associated with cisplatinumresistance. miRNA-152 regulates autophagy by targeting ATG14 the key player in orchestrating autophagy. Thus over-expression of miRNA-152 sensitized cisplatinumresistant ovarian cancer cells by reducing cisplatinum-induced autophagy, enhancing cispla‐ tinum-induced apoptosis and by inhibition of cell proliferation [69]. Microarray analyses have been used to identify miRNAs involved in cisplatinum-resistance and it was demonstrated that miRNA-21-3p over-expression, the passenger strand of the known oncomiR 5p, increased resistance to cisplatinum in a range of ovarian cell lines [66]. Furthermore a high level of miRNA-490-3p expression was identified as involved in the development of drug resistance against paclitaxel [68]. Another miRNA, miRNA-449a, was found to be down-regulated in cisplatinum-resistant ovarian cancer cells and NOTCH1 was identified as direct target of its modulation [67]. Therefore it is evident that down-regulation as well as over-expression of miRNAs can result in resistance to anti-tumour agents. Recently it was demonstrated that miRNAs involved in platinum-resistance are directly involved in regulation of PTEN, AKT or other downstream molecules of the PI3K/AKT pathway [71-79].

The evidence that members of the PI3K/AKT/mTOR pathway are regulated by miRNAs involved in platinum-resistance increases the importance of the PI3K/AKT/mTOR signalling cascade as therapeutic target. Therefore inhibition of PI3K/AKT/mTOR signalling in ovarian carcinomas appears a promising target to enhance the efficacy of anticancer agents such as cisplatinum and to overcome the resistance of tumour cells against therapy. This hypothesis was tested in different preclinical *in-vitro* studies. Cancer cell lines are frequently used as *invitro* tumour models especially for analyzing and studying the effects related to a single gene modification. Nowadays approximately 100 ovarian cancer cell lines are publicly available [80]. Some of these cell lines are known to be platinum resistant e.g. SKOV-3/DDP and Caov-3. Among different ovarian cancer cell lines established there are also the parental A2780 cells and the cisplatinum-resistant A2780cis cells [81]. Both cell lines are p53 and K-Ras wild-type and they share the same genetic background. The cisplatinum-resistant A2780cis cell line has been developed by chronic exposure of the parental cisplatinum-sensitive A2780 cell line to increasing concentrations of the chemotherapeutic agent [81]. These cell lines are excellent models for analyzing the molecular basis for cisplatinum resistance in ovarian cancer [47-49, 82-85]. According to these studies AKT over-expression in ovarian cancer is strongly related to platinum resistance in this specific tumour [37, 47, 86]. It was shown that high AKT protein expression is strongly associated to cisplatinum-resistant A2780cis cell line compared to the parental A2780 cell lines [47, 48]. The platinum resistance in A2780cis cell line could be overcome by AKT down-regulation via siRNA [47]. This was demonstrated in several functional *in-vitro* assays, e.g. clonogenicity assays and irradiation assays (Figure 1), as by determination of the apoptosis rate. Furthermore the cytotoxicity of cisplatinum was ad‐ dressed in proliferation assays. Stable increase of AKT amount in the cell lines results in an increased IC50 value for cisplatinum whereas a stable decrease of AKT results in an increased accessibility for cisplatinum treatment [47].

However in the same isogenic model it was shown that AKT-over-expression was able to transform platinum-sensitive A2780 cells into platinum-resistant. On the contrary, platinumresistance of A2780cis cells could be reversed by down-regulation of AKT [47]. FACS analysis demonstrated also that cisplatinum induces cell cycle arrest predominantly in the S and the G2/M phase but also in the G1 phase regardless of the AKT-expression status (Figure 2). However, required doses of cisplatinum to induce cell cycle arrest were progressively higher in cell lines with AKT over-expressed [47, 87].

As already mentioned above the sensitivity of cells to radiation and drug-induced apoptosis is determined by the balanced expression between pro-apoptotic and anti-apoptotic proteins [5, 12]. Therefore the effect of the PI3K/AKT cascade on pro-apoptotic protein like BAD, a known substrate of AKT, has been studied in both cisplatinum-resistant Caov-3 and sensitive A2780 human ovarian cancer cells [88]. Treatment of Caov-3 and A2780 cells with cisplatinum was able to stimulate the activation of AKT, whereas the PI3K inhibitor wortmannin blocked the cisplatinum-induced AKT activation. Cisplatinum treatment was capable to activate phosphorylation of BAD at Ser-112 and Ser-136 sites in Caov-3 and A2780 cells. While phosphorylation of BAD at Ser-136 was blocked by treatment with wortmannin, its phos‐ phorylation at Ser-112 was blocked by a MAP/ERK kinase inhibitor PD98059 [89]. Transient exogenous expression of a dominant-negative AKT in both Caov-3 and A2780 cells decreased cell viability after treatment with cisplatinum. In contrast, no sensitization to cisplatinum was

cascade as therapeutic target. Therefore inhibition of PI3K/AKT/mTOR signalling in ovarian carcinomas appears a promising target to enhance the efficacy of anticancer agents such as cisplatinum and to overcome the resistance of tumour cells against therapy. This hypothesis was tested in different preclinical *in-vitro* studies. Cancer cell lines are frequently used as *invitro* tumour models especially for analyzing and studying the effects related to a single gene modification. Nowadays approximately 100 ovarian cancer cell lines are publicly available [80]. Some of these cell lines are known to be platinum resistant e.g. SKOV-3/DDP and Caov-3. Among different ovarian cancer cell lines established there are also the parental A2780 cells and the cisplatinum-resistant A2780cis cells [81]. Both cell lines are p53 and K-Ras wild-type and they share the same genetic background. The cisplatinum-resistant A2780cis cell line has been developed by chronic exposure of the parental cisplatinum-sensitive A2780 cell line to increasing concentrations of the chemotherapeutic agent [81]. These cell lines are excellent models for analyzing the molecular basis for cisplatinum resistance in ovarian cancer [47-49, 82-85]. According to these studies AKT over-expression in ovarian cancer is strongly related to platinum resistance in this specific tumour [37, 47, 86]. It was shown that high AKT protein expression is strongly associated to cisplatinum-resistant A2780cis cell line compared to the parental A2780 cell lines [47, 48]. The platinum resistance in A2780cis cell line could be overcome by AKT down-regulation via siRNA [47]. This was demonstrated in several functional *in-vitro* assays, e.g. clonogenicity assays and irradiation assays (Figure 1), as by determination of the apoptosis rate. Furthermore the cytotoxicity of cisplatinum was ad‐ dressed in proliferation assays. Stable increase of AKT amount in the cell lines results in an increased IC50 value for cisplatinum whereas a stable decrease of AKT results in an increased

However in the same isogenic model it was shown that AKT-over-expression was able to transform platinum-sensitive A2780 cells into platinum-resistant. On the contrary, platinumresistance of A2780cis cells could be reversed by down-regulation of AKT [47]. FACS analysis demonstrated also that cisplatinum induces cell cycle arrest predominantly in the S and the G2/M phase but also in the G1 phase regardless of the AKT-expression status (Figure 2). However, required doses of cisplatinum to induce cell cycle arrest were progressively higher

As already mentioned above the sensitivity of cells to radiation and drug-induced apoptosis is determined by the balanced expression between pro-apoptotic and anti-apoptotic proteins [5, 12]. Therefore the effect of the PI3K/AKT cascade on pro-apoptotic protein like BAD, a known substrate of AKT, has been studied in both cisplatinum-resistant Caov-3 and sensitive A2780 human ovarian cancer cells [88]. Treatment of Caov-3 and A2780 cells with cisplatinum was able to stimulate the activation of AKT, whereas the PI3K inhibitor wortmannin blocked the cisplatinum-induced AKT activation. Cisplatinum treatment was capable to activate phosphorylation of BAD at Ser-112 and Ser-136 sites in Caov-3 and A2780 cells. While phosphorylation of BAD at Ser-136 was blocked by treatment with wortmannin, its phos‐ phorylation at Ser-112 was blocked by a MAP/ERK kinase inhibitor PD98059 [89]. Transient exogenous expression of a dominant-negative AKT in both Caov-3 and A2780 cells decreased cell viability after treatment with cisplatinum. In contrast, no sensitization to cisplatinum was

accessibility for cisplatinum treatment [47].

172 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

in cell lines with AKT over-expressed [47, 87].

**Figure 1.** Clonogenicity-Assays. (A) Cells were treated with different concentrations of cisplatin (cis-Pt) for 6 days and (B) cells were first irradiated with 2.5 Gray and then treated with 3.5 nM cisplatin (cis-Pt) for 11 days. Cells were stained and fixed with crystal violet. The formed cell colonies were counted. The figure shows the colony numbers in relation to the colonies formed by untreated A2780 (set to 100%). Three independent experiments were performed, and each experiment was carried out in triplicate. Statistically significant difference (p<0.05) between a sample and the rele‐ vant control is indicated by \*. All data were previously published in "Oncology Reports" [47] and is reprinted by per‐ mission of Spandidos Publications ©2012.

observed in cells expressing wild-type AKT. These findings suggested that cisplatinuminduced DNA damage causes phosphorylation of BAD via an extracellular signal-regulated protein kinase (ERK) cascade and via a PI3K/AKT/mTOR cascade. Inhibition of both cascades enhance the sensitivity of ovarian cancer cells to cisplatinum thus providing further evidence that AKT-pathway is involved in cisplatinum resistance in ovarian cancers [88]. Additional

**Figure 2.** Effect of cisplatin (cis-Pt) on the cell cycle distribution. A2780 (A), A2780cis (B), A2780cis AKT+ (C) and A2780cis AKT- (D) cells were treated with different concentrations of cisplatin (cis-Pt) for 24 h, fixed, permeabilzed, stained with propidium iodide and analysed by flow cytometry. The figure shows the distribution of the cells to the different phases of cell cycle (%). All data were previously published in "Oncology Reports" [47] and is reprinted by permission of Spandidos Publications ©2012.

results suggest that AKT confers platinum-resistance in part by modulating the direction of p53 on the caspase-dependent mitochondrial death pathway [90]. Thus in ovarian cancers while p53 is a determinant for platinum sensitivity AKT contributes to chemoresistance in part by attenuating p53-mediated PUMA upregulation and phosphorylation of p53 [91]. Recent results suggest that in platinum sensitive ovarian cancer cells cisplatinum-induced apoptosis can also proceed to a lesser extent via a caspase-independent mechanism involving apoptosis inducing factor (AIF) and that AKT activation additionally confers resistance to cisplatinuminduced apoptosis by blocking this pathway [90].

A recent work evaluated the anti-tumour efficacy of the AKT inhibitor perifosine in platinumsensitive and –resistant human ovarian cancer cells [45, 92]. In different ovarian cancer cell lines and *in-vivo* experiments it has been possible to show that cells with higher levels of phospho-AKT are more sensitive to treatment with AKT-inhibitor perifosine. Furthermore, coincubation with perifosine sensitized A2780cis cells to treatment with cisplatinum. AKTinhibitor perifosine has already been tested in phase II studies in patients with breast, prostate, pancreatic, head and neck, colorectal cancer, malignant melanoma, multiple myeloma, and soft tissue sarcoma [93-99]. A recent phase I study with perifosine combined with radiotherapy performed in patients with advanced solid tumours has shown preliminary evidence of anticancer activity, including complete responses [100]. Thus, perifosine seems to be an attractive compound for further clinical studies in particular phenotypes tumour like platinum-resistant ovarian cancers.

New attractive therapeutic targets are presented by the PI3K/AKT/mTOR-pathway activating cell surface receptors like vascular endothelial growth factor (VEGF) receptors [101]. VEGF is a key activator of angiogenesis, a physiological multi-step process that includes endothelial cell growth and movement [102]. It plays important roles in wound healing and endothelialcell-mediated degradation of the extracellular matrix, as well as the transition of benign tissue into solid tumours [102-104]. Recent studies have suggested that the PI3K/AKT signalling cascade may be implicated in tumour angiogenesis [105-107]. In clinical trial studies, high VEGF levels have been negatively correlated with survival of patients. Ovarian cancer cells over-expressing VEGF own a metastatic advantage over those VEGF low expressing [108, 109] and even more higher levels of serum VEGF are found in patients with metastasis if compared to metastasis-free patients [110]. Moreover, down-modulation of VEGF has been shown to inhibit tumour growth and to suppress tumour invasion and metastasis. These findings have laid the basis for the clinical evaluation of agents targeting VEGF signaling pathway in patients with ovarian cancer [101]. Bevacizumab (Avastin) has been the first and most studied anti-VEGF agent in clinical evaluation for ovarian cancer [111]. Bevacizumab showed additive or synergistic effects in combinational therapy with paclitaxel and marked reduction of tumour growth and ascites formation [112]. Using a murine ovarian cancer model a significant antitumour activity of Bevacizumab as a single agent or in combination with cisplatinum was demonstrated [113]. In the meantime Bevacizumab was approved as a treatment in combination with paclitaxel, topotecan, or pegylated liposomal doxorubicin chemotherapy for women with recurrent ovarian cancer that are resistant to platinum-based chemotherapy [114-117].

Furthermore, other agents targeting VEGF receptors have also been evaluated for the use in treatment of ovarian cancer as Ramucirumab, a fully humanized monoclonal antibody, that specifically block VEGFR-2 resulting in reduced tumour growth, increased apoptosis and decreased tumour microvessel proliferation and density [118]. Following a phase I evaluation [119], it is currently being assessed in a phase II trial as monotherapy in patients with platinumrefractory persistent or recurrent epithelial ovarian cancer.

results suggest that AKT confers platinum-resistance in part by modulating the direction of p53 on the caspase-dependent mitochondrial death pathway [90]. Thus in ovarian cancers while p53 is a determinant for platinum sensitivity AKT contributes to chemoresistance in part by attenuating p53-mediated PUMA upregulation and phosphorylation of p53 [91]. Recent results suggest that in platinum sensitive ovarian cancer cells cisplatinum-induced apoptosis can also proceed to a lesser extent via a caspase-independent mechanism involving apoptosis inducing factor (AIF) and that AKT activation additionally confers resistance to cisplatinum-

**Figure 2.** Effect of cisplatin (cis-Pt) on the cell cycle distribution. A2780 (A), A2780cis (B), A2780cis AKT+ (C) and A2780cis AKT- (D) cells were treated with different concentrations of cisplatin (cis-Pt) for 24 h, fixed, permeabilzed, stained with propidium iodide and analysed by flow cytometry. The figure shows the distribution of the cells to the different phases of cell cycle (%). All data were previously published in "Oncology Reports" [47] and is reprinted by

A recent work evaluated the anti-tumour efficacy of the AKT inhibitor perifosine in platinumsensitive and –resistant human ovarian cancer cells [45, 92]. In different ovarian cancer cell lines and *in-vivo* experiments it has been possible to show that cells with higher levels of phospho-AKT are more sensitive to treatment with AKT-inhibitor perifosine. Furthermore, coincubation with perifosine sensitized A2780cis cells to treatment with cisplatinum. AKTinhibitor perifosine has already been tested in phase II studies in patients with breast, prostate, pancreatic, head and neck, colorectal cancer, malignant melanoma, multiple myeloma, and soft tissue sarcoma [93-99]. A recent phase I study with perifosine combined with radiotherapy performed in patients with advanced solid tumours has shown preliminary evidence of anticancer activity, including complete responses [100]. Thus, perifosine seems to be an attractive compound for further clinical studies in particular phenotypes tumour like platinum-resistant

induced apoptosis by blocking this pathway [90].

174 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

permission of Spandidos Publications ©2012.

ovarian cancers.

#### **5. Role of AKT expression level in tumour cells in regard to NK killing**

Another important aspect in cancer development and progression is the role of the immune system. Since survival is strongly influenced by immunological parameters, immunothera‐ peutic strategies appear promising and for this reason during the last years the interest in tumour immunology has constantly increased. A necessary prerequisite for immunotherapy in patients is a better understanding of the interaction between ovarian tumour cells and cells of the immune system especially with natural-killer (NK)-cells. NK-cells are a critical compo‐ nent of the innate immune response against infectious pathogens and malignant transforma‐ tion [120, 121]. NK-cells mediate this activity through the elaboration of various cytokines as well as through direct cytolytic activity. However, unlike adaptive immune cells, which utilize specific clonal recognition receptors, NK-cell activation depends on a complex balance between activating and inhibitory signals [122, 123]. Nevertheless, NK-cells play an important role in immune surveillance and coordinating responses of other immune cells. Most tumour cells express surface molecules that can be recognized by activating receptors on NK-cells [124]. The expression of these receptors make such cells susceptible to endogenous NK-cells, but malignant cells have developed mechanisms to evade these mechanisms of innate immune surveillance [125-127]. In patients with cancer, it is presumed that tumour cells have developed mechanisms to suppress NK-cell activation and resist lysis by endogenous NK-cells, but the molecular basis for target resistance is not well understood. Recent studies have suggested that AKT can regulate the development and functions of innate immune cells [128] thus providing evidence that AKT plays also an important role in immune modulation. However in this chapter will be addressed only the role of activated AKT in tumour cells in regard to NK-cells.

Dysregulated cytokine release can either lead to or be associated with a failure in cell-cell recognition thus allowing cancer cells to evade the killing system. The PI3K/AKT/mTOR pathway regulates multiple cellular processes which underlie immune responses against pathogens or malignant cells [129, 130]. Conversely, there is accumulating evidence that the PI3K/AKT/mTOR pathway is involved in the development of several malignant traits of cancer cells as well as their escape from immunity [131]. In some studies the interactions between cancer cells and natural-killer (NK)-cells have been enlightened [48, 82, 132-134]. Modified FATAL assay was used for determining the killing efficiency of NK-cells in regard to ovarian cancer cell models *in-vitro* (Figure 3).

**Figure 3.** Lytic activity of polyclonal natural-killer (NK)-cells. A2780 and A2780cis cells (105 cells/well), respectively, were used as targets in a modified 5 h FATAL assay using various tumour cell : NK-cell ratios. Target cell lysis was determined by flow cytometric analysis. The percentage of tumour cell lysis was determined in relation to a control containing tumour cells with medium alone. A representative of three independent experiments is shown. All data were previously published in "International Journal of Oncology" [48] and is reprinted by permission of Spandidos Publications ©2013.

In this model parental A2780 cells and the cisplatinum resistant A2780cis human ovarian cancer cells have been used. The efficiency of NK-cell mediated cell lysis differs between A2780 cells and the cisplatinum-resistant A2780cis cells. The A2780cis cells are less accessible for NKcell mediated killing [48, 82] and this findings are in agreement with a report by Bellucci *et al.* [135]. Using a lentiviral shRNA library targeting >1,000 human genes they identified 83 genes that promote target cell resistance to human NK-cell-mediated killing [135]. Many of the genes discovered by this screening belong to common signalling pathways including multiple members of the AKT/PI3K/mTOR pathway as PIK3CA and PIK3CB [135]. The comparison of the cancer cell lines A2780 and A2780cis revealed that the differences observed with regard to NK-cell mediated killing rely mainly on two mechanisms. Firstly, the observed increased expression of anti-apoptotic genes (especially *ciap-1* and *-2*) in A2780cis cells compared to A2780 cells is able to confer resistance to A2780cis cells to apoptosis. Second, the CD112 ligand for NK-cell receptor DNAM-1 was expressed at a lower level in A2780cis cells though ligands for the NK-cell receptor NKG2D, e.g. MICA/B, were more strongly expressed in the platinum-resistant cells than in the parental A2780 cells [48]. Moreover A2780cis cells expressed lower levels of TIMP-3, the inhibitor of MICA/B shedding, whereas specific proteases for shedding were also found expressed and this resulted in a net increase of soluble MICA/B in A2780cis cell lines [48]. It is well known that cleaved MICA/B protects cells against NK mediated cell killing [48, 136, 137]. Therefore, it is reasonable to speculate that the increased amount of soluble MICA/B is responsible for the lower killing rate of platinum-resistant A2780cis cells compared to their parental A2780 cells [48]. It was previously well demonstrated that PI3K/AKT/mTOR pathway is involved in inducing MICA/B expression in breast cancer cells [138]. Overall these findings indicate a more general effect of induced PI3K/AKT/mTOR signal transduction pathway. As well as in breast, in ovarian cancer cells with an increase of phosphorylated AKT-activated, PI3K/AKT/mTOR pathway higher MICA/B expression has been also detected [48]. Recently it has been demonstrated that treatment of tumour cells with JAK inhibitors increased their susceptibility to NK-cell mediated killing [135]. The authors suggested that common signalling pathways can regulate susceptibility of human tumour cells to the surveillance and killing ability of the immunologic effector cells and that small molecules inhibitors of JAK may have promising immunologic effects *in-vivo* [135]. Whether or not inhibition of PI3K/AKT/mTOR pathway might render the platinum-resistant A2780cis cells accessible for NK-cell mediated killing must be evaluated in further studies. Only the few first steps towards the characterization of the molecular basis of resistance mechanisms in ovarian cancer with different AKT expression levels in the context of NK-cell mediated killing are being explored [48, 82].

#### **Author details**

role in immune surveillance and coordinating responses of other immune cells. Most tumour cells express surface molecules that can be recognized by activating receptors on NK-cells [124]. The expression of these receptors make such cells susceptible to endogenous NK-cells, but malignant cells have developed mechanisms to evade these mechanisms of innate immune surveillance [125-127]. In patients with cancer, it is presumed that tumour cells have developed mechanisms to suppress NK-cell activation and resist lysis by endogenous NK-cells, but the molecular basis for target resistance is not well understood. Recent studies have suggested that AKT can regulate the development and functions of innate immune cells [128] thus providing evidence that AKT plays also an important role in immune modulation. However in this chapter will be addressed only the role of activated AKT in tumour cells in regard to

176 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

Dysregulated cytokine release can either lead to or be associated with a failure in cell-cell recognition thus allowing cancer cells to evade the killing system. The PI3K/AKT/mTOR pathway regulates multiple cellular processes which underlie immune responses against pathogens or malignant cells [129, 130]. Conversely, there is accumulating evidence that the PI3K/AKT/mTOR pathway is involved in the development of several malignant traits of cancer cells as well as their escape from immunity [131]. In some studies the interactions between cancer cells and natural-killer (NK)-cells have been enlightened [48, 82, 132-134]. Modified FATAL assay was used for determining the killing efficiency of NK-cells in regard to ovarian

**Figure 3.** Lytic activity of polyclonal natural-killer (NK)-cells. A2780 and A2780cis cells (105 cells/well), respectively, were used as targets in a modified 5 h FATAL assay using various tumour cell : NK-cell ratios. Target cell lysis was determined by flow cytometric analysis. The percentage of tumour cell lysis was determined in relation to a control containing tumour cells with medium alone. A representative of three independent experiments is shown. All data were previously published in "International Journal of Oncology" [48] and is reprinted by permission of Spandidos

In this model parental A2780 cells and the cisplatinum resistant A2780cis human ovarian cancer cells have been used. The efficiency of NK-cell mediated cell lysis differs between A2780 cells and the cisplatinum-resistant A2780cis cells. The A2780cis cells are less accessible for NKcell mediated killing [48, 82] and this findings are in agreement with a report by Bellucci *et al.* [135]. Using a lentiviral shRNA library targeting >1,000 human genes they identified 83

NK-cells.

Publications ©2013.

cancer cell models *in-vitro* (Figure 3).

Jens C. Hahne1\*, Arnd Honig2 , Jörg B. Engel3 , Andrea Lampis1 and Nicola Valeri1

\*Address all correspondence to: jens.hahne@icr.ac.uk


#### **References**


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## **Recurrent Ovarian Cancer — Basic Knowledge, Current Management, and Future Directions**

Bernardo L. Rapoport

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Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60609

#### **Abstract**

Recurrent ovarian cancer is incurable. Chemotherapy is indicated to control disease-relat‐ ed symptoms. The benefit from chemotherapy in these patients depends on the platinumfree interval. Patients with platinum-resistant disease (a relapse of less than six months from the completion of platinum treatment) are managed with non-platinum agents. Pa‐ tients with platinum semi-sensitive relapse (six to 12 months from the completion of treatment) have a response rate of 30% to second-line platinum treatment. In patients with platinum-sensitive relapse (more than 12 months from the completion of treatment), the response rate to platinum is 60–70%. Limited data is available regarding the benefits of secondary cytoreductive surgery. GOG 213 and the AGO Desktop III studies will de‐ fine the role of this procedure in patients with recurrent disease. Two studies have shown benefit of bevacizumab in the treatment of patients with platinum-sensitive (Oceans) and refractory disease (Aurelia). Additional studies are needed to establish the optimal dura‐ tion and timing of treatment. Cediranib has shown activity in patients with recurrent platinum-sensitive ovarian cancer (ICON 6 trial). Numerous novel biological agents are being investigated in relapsed ovarian cancer. This chapter focuses on current manage‐ ment and future directions in patients with relapsed ovarian cancer.

**Keywords:** Ovarian Cancer, relapsed, platinum sensitive, targeted therapy

#### **1. Introduction**

The vast majority of patients with advanced ovarian cancer will recur after first-line chemo‐ therapy. [1] A common sign of relapse is a rise in the serum CA-125 level in the absence of symptoms (defined as marker-only relapse) or objective evidence of disease as assessed by physical or radiological examinations. Recurrent disease is not curable, and the majority of patients with recurrent disease will succumb to their disease irrespective of the second-line

© 2016 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.

treatment modality used. As there is no compelling evidence that early treatment with chemotherapy is beneficial in relapsed asymptomatic disease, patients with marker-onlyrelapse are often observed. The MRC OV05/EORTC 55955 did not show a survival benefit with early treatment of relapse on the basis of a raised CA125 concentration only. [2] Some patients with asymptomatic disease with tumour-marker elevation only may develop symptomatic disease within months, while others may take years. [2] Assessment of the rate of progression by CA 125 is essential and useful for most patients with advanced ovarian cancer in remission. CA 125 is elevated in most patients with documented progressive disease. Serial measurement is a useful marker to assess the response to chemotherapy according to GCIG criteria. [3] Clinicians should keep in mind that CA 125 is not specific for ovarian cancer. Raised CA 125 levels may also be found in non-gynaecological malignancies (breast cancer and lung cancer, as well as colon and pancreatic malignancies). An elevated CA 125 can be found in patients with benign conditions such as endometriosis, pelvic inflammatory disease, and ovarian cysts.

**Figure 1.** Progressive disease — shows multiple complex cystic/solid peritoneal deposits on the liver's surface (visceral peritoneum) and elsewhere in the peritoneal cavity.

#### **2. Chemotherapy**

The role of chemotherapy in the management of recurrent ovarian cancer is palliative and is usually indicated for ovarian cancer-related symptoms, or for patients with objective evidence of significant disease progression on physical or radiological examination.

The likelihood of benefit from chemotherapy treatment in patients with relapsed ovarian cancer depends on the platinum-free interval (PFI). PFI is defined as the interval between the last dose of platinum and the time of relapse. Patients with platinum-resistant disease (PFI of less than six months) are unlikely to respond to second-line platinum agents and are often managed with an alternative agent (see Table 1). Patients with platinum semi-sensitive disease (PFI of between six and 12 months) have a response rate of approximately 30% to second-line platinum treatment. In patients with fully platinum-sensitive disease (PFI more than 12 months with a subset greater than 24 months), the response rate to second-line platinum may be as high as 60–70%. [4]

The role of combination chemotherapy has been assessed in randomized trials in the setting of platinum semi-sensitive and fully sensitive relapses. Patients with fully sensitive disease should be re-challenged with a platinum-based (cisplatin or carboplatin) chemotherapy regime. [5] Patients with platinum semi-sensitive disease should be treated with a platinumbased doublet combination. It has been demonstrated that retreatment results in valuable responses that translate into improvement in quality of life and survival. Patients with a PFI of greater than six months usually receive treatment with a platinum-based regimen either as a single-agent or in combination with agents like paclitaxel [6], gemcitabine [7], or pegylated liposomal doxorubicin (PLD) [8]


**Table 1.** Platinum refractory.

treatment modality used. As there is no compelling evidence that early treatment with chemotherapy is beneficial in relapsed asymptomatic disease, patients with marker-onlyrelapse are often observed. The MRC OV05/EORTC 55955 did not show a survival benefit with early treatment of relapse on the basis of a raised CA125 concentration only. [2] Some patients with asymptomatic disease with tumour-marker elevation only may develop symptomatic disease within months, while others may take years. [2] Assessment of the rate of progression by CA 125 is essential and useful for most patients with advanced ovarian cancer in remission. CA 125 is elevated in most patients with documented progressive disease. Serial measurement is a useful marker to assess the response to chemotherapy according to GCIG criteria. [3] Clinicians should keep in mind that CA 125 is not specific for ovarian cancer. Raised CA 125 levels may also be found in non-gynaecological malignancies (breast cancer and lung cancer, as well as colon and pancreatic malignancies). An elevated CA 125 can be found in patients with benign conditions such as endometriosis, pelvic inflammatory disease, and ovarian cysts.

190 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

**Figure 1.** Progressive disease — shows multiple complex cystic/solid peritoneal deposits on the liver's surface (visceral

The role of chemotherapy in the management of recurrent ovarian cancer is palliative and is usually indicated for ovarian cancer-related symptoms, or for patients with objective evidence

The likelihood of benefit from chemotherapy treatment in patients with relapsed ovarian cancer depends on the platinum-free interval (PFI). PFI is defined as the interval between the

of significant disease progression on physical or radiological examination.

peritoneum) and elsewhere in the peritoneal cavity.

**2. Chemotherapy**

The International Collaborative Ovarian Neoplasm-4 (ICON-4) trial compared combination chemotherapy with paclitaxel and platinum to single-agent platinum in patients with plati‐ num-sensitive disease. In this study, most patients had a PFI of 12 months or greater. ICON-4 showed a statistically significant improvement in overall survival (OS) in favour of combina‐ tion chemotherapy for recurrent ovarian cancer, with a 7% absolute increase at two years (P = 0.023). Although the ICON-4 trial showed a positive outcome, the results remain controversial because of methodological limitations. Around, 40% of patients randomized to the platinum single-agent arm never received a taxane during the course of their disease, including firstline therapy and at disease progression, raising the possibility that the sequential use of platinum followed by paclitaxel at disease progression might have conferred the same survival advantage. [6]

Docetaxel has been investigated in the treatment of metastatic ovarian cancer, in both the frontline and relapsed setting. In the front-line setting, docetaxel was shown to be equivalent to paclitaxel. [9] Docetaxel may also be a useful choice for patients at risk of developing peripheral neuropathy. [9–11]

In a subsequent study, the AGO (Arbeitsgemeinschaft Gynaekologische Onkologie) from Germany conducted a randomized Phase III trial in patients with platinum-sensitive relapse to either gemcitabine and carboplatin, or carboplatin alone. [7] PFS was 8.6 months for the combination versus 5.8 months for single-agent carboplatin (P =.0038), with no improvement in OS. Quality of life was similar between the two arms, despite a higher incidence of throm‐ bocytopoenia, neutropoenia, and anaemia with the combination.

Rapoport et al. investigated pegylated liposomal doxorubicin (PLD) in combination with carboplatin in a Phase II trial in patients with relapsed ovarian cancer with semi-sensitive and fully sensitive relapse. Results were encouraging, with a complete response rate of 35% and a partial response rate of 32.5% (overall response, 67.5%). Median time to progression was 11.9 months, and median survival was 30.0 months. Overall responses were higher in the platinum fully sensitive subgroup as opposed to the semi-sensitive group. [12] The GCIG (Gynecologic Cancer Intergroup) conducted a Phase III study (CALYPSO trial) comparing paclitaxel and carboplatin with PLD and carboplatin in patients presenting with platinum-sensitive relapse. There was a significant improvement in median progression-free survival (PFS) (11.3 months vs. 9.4 months; P =.005), with a lower incidence of severe hypersensitivity reactions (5% vs. 18%), in favour of the PLD-containing arm. No difference in OS was noted. Toxicities were similar to the toxicities reported in the Phase II study by Rapoport et al. and included grade 2 or greater alopecia (83.6% vs. 7%) and sensory neuropathy (26.9% vs. 4.9%) in the paclitaxelcontaining arm, and with more hand-foot syndrome (grades 2 to 3, 12.0% vs. 2.2%), nausea (35.2% vs. 24.2%), and mucositis (grades 2 to 3, 13.9% vs. 7%) in the PLD containing arm. [8]

As recurrent ovarian cancer is incurable, palliation and symptom control is the goal of secondline treatment. Choosing the most appropriate agent for use in the recurrent disease setting, therefore, involves balancing the need to attain a response to treatment against maintenance of reasonable quality of life. The decision to use platinum-based chemotherapy combinations or single-agent platinum in this setting should be based on a number of factors. These factors include patient age, disease burden, rate of relapse, and patient preference. For elderly patients who require chemotherapy for mild symptomatic and low tumour burden, platinum-sensitive relapse, the usage of single-agent carboplatin is a reasonable approach. PLD is a well-tolerated alternative in patients that develop an allergy to carboplatin during the course of treatment or if further use of carboplatin is contraindicated. Both agents are associated with a good quality of life as well as acceptable toxicity profiles in terms of alopecia or severe myelosuppression. A more aggressive approach is needed for younger patients with rapidly growing cancer and platinum-sensitive relapse. Combination chemotherapy with either paclitaxel and carboplatin, docetaxel and carboplatin [13], gemcitabine and carboplatin [7], or PLD and carboplatin are reasonable [8].

Patients with platinum/taxane-resistant disease (defined by a short PFI of less than six months, or progression during platinum-based chemotherapy) are best treated with agents who lack cross-resistance to platinum compounds or are not susceptible to the common resistance mechanisms.

Potentially non-cross-resistant drugs with activity in the platinum-resistant setting include PLD [14], paclitaxel [15–16], docetaxel [17], topotecan [18], gemcitabine [19], pemetrexed [20], ixabepilone [21], or oral etoposide [22] (Table 1). In the platinum-resistant setting, the overall response rate to any of these agents is approximately 20%. Responses are short, with a median PFS of four to six months. These responses are progressively shorter with each subsequent regimen.

In the setting of platinum-resistant relapse, PLD is well tolerated at doses of 40 mg/m2 given every four weeks. Common toxicities include palmer-plantar erythrodysesthesia (hand-foot syndrome) and mucositis. Topotecan may cause significant myelosuppression and fatigue. A recently reported Phase II randomized multicenter study however showed that weekly topotecan has a favourable toxicity profile compared to the conventional 5-day schedule of topotecan with similar OS. [23]

The Cochrane Gynaecological Cancer Group conducted a meta-analysis of 14 randomized trials evaluating the usefulness of PLD in relapsed epithelial ovarian cancer. Results of this meta-analysis concluded that in patients with platinum-sensitive disease, PLD and carboplatin is more effective than paclitaxel and carboplatin, and is better tolerated. Therefore, PLD and carboplatin should be considered as the first option of treatment in women with platinumsensitive relapse. PLD alone is also a useful agent for platinum-resistant disease. It remains unclear, however, how it compares with other single agents in this subgroup, and in which order these agents should be used. There is no data available to support the use of PLD in combination with other agents in patients with platinum-resistant relapse. [24]

As a general rule, combination chemotherapy regimens are not superior to single agents in the management of patients with platinum-resistant relapse. Combination regimens are also more toxic and should not be used in this palliative setting.

#### **3. The role of surgery in the treatment of relapsed ovarian cancer**

#### **3.1. Secondary cytoreductive surgery**

Docetaxel has been investigated in the treatment of metastatic ovarian cancer, in both the frontline and relapsed setting. In the front-line setting, docetaxel was shown to be equivalent to paclitaxel. [9] Docetaxel may also be a useful choice for patients at risk of developing peripheral

In a subsequent study, the AGO (Arbeitsgemeinschaft Gynaekologische Onkologie) from Germany conducted a randomized Phase III trial in patients with platinum-sensitive relapse to either gemcitabine and carboplatin, or carboplatin alone. [7] PFS was 8.6 months for the combination versus 5.8 months for single-agent carboplatin (P =.0038), with no improvement in OS. Quality of life was similar between the two arms, despite a higher incidence of throm‐

Rapoport et al. investigated pegylated liposomal doxorubicin (PLD) in combination with carboplatin in a Phase II trial in patients with relapsed ovarian cancer with semi-sensitive and fully sensitive relapse. Results were encouraging, with a complete response rate of 35% and a partial response rate of 32.5% (overall response, 67.5%). Median time to progression was 11.9 months, and median survival was 30.0 months. Overall responses were higher in the platinum fully sensitive subgroup as opposed to the semi-sensitive group. [12] The GCIG (Gynecologic Cancer Intergroup) conducted a Phase III study (CALYPSO trial) comparing paclitaxel and carboplatin with PLD and carboplatin in patients presenting with platinum-sensitive relapse. There was a significant improvement in median progression-free survival (PFS) (11.3 months vs. 9.4 months; P =.005), with a lower incidence of severe hypersensitivity reactions (5% vs. 18%), in favour of the PLD-containing arm. No difference in OS was noted. Toxicities were similar to the toxicities reported in the Phase II study by Rapoport et al. and included grade 2 or greater alopecia (83.6% vs. 7%) and sensory neuropathy (26.9% vs. 4.9%) in the paclitaxelcontaining arm, and with more hand-foot syndrome (grades 2 to 3, 12.0% vs. 2.2%), nausea (35.2% vs. 24.2%), and mucositis (grades 2 to 3, 13.9% vs. 7%) in the PLD containing arm. [8] As recurrent ovarian cancer is incurable, palliation and symptom control is the goal of secondline treatment. Choosing the most appropriate agent for use in the recurrent disease setting, therefore, involves balancing the need to attain a response to treatment against maintenance of reasonable quality of life. The decision to use platinum-based chemotherapy combinations or single-agent platinum in this setting should be based on a number of factors. These factors include patient age, disease burden, rate of relapse, and patient preference. For elderly patients who require chemotherapy for mild symptomatic and low tumour burden, platinum-sensitive relapse, the usage of single-agent carboplatin is a reasonable approach. PLD is a well-tolerated alternative in patients that develop an allergy to carboplatin during the course of treatment or if further use of carboplatin is contraindicated. Both agents are associated with a good quality of life as well as acceptable toxicity profiles in terms of alopecia or severe myelosuppression. A more aggressive approach is needed for younger patients with rapidly growing cancer and platinum-sensitive relapse. Combination chemotherapy with either paclitaxel and carboplatin, docetaxel and carboplatin [13], gemcitabine and carboplatin [7], or PLD and carboplatin are

Patients with platinum/taxane-resistant disease (defined by a short PFI of less than six months, or progression during platinum-based chemotherapy) are best treated with agents who lack cross-resistance to platinum compounds or are not susceptible to the common resistance

bocytopoenia, neutropoenia, and anaemia with the combination.

192 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

neuropathy. [9–11]

reasonable [8].

mechanisms.

Surgery for the debulking of disease at the time of relapse, referred to as secondary cytore‐ ductive surgery, is performed in selected patients prior to second-line chemotherapy. [25]

Due to a lack of large randomized trials, conclusive data are limited regarding the benefits of secondary cytoreductive surgery. The ability to complete a successful secondary cytoreduction may identify patients presenting with a biologically less aggressive disease or those patients who have a lower tumour burden at the time of relapse. A prospective randomized trial of secondary cytoreduction is required to determine whether this procedure improves survival in these patients. The value of secondary cytoreduction is currently being investigated in two prospective, randomized trials, GOG 213 and the AGO Desktop III study.

The Gynaecologic Oncology group (GOG) currently defines 'optimal' cytoreductive surgery as having residual tumours having a maximum diameter of 1 cm or less. Complete cytore‐ duction is the ideal surgical outcome in the form of microscopic disease. [26]

and the AGO Desktop III study.

form of microscopic disease. [26]

Secondary cytoreduction might be considered for the subgroup of patients with a progressionfree interval of more than 12 to 18 months from the time of completion of adjuvant chemo‐ therapy, localized recurrence amenable to complete cytoreduction, potentially chemosensitive disease, and good performance status. [27–31] completion of adjuvant chemotherapy, localized recurrence amenable to complete cytoreduction, potentially chemosensitive disease, and good performance status. [27–31]

Secondary cytoreduction might be considered for the subgroup of patients with a progression-free interval of more than 12 to 18 months from the time of

The Gynaecologic Oncology group (GOG) currently defines 'optimal' cytoreductive surgery as having residual tumours having a maximum diameter of 1 cm or less. Complete cytoreduction is the ideal surgical outcome in the

Due to a lack of large randomized trials, conclusive data are limited regarding the benefits of secondary cytoreductive surgery. The ability to complete a successful secondary cytoreduction may identify patients presenting with a biologically less aggressive disease or those patients who have a lower tumour burden at the time of relapse. A prospective randomized trial of secondary cytoreduction is required to determine whether this procedure improves survival in these patients. The value of secondary cytoreduction is currently being investigated in two prospective, randomized trials, GOG 213

On the other hand, a patient with a rapid, multifocal recurrence is unlikely to obtain any clinical benefit from surgery. [26] On the other hand, a patient with a rapid, multifocal recurrence is unlikely to obtain any clinical benefit from surgery. [26]

**Figure 2.** Pleural and peritoneal recurrence.

#### Picture 2. Pleural and peritoneal recurrence. **3.2. Palliative surgery**

3.2. Palliative Surgery Palliative surgery may be indicated in patients with recurrent ovarian cancer. Standard operations performed in this setting include colostomy for relief of a large bowel obstruction, treatment of peritoneal adhesions, and management of small bowel obstruction. [32] Surgery to relieve small bowel obstruction should take into account the likelihood of continued responsiveness to chemotherapy postoperatively (platinum-sensitive as opposed to platinumrefractory disease). Women who develop a small bowel obstruction during Palliative surgery may be indicated in patients with recurrent ovarian cancer. Standard operations performed in this setting include colostomy for relief of a large bowel obstruction, treatment of peritoneal adhesions, and management of small bowel obstruction. [32] Surgery to relieve small bowel obstruction should take into account the likelihood of continued responsiveness to chemotherapy postoperatively (platinum-sensitive as opposed to platinumrefractory disease). Women who develop a small bowel obstruction during first-line chemo‐ therapy have aggressive and resistant ovarian cancer, and the procedure is not beneficial in this subset of patients. A palliative gastrostomy tube may be most appropriate in this situation. Best outcomes are seen in women who have had prolonged PFI, usually lasting more than one year.

Surgery is not indicated in the management of patients with a pseudo-obstruction due to an intra-abdominal carcinomatosis and infiltration of the myoenteric plexus of the small bowel. Pharmacological treatment with metoclopramide (an agent that improves motility of the upper gastrointestinal tract without stimulating gastric, biliary, or pancreatic secretions) may be helpful to treat this complication. A palliative colostomy may be indicated for patients developing a large bowel obstruction. This type of surgery can provide significant prolonga‐ tion of survival and improved quality of life in selected patients.

#### **3.3. Radiation therapy**

Secondary cytoreduction might be considered for the subgroup of patients with a progressionfree interval of more than 12 to 18 months from the time of completion of adjuvant chemo‐ therapy, localized recurrence amenable to complete cytoreduction, potentially chemosensitive

Secondary cytoreduction might be considered for the subgroup of patients with a progression-free interval of more than 12 to 18 months from the time of completion of adjuvant chemotherapy, localized recurrence amenable to complete cytoreduction, potentially chemosensitive disease, and good

The Gynaecologic Oncology group (GOG) currently defines 'optimal' cytoreductive surgery as having residual tumours having a maximum diameter of 1 cm or less. Complete cytoreduction is the ideal surgical outcome in the

Due to a lack of large randomized trials, conclusive data are limited regarding the benefits of secondary cytoreductive surgery. The ability to complete a successful secondary cytoreduction may identify patients presenting with a biologically less aggressive disease or those patients who have a lower tumour burden at the time of relapse. A prospective randomized trial of secondary cytoreduction is required to determine whether this procedure improves survival in these patients. The value of secondary cytoreduction is currently being investigated in two prospective, randomized trials, GOG 213

On the other hand, a patient with a rapid, multifocal recurrence is unlikely to obtain any clinical

On the other hand, a patient with a rapid, multifocal recurrence is unlikely to

Palliative surgery may be indicated in patients with recurrent ovarian cancer. Standard operations performed in this setting include colostomy for relief of a large bowel obstruction, treatment of peritoneal adhesions, and management of small bowel obstruction. [32] Surgery to relieve small bowel obstruction should take into account the likelihood of continued responsiveness to chemotherapy postoperatively (platinum-sensitive as opposed to platinumrefractory disease). Women who develop a small bowel obstruction during

Surgery is not indicated in the management of patients with a pseudo-obstruction due to an intra-abdominal carcinomatosis and infiltration of the myoenteric plexus of the small bowel. Pharmacological treatment with metoclopramide (an agent that improves motility of the upper gastrointestinal tract without stimulating gastric, biliary, or pancreatic secretions) may be helpful to treat this complication. A palliative colostomy may be indicated for patients developing a large bowel obstruction. This type of surgery can provide significant prolonga‐

Palliative surgery may be indicated in patients with recurrent ovarian cancer. Standard operations performed in this setting include colostomy for relief of a large bowel obstruction, treatment of peritoneal adhesions, and management of small bowel obstruction. [32] Surgery to relieve small bowel obstruction should take into account the likelihood of continued responsiveness to chemotherapy postoperatively (platinum-sensitive as opposed to platinumrefractory disease). Women who develop a small bowel obstruction during first-line chemo‐ therapy have aggressive and resistant ovarian cancer, and the procedure is not beneficial in this subset of patients. A palliative gastrostomy tube may be most appropriate in this situation. Best outcomes are seen in women who have had prolonged PFI, usually lasting more than one

disease, and good performance status. [27–31]

obtain any clinical benefit from surgery. [26]

194 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

Picture 2. Pleural and peritoneal recurrence.

tion of survival and improved quality of life in selected patients.

and the AGO Desktop III study.

form of microscopic disease. [26]

benefit from surgery. [26]

3.2. Palliative Surgery

year.

**3.2. Palliative surgery**

**Figure 2.** Pleural and peritoneal recurrence.

performance status. [27–31]

Radiation therapy may be useful in the palliation of patients with recurrent ovarian cancer. Symptomatic pelvic masses may cause bleeding, pain, and rectal narrowing. Palliative pelvic radiotherapy can offer symptom relief. Cerebral or bone metastases are unusual complications of ovarian cancer and can be successfully palliated with radiotherapy.

#### **4. Investigational Agents**

Several investigational agents are being studied in the relapse setting.

#### **4.1. Bevacizumab**

#### *4.1.1. Bevacizumab single-agent activity*

Bevacizumab is a humanized antibody that recognizes and neutralizes vascular endothelial growth factor (VEGF). VEGF is a pro-angiogenic factor that is secreted by ovarian cancer cells. Randomized data in other metastatic malignant diseases have shown a survival advantage for the use of bevacizumab in combination with chemotherapy [33–35].

Single-agent bevacizumab has been shown by the GOG to induce a response rate of 18% in patients with relapsed ovarian cancer. In this GOG trial, there were two complete and 11 partial responses, with a median response duration of 10 months, and 25 patients (40%) survived progression-free for at least six months. Median PFS was 4.7 months and OS was 17 months. This study did not show a significant association with prior platinum sensitivity, age, number of prior chemotherapeutic regimens, or performance status. [36]

Cannistra et al. reported a risk of life-threatening bowel perforation in patients with ovarian cancer treated with bevacizumab. All patients in this trial were heavily pre-treated, with 50% having received three prior regimens. Partial responses were observed in seven patients (15.9%). Median PFS was 4.4 months (95% CI, 3.1 to 5.5 months), with a median survival duration of 10.7 months at study termination. Bevacizumab-associated grades 3 to 4 events included hypertension (9.1%), proteinuria (15.9%), bleeding (2.3%), and wound-healing complications (2.3%). The incidence of bowel perforation was 11.4%. This was higher than reported in bevacizumab trials of other tumour types. Risk factors for bevacizumab-induced bowel perforation included a higher number of prior chemotherapy regimens, radiographic presence of bowel wall involvement by tumour, or evidence of bowel obstruction. [37]

#### *4.1.2. Bevacizumab in combination with chemotherapy in newly diagnosed ovarian cancer patients*

Two randomized studies (GOG-0218 and ICON7) have shown improvement in the PFS in patients with advanced ovarian cancer treated with chemotherapy and bevacizumab. [38–39] In these trials, bevacizumab was evaluated in combination with standard paclitaxel plus carboplatin as part of initial treatment for women with ovarian cancer. Both these trials met their primary endpoints and demonstrated an improvement in PFS.

In the GOG-0218 trial, 1,873 women with newly diagnosed stage III (incompletely resectable) or stage IV epithelial ovarian cancer which had undergone debulking surgery were random‐ ized to receive one of three treatments in a double-blind, placebo-controlled trial. Each of the three study regimens comprised 22 3-week cycles of intravenous infusions on day one, with the first six cycles consisting of standard chemotherapy with carboplatin and paclitaxel. [38]

Arm 1 — Control: chemotherapy with a placebo added in cycles 2 to 22.

Arm 2 — Chemotherapy with bevacizumab (15mg per kilogram of body weight) added in cycles 2 to 6, and a placebo from cycles 7 to 22.

Arm 3 — Chemotherapy with bevacizumab added in cycles 2 to 22.

At a median follow-up of 17.4 months, the median PFS was 10.3, 11.2, and 14.1 months in the control group, the bevacizumab-initiation group, and the bevacizumab-throughout group, respectively. No significant difference in OS was reported. The potential to detect a difference in survival is likely to be limited by lack of control for multiple subsequent regimens, including crossover to bevacizumab or other anti-VEGF agents. [38]

Although bevacizumab use resulted in additional toxicity, it was not associated with a decrease in quality of life. Grade 2 or greater hypertension was significantly more frequent with use of bevacizumab than a placebo. [38]

The second trial, ICON 7 was led by the U.K. Medical Research Council Clinical Trials Unit. This trial enrolled 1528 women with histologically confirmed, high-risk, early-stage disease (FIGO stage I or IIA and clear-cell or grade 3 tumours), or advanced (FIGO stage IIB to IV) epithelial ovarian cancer, primary peritoneal cancer, or fallopian-tube cancer. Patients were randomized to receive carboplatin and paclitaxel given every three weeks for six cycles, or to the same regimen plus bevacizumab (7.5mg per kilogram) given concurrently every three weeks for five or six cycles and continued for 12 additional cycles or until disease progression. Complete or partial response rates were reported in 67% of patients in the bevacizumab group and 48% in the control group (p = 0.001). With a median follow-up of 19.4 months, the data provide clear evidence of the biologic activity of bevacizumab with a median PFS of 19 months compared to 17.4 months in the standard therapy group (HR 0.81, CI, 0.70 to 0.94; p = 004). Final survival data are expected soon. Bevacizumab treatment did not affect the delivery of chemotherapy; it was, however, associated with a significant increase in side effects, including grade 2 or greater hypertension and bowel perforation. [39]

#### *4.1.3. Bevacizumab in combination with chemotherapy in patients with recurrent platinum-sensitive disease*

The OCEANS (Carboplatin and Gemcitabine plus Bevacizumab in Patients with Ovary, Peritoneal, or Fallopian Tube Carcinoma) study showed a benefit for the addition of bevaci‐ zumab to platinum-based chemotherapy in terms of PFS and a trend towards a benefit in OS.

Updated data by Aghajanian et al. indicate a median PFS advantage of four months (12.4 vs. 8.4 months) (hazard ratio 0.484) and overall response rate by RECIST of 21% (response rate 78.5% vs. 57.4%) were seen when bevacizumab was added to carboplatin and gemcitabine chemotherapy. Nevertheless, no benefit in OS was seen, but the data is still immature. [40]


VEGFR: Vascular endothelial growth factor receptor

PDGFR: platelet-derived growth factor receptor

FGFR: fibroblast growth factor receptor

In the GOG-0218 trial, 1,873 women with newly diagnosed stage III (incompletely resectable) or stage IV epithelial ovarian cancer which had undergone debulking surgery were random‐ ized to receive one of three treatments in a double-blind, placebo-controlled trial. Each of the three study regimens comprised 22 3-week cycles of intravenous infusions on day one, with the first six cycles consisting of standard chemotherapy with carboplatin and paclitaxel. [38]

Arm 2 — Chemotherapy with bevacizumab (15mg per kilogram of body weight) added in

At a median follow-up of 17.4 months, the median PFS was 10.3, 11.2, and 14.1 months in the control group, the bevacizumab-initiation group, and the bevacizumab-throughout group, respectively. No significant difference in OS was reported. The potential to detect a difference in survival is likely to be limited by lack of control for multiple subsequent regimens, including

Although bevacizumab use resulted in additional toxicity, it was not associated with a decrease in quality of life. Grade 2 or greater hypertension was significantly more frequent with use of

The second trial, ICON 7 was led by the U.K. Medical Research Council Clinical Trials Unit. This trial enrolled 1528 women with histologically confirmed, high-risk, early-stage disease (FIGO stage I or IIA and clear-cell or grade 3 tumours), or advanced (FIGO stage IIB to IV) epithelial ovarian cancer, primary peritoneal cancer, or fallopian-tube cancer. Patients were randomized to receive carboplatin and paclitaxel given every three weeks for six cycles, or to the same regimen plus bevacizumab (7.5mg per kilogram) given concurrently every three weeks for five or six cycles and continued for 12 additional cycles or until disease progression. Complete or partial response rates were reported in 67% of patients in the bevacizumab group and 48% in the control group (p = 0.001). With a median follow-up of 19.4 months, the data provide clear evidence of the biologic activity of bevacizumab with a median PFS of 19 months compared to 17.4 months in the standard therapy group (HR 0.81, CI, 0.70 to 0.94; p = 004). Final survival data are expected soon. Bevacizumab treatment did not affect the delivery of chemotherapy; it was, however, associated with a significant increase in side effects, including

*4.1.3. Bevacizumab in combination with chemotherapy in patients with recurrent platinum-sensitive*

The OCEANS (Carboplatin and Gemcitabine plus Bevacizumab in Patients with Ovary, Peritoneal, or Fallopian Tube Carcinoma) study showed a benefit for the addition of bevaci‐ zumab to platinum-based chemotherapy in terms of PFS and a trend towards a benefit in OS. Updated data by Aghajanian et al. indicate a median PFS advantage of four months (12.4 vs. 8.4 months) (hazard ratio 0.484) and overall response rate by RECIST of 21% (response rate 78.5% vs. 57.4%) were seen when bevacizumab was added to carboplatin and gemcitabine chemotherapy. Nevertheless, no benefit in OS was seen, but the data is still immature. [40]

Arm 1 — Control: chemotherapy with a placebo added in cycles 2 to 22.

Arm 3 — Chemotherapy with bevacizumab added in cycles 2 to 22.

crossover to bevacizumab or other anti-VEGF agents. [38]

grade 2 or greater hypertension and bowel perforation. [39]

cycles 2 to 6, and a placebo from cycles 7 to 22.

196 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

bevacizumab than a placebo. [38]

*disease*

PARP: poly-adenosine diphosphate [ADP]-ribose polymerase

TIE-2 receptor: receptor tyrosine kinase expressed predominantly on endothelial cells

c-Kit: trans-membrane receptor tyrosine kinase KIT, which is defined by the CD117 antigen

**Table 2.** Antiangiogenesis agents under investigation for the treatment of relapsed ovarian cancer.

#### *4.1.4. Bevacizumab in combination with chemotherapy in patients with platinum-resistant disease*

The AURELIA randomized Phase III study showed that addition of bevacizumab to standard chemotherapy with either PLD, topotecan, or weekly paclitaxel was associated with an improvement in PFS of 3.3 months and overall response rate by RECIST of 18%. OS and quality of life data from this study are still immature. [41]

Bevacizumab is an active agent in advanced and recurrent ovarian cancer. Large clinical trials are needed to improve the knowledge of the safety and effectiveness of bevacizumab, the duration and timing of treatment, and activity of this agent when given in combination with other chemotherapeutic agents. There is also an urgent need to identify biologic predictive factors of efficacy. When to start and end anti-angiogenesis therapy remain controversial questions, and further evaluation of personalized novel angiogenesis-based therapy is needed.

#### **4.2. Cediranib**

Cediranib (AZD2171) is a highly potent, small-molecule, oral tyrosine kinase inhibitor of VEGFR-1, -2, and -3, and c-Kit, which compete for the ATP-binding site within the receptor kinase domain. [42–43] It is postulated that cediranib is useful in the prevention of tumour progression, by inhibiting VEGFR-2 activity and angiogenesis, and also by concomitantly inhibiting VEGFR-3 activity and lymphangiogenesis.

Cediranib has been shown to be an active drug in recurrent ovarian cancer, fallopian tube, and peritoneal cancer with the predictable toxicities observed with other tyrosine kinase inhibitors. In a Phase II trial, partial responses were seen in eight of the 46 treated patients (17.4%). [44] The original dose was 45 mg/d, but the dose was lowered to 30 mg because of toxicity observed in the first 11 patients. Major grade 3 toxicities included hypertension (46%), fatigue (24%), and diarrhoea (13%). Grade 4 toxicities included central nervous system haemorrhage (n = 1), hypertriglyceridaemia/hypercholesterolaemia/elevated lipase (n = 1), and dehydration/ elevated creatinine (n = 1). No GI perforations or fistulas occurred. [44]

Initial results of the international three-arm Phase III randomized trial (ICON 6) showed that the addition of cediranib to chemotherapy (carboplatin and paclitaxel) increased PFS by about three months in women with recurrent platinum-sensitive ovarian cancer. Additional benefit was obtained when cediranib was used as maintenance therapy, increasing overall PFS over chemotherapy alone. The time to disease progression increased from 9.4 to 12.6 months and OS was extended from 17.6 to 20.3 months over a follow-up period of two years. [45, 46]

#### **5. Other investigational agents**

Other anti-angiogenic agents have also been evaluated in recurrent ovarian cancer.

Aflibercept is a potent inhibitor of both VEGF and placental growth factor. Aflibercept has shown anti-tumour activity in combination with docetaxel and is useful as a single agent in the reduction of malignant ascites. The combination was tested in a Phase I/II study in patients with measurable, recurrent, or persistent epithelial ovarian cancer. The confirmed objective response rate was 54% (25 of 46 patients responded to treatment, with 11 patients achieving a complete response and 14 a partial response). [47]

AMG 386 (trebananib) is a peptide-Fc fusion protein that inhibits angiogenesis by neutralizing the interaction between the Tie2 receptor and angiopoietin 1 and 2. Targeting of the angio‐ poietins/Tie2 pathway as a strategy to overcome bevacizumab resistance and toxicities has gained increasing interest in recent years. A randomized study of 161 patients with recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer evaluated weekly treatment with paclitaxel plus intravenous AMG 386 at a dose of 10mg/kg week (arm A), weekly paclitaxel plus AMG 386 at a dose of 10mg/kg week (arm B), or weekly paclitaxel plus placebo (arm C). Median PFS was 7.2 months in arm A, 5.7 months in arm B, and 4.6 months in arm C. The study has shown promising anti-cancer activity with a manageable safety profile when combined with weekly paclitaxel and a dose-response effect. [48] A recent publication of 919 enrolled patients, of whom 461 were randomly assigned to the trebananib group and 458 to the placebo group, was associated with a significantly longer median progression-free survival in favour of trebananib compared to placebo (7 2 months vs. 5 4 months; hazard ratio 0.66, p<0 0001). There was no significant increase in the incidence of grade 3 or higher adverse events between treatment groups (244 [54%] of 452 patients) for the placebo group compared to 258 [56%] of 461 patients) in the trebananib group. In this Phase III study, inhibition of angiopoie‐ tins 1 and 2 with trebananib resulted in a significant prolongation in progression-free survival. The results of the ongoing TRINOVA-1, -2, and -3 trials will define the role of trebananib in the management of patients with advanced epithelial ovarian cancer. [49]

BIBF-1120 is a triple angiokinase inhibitor of VEGFR, PDGFR, and FGFR. This agent has shown promising activity in a randomized Phase II placebo-controlled trial in relapsed ovarian cancer in the maintenance setting. The study showed a 36 week PFS of 16.3% vs. 5.0% in favour of BIBF 1120 compared to the placebo group with a hazard ratio of 0.65 (95% CI, 0.42 to 1.02; P = 0.06). [50]

#### **5.1. Pazopanib**

*4.1.4. Bevacizumab in combination with chemotherapy in patients with platinum-resistant disease*

of life data from this study are still immature. [41]

198 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

inhibiting VEGFR-3 activity and lymphangiogenesis.

**5. Other investigational agents**

elevated creatinine (n = 1). No GI perforations or fistulas occurred. [44]

**4.2. Cediranib**

The AURELIA randomized Phase III study showed that addition of bevacizumab to standard chemotherapy with either PLD, topotecan, or weekly paclitaxel was associated with an improvement in PFS of 3.3 months and overall response rate by RECIST of 18%. OS and quality

Bevacizumab is an active agent in advanced and recurrent ovarian cancer. Large clinical trials are needed to improve the knowledge of the safety and effectiveness of bevacizumab, the duration and timing of treatment, and activity of this agent when given in combination with other chemotherapeutic agents. There is also an urgent need to identify biologic predictive factors of efficacy. When to start and end anti-angiogenesis therapy remain controversial questions, and further evaluation of personalized novel angiogenesis-based therapy is needed.

Cediranib (AZD2171) is a highly potent, small-molecule, oral tyrosine kinase inhibitor of VEGFR-1, -2, and -3, and c-Kit, which compete for the ATP-binding site within the receptor kinase domain. [42–43] It is postulated that cediranib is useful in the prevention of tumour progression, by inhibiting VEGFR-2 activity and angiogenesis, and also by concomitantly

Cediranib has been shown to be an active drug in recurrent ovarian cancer, fallopian tube, and peritoneal cancer with the predictable toxicities observed with other tyrosine kinase inhibitors. In a Phase II trial, partial responses were seen in eight of the 46 treated patients (17.4%). [44] The original dose was 45 mg/d, but the dose was lowered to 30 mg because of toxicity observed in the first 11 patients. Major grade 3 toxicities included hypertension (46%), fatigue (24%), and diarrhoea (13%). Grade 4 toxicities included central nervous system haemorrhage (n = 1), hypertriglyceridaemia/hypercholesterolaemia/elevated lipase (n = 1), and dehydration/

Initial results of the international three-arm Phase III randomized trial (ICON 6) showed that the addition of cediranib to chemotherapy (carboplatin and paclitaxel) increased PFS by about three months in women with recurrent platinum-sensitive ovarian cancer. Additional benefit was obtained when cediranib was used as maintenance therapy, increasing overall PFS over chemotherapy alone. The time to disease progression increased from 9.4 to 12.6 months and OS was extended from 17.6 to 20.3 months over a follow-up period of two years. [45, 46]

Other anti-angiogenic agents have also been evaluated in recurrent ovarian cancer.

Aflibercept is a potent inhibitor of both VEGF and placental growth factor. Aflibercept has shown anti-tumour activity in combination with docetaxel and is useful as a single agent in the reduction of malignant ascites. The combination was tested in a Phase I/II study in patients Pazopanib inhibits this signalling pathway via ATP-competitive inhibition of VEGFR-1, VEGFR-2, and VEGFR-3; platelet-derived growth factor receptor (PDGFR)-α, PDGFR-β; fibroblast growth factor receptor (FGFR)-1, FGFR-3, and c-Kit. [51] Friedlander et al. reported responses to pazopanib in 11 of 36 patients (31%) with advanced ovarian cancer. The median time to response was 29 days and median response duration 113 days. The overall response rate was 18% in patients with measurable disease at baseline. [52]

De Bois et al. reported results of the AGO OVAR16 trial investigating the role of pazopanib as maintenance treatment for patients with advanced ovarian cancer in the front-line setting following induction chemotherapy. A total of 940 patients with stage 3 or 4 disease were randomized. The median time from diagnosis to randomization was 7.1 months in the placebo arm and 7.0 months in the pazopanib arm. At a median follow-up of 24 months, patients in the pazopanib arm had a prolonged PFS compared to a placebo, 17.9 versus 12.3 months, respectively (HR = 0.766; 95% CI: 0.64-0.91; p = 0.0021). Sensitivity and subgroup analyses of PFS, and analysis of PFS by GCIG criteria, were consistent with the primary analysis. The first interim analysis for OS (only 189 OS events = 20.1% of the population) showed no difference between the arms. Pazopanib treatment was associated with a higher incidence of adverse and serious adverse events (26% vs. 11%). The most common toxicities included hypertension, diarrhoea, nausea, headache, fatigue, and neutropoenia. The AGO investigators concluded that pazopanib maintenance therapy provided a statistically significant and clinically mean‐ ingful PFS benefit in patients with advanced ovarian cancer. It is possible that pazopanib will be incorporated into the armamentarium of ovarian cancer drugs in routine practice in the near future. The OS data are not mature. [53–54]

#### **5.2. PARP inhibitors**

A new class of agents that inhibit poly-adenosine diphosphate [ADP]-ribose polymerase (PARP) demonstrated significant activity in patients with recurrent disease, especially those with a germline mutation in BRCA1 or BRCA2. Several recent ongoing studies are evaluating the activity of PARP inhibitors in epithelial ovarian cancer, primarily in BRCA-mutation carriers. A proof-of-concept Phase I study of olaparib was conducted in 50 BRCA-carrier patients with relapsed ovarian cancer. This study showed a 40% objective response rate judged by RECIST criteria and/or a CA125 response assessed by a greater than 50% decline in CA125. Patients that were platinum sensitive had a higher chance of achieving a response to olaparib. [55] In an international multicenter Phase II study, 57 BRCA-carriers patients with recurrent ovarian cancer were enrolled in two sequential cohorts of two doses of olaparib (400 mg and 100 mg orally twice daily). The overall response rate as per the RECIST criteria was 33% in the 400 mg and 13% in the 100 mg cohort. These results suggest a possible dose-response effect. [56] Finally, PARP inhibitors may also show activity in patients with sporadic disease without germline BRCA1 or BRCA2 mutations. Gelmon et al. had shown a 24% objective response rate in relapsed ovarian cancer in the absence of BRCA1 or BRCA2 mutations to PARP inhibitors. Olaparib was well tolerated with the most common adverse events being fatigue in 70% of patients, nausea in 66%, vomiting in 39%, and decreased appetite in 36%. [57]

Ledermann et al. studied the role of olaparib maintenance in the subset of patients with relapsed platinum-sensitive high-grade serous ovarian cancer. The study was a double-blind, placebo-controlled, Phase II study. Results showed a significantly higher PFS rate compared to a placebo (median, 8.4 months vs. 4.8 months, with a hazard ratio for progression of 0.35, P < 0.001). OS analysis is not yet mature. [58]

Olaparib was also investigated in combination with chemotherapy in patients with advanced ovarian cancer. In a randomized, open-label, Phase II study, adult patients with platinumsensitive, recurrent, high-grade serous ovarian cancer who had received up to three previous courses of platinum-based chemotherapy and who were progression-free for at least six months before randomization received either olaparib (200 mg capsules twice daily, admin‐ istered orally on days one to 10 of each 21-day cycle) plus paclitaxel (175 mg/m2, administered intravenously on day one) and carboplatin (area under the curve [AUC] 4 mg/mL per min, according to the Calvert formula, administered intravenously on day one). Subsequently, patients received either olaparib monotherapy (400 mg capsules twice daily, given continu‐ ously) until disease progression in the olaparib plus chemotherapy group, or paclitaxel (175 mg/m2 on day one) and carboplatin (AUC 6 mg/mL per min on day one) then no further treatment in the chemotherapy alone group. The progression-free survival was significantly longer in the olaparib plus chemotherapy group (median 12.2 months [95% CI 9.7–15.0]) compared to chemotherapy alone group (median 9.6 months; 95% CI 9.1–9.7 and a HR 0.51; 95% CI 0.34–0.77; p=0.0012) — the difference was more pronounced in patients with BRCA mutations (HR 0.21 [0.08–0.55]; p=0.0015). [59]

Both the U.S. Food and Drug Administration and the EMA granted accelerated approval for olaparib (Lynparza) in the treatment of women with advanced ovarian cancer associated with defective BRCA genes, as detected by an FDA-approved test. [60]

#### **6. Summary**

PFS, and analysis of PFS by GCIG criteria, were consistent with the primary analysis. The first interim analysis for OS (only 189 OS events = 20.1% of the population) showed no difference between the arms. Pazopanib treatment was associated with a higher incidence of adverse and serious adverse events (26% vs. 11%). The most common toxicities included hypertension, diarrhoea, nausea, headache, fatigue, and neutropoenia. The AGO investigators concluded that pazopanib maintenance therapy provided a statistically significant and clinically mean‐ ingful PFS benefit in patients with advanced ovarian cancer. It is possible that pazopanib will be incorporated into the armamentarium of ovarian cancer drugs in routine practice in the

A new class of agents that inhibit poly-adenosine diphosphate [ADP]-ribose polymerase (PARP) demonstrated significant activity in patients with recurrent disease, especially those with a germline mutation in BRCA1 or BRCA2. Several recent ongoing studies are evaluating the activity of PARP inhibitors in epithelial ovarian cancer, primarily in BRCA-mutation carriers. A proof-of-concept Phase I study of olaparib was conducted in 50 BRCA-carrier patients with relapsed ovarian cancer. This study showed a 40% objective response rate judged by RECIST criteria and/or a CA125 response assessed by a greater than 50% decline in CA125. Patients that were platinum sensitive had a higher chance of achieving a response to olaparib. [55] In an international multicenter Phase II study, 57 BRCA-carriers patients with recurrent ovarian cancer were enrolled in two sequential cohorts of two doses of olaparib (400 mg and 100 mg orally twice daily). The overall response rate as per the RECIST criteria was 33% in the 400 mg and 13% in the 100 mg cohort. These results suggest a possible dose-response effect. [56] Finally, PARP inhibitors may also show activity in patients with sporadic disease without germline BRCA1 or BRCA2 mutations. Gelmon et al. had shown a 24% objective response rate in relapsed ovarian cancer in the absence of BRCA1 or BRCA2 mutations to PARP inhibitors. Olaparib was well tolerated with the most common adverse events being fatigue in 70% of

patients, nausea in 66%, vomiting in 39%, and decreased appetite in 36%. [57]

Ledermann et al. studied the role of olaparib maintenance in the subset of patients with relapsed platinum-sensitive high-grade serous ovarian cancer. The study was a double-blind, placebo-controlled, Phase II study. Results showed a significantly higher PFS rate compared to a placebo (median, 8.4 months vs. 4.8 months, with a hazard ratio for progression of 0.35, P

Olaparib was also investigated in combination with chemotherapy in patients with advanced ovarian cancer. In a randomized, open-label, Phase II study, adult patients with platinumsensitive, recurrent, high-grade serous ovarian cancer who had received up to three previous courses of platinum-based chemotherapy and who were progression-free for at least six months before randomization received either olaparib (200 mg capsules twice daily, admin‐ istered orally on days one to 10 of each 21-day cycle) plus paclitaxel (175 mg/m2, administered intravenously on day one) and carboplatin (area under the curve [AUC] 4 mg/mL per min, according to the Calvert formula, administered intravenously on day one). Subsequently, patients received either olaparib monotherapy (400 mg capsules twice daily, given continu‐

near future. The OS data are not mature. [53–54]

200 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

< 0.001). OS analysis is not yet mature. [58]

**5.2. PARP inhibitors**

Recurrent ovarian cancer is not curable. The goals of therapy should focus on palliation of cancer-related symptoms, extension of life, and maintenance of quality of life. The outlook has clearly improved over the last decade, due to an increase in options for the management of recurrent disease. Secondary cytoreduction has been advocated, but it remains controversial. For patients with platinum-sensitive disease retreatment with a platinum or a platinumcontaining combination, such as carboplatin, should be considered. For patients with plati‐ num-refractory or platinum-resistant disease, clinical trials should be considered. For patients who are not entering a trial, treatment with agents like PLD, paclitaxel, docetaxel, topotecan, gemcitabine, pemetrexed, ixabepilone, or oral etoposide can be considered. Despite the advances made in biological and targeted therapies like bevacizumab and pazopanib in extending disease-free survival in patients with recurrent advanced ovarian cancer, further research is needed to better understand the safety and effectiveness, the optimal duration and timing of treatment, and activity in association with other chemotherapeutic agents.

#### **Author details**

Bernardo L. Rapoport

Address all correspondence to: brapoport@rosebankoncology.co.za

The Medical Oncology Center of Rosebank, Saxonwold, Johannesburg, South Africa

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**Chapter 9**

## **Management of Ovarian Cancer — Is There a Role for Cytoreductive Surgery (CRS) and Hyperthermic Intraperitoneal Chemotherapy (HIPEC)?**

#### Melissa Ching Ching Teo and Grace Hwei Ching Tan

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60601

#### **Abstract**

Ovarian cancer is one of the commonest malignancy in women worldwide, and is the most lethal of all the gynaecological malignancies. Ovarian cancer often presents at an advanced stage, with the involvement of the peritoneal surface either at the initial diagnosis or at recurrence. Despite the advances made in the surgical techniques and chemotherapeutic options regarding agents, schedule, and route of administration, majority of the patients recur and eventually succumb to their disease. The change in the surgical approach supporting more radical and extensive surgical procedures, in a bid to attain optimal cytoreduction with no gross residual disease, has seen improvement in the survival, as has the use of intraperitoneal chemotherapy in combination with i.v. agents. Although peritoneal carcinomatosis has always been a poor prognostic factor, it ceases to be a factor of much importance if complete cytoreduction can be achieved. Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) provide the combined benefits of surgical eradication and effective chemotherapy, and can be performed with acceptable morbidity and mortality. Further trials are being undertaken to examine its role in the primary, as well as recurrent settings of advanced ovarian cancer and to determine the ideal drug combinations and dosages. We aim to discuss the role of CRS and HIPEC in the treatment of ovarian cancer.

**Keywords:** Ovarian Cancer, CRS, HIPEC

© 2016 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.

#### **1. Introduction**

Ovarian cancer one of the commonest malignancies in women worldwide, with an annual incidence of 239,000 [1, 2]. It is the most lethal of all the gynaecological malignancies, the fifth leading cause of cancer deaths, and claimed 151,917 lives in [1, 2]. Most patients with ovarian cancer present with ostensibly innocuous symptoms of abdominal bloatedness and discom‐ fort, and hence are often diagnosed at an advanced stage, with 60–70% of patients having stage 3 or 4 disease at diagnosis [3]. The standard approach is optimal debulking and adjuvant chemotherapy with platinum-based and taxol-based chemotherapy. Even with optimal treatment, the median five-year survival is less than 50% [4] and in advanced ovarian cancer, this drops to less than 25% [4]. Up to 70% of all patients diagnosed with ovarian cancer relapse and ultimately succumb to their disease.

#### **2. Optimal debulking**

Surgery remains the foundation of the management of ovarian cancers, as it is often required initially to attain a diagnosis, to formally stage the patient, and is the mainstay of treatment in the majority of diagnosed cases [5]. The definition of optimal cytoreduction has evolved over the years, but was originally defined as residual disease less than 2 cm in size. This was further altered with evidence establishing the significantly superior survival of those with residual disease measuring less than 1 cm and subsequently 5 mm in size [6–8]. Even in those with traditionally defined optimal cytoreduction with residual disease measuring less than 1 cm, the risk of death increases considerably when compared with those with no residual disease [9]. Over the years, there has been a glut of data concluding that complete cytoreduction, with no gross residual disease, yields the best results in terms of survival [10]. Patients with no gross residual disease, 0.1–2 cm residual disease, and more than 2 cm residual disease had five-year survivals of 60,35, and less than 20%, respectively [11, 12].

#### **3. What happens after debulking?**

In the bulk of patients with ovarian cancer, adjuvant chemotherapy is required [13]. Initially, the chemotherapeutic agents of choice included a platinum-based chemotherapy and a classic alkylating agent, and common agents used were cisplatin and cyclophosphamide [14]. After the Gynaecological Oncology Group (GOG) 111 and OV10 trials, looking specifically at the combinations of cisplatin with either cyclophosphamide or paclitaxel, were performed, the standard of care following surgery for stage 3 and 4 ovarian cancer was a combination of a platinum-based agent and a taxane, with intravenous (i.v.) cisplatin and paclitaxel being the agents of choice. Subsequently, a combination of paclitaxel and carboplatin showed similar results for response and survival rates, but without the toxicity often related to cisplatin treatment, and with a better quality of life. The standard approach is six cycles of paclitaxel 175mg/m2 administered every three weeks, in combination with carboplatin area under the curve (AUC) 5–6 [15].

The response and survival rates vary between the different histological types, with the clear cell and endometrioid subtypes having the worst and best prognoses, respectively, and mucinous and serous subtypes having intermediate prognoses [16, 17]. High-grade features likewise affect the prognosis unfavourably and such diseases often take on a much more aggressive course [16, 17].

**1. Introduction**

and ultimately succumb to their disease.

210 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

survivals of 60,35, and less than 20%, respectively [11, 12].

**3. What happens after debulking?**

curve (AUC) 5–6 [15].

**2. Optimal debulking**

Ovarian cancer one of the commonest malignancies in women worldwide, with an annual incidence of 239,000 [1, 2]. It is the most lethal of all the gynaecological malignancies, the fifth leading cause of cancer deaths, and claimed 151,917 lives in [1, 2]. Most patients with ovarian cancer present with ostensibly innocuous symptoms of abdominal bloatedness and discom‐ fort, and hence are often diagnosed at an advanced stage, with 60–70% of patients having stage 3 or 4 disease at diagnosis [3]. The standard approach is optimal debulking and adjuvant chemotherapy with platinum-based and taxol-based chemotherapy. Even with optimal treatment, the median five-year survival is less than 50% [4] and in advanced ovarian cancer, this drops to less than 25% [4]. Up to 70% of all patients diagnosed with ovarian cancer relapse

Surgery remains the foundation of the management of ovarian cancers, as it is often required initially to attain a diagnosis, to formally stage the patient, and is the mainstay of treatment in the majority of diagnosed cases [5]. The definition of optimal cytoreduction has evolved over the years, but was originally defined as residual disease less than 2 cm in size. This was further altered with evidence establishing the significantly superior survival of those with residual disease measuring less than 1 cm and subsequently 5 mm in size [6–8]. Even in those with traditionally defined optimal cytoreduction with residual disease measuring less than 1 cm, the risk of death increases considerably when compared with those with no residual disease [9]. Over the years, there has been a glut of data concluding that complete cytoreduction, with no gross residual disease, yields the best results in terms of survival [10]. Patients with no gross residual disease, 0.1–2 cm residual disease, and more than 2 cm residual disease had five-year

In the bulk of patients with ovarian cancer, adjuvant chemotherapy is required [13]. Initially, the chemotherapeutic agents of choice included a platinum-based chemotherapy and a classic alkylating agent, and common agents used were cisplatin and cyclophosphamide [14]. After the Gynaecological Oncology Group (GOG) 111 and OV10 trials, looking specifically at the combinations of cisplatin with either cyclophosphamide or paclitaxel, were performed, the standard of care following surgery for stage 3 and 4 ovarian cancer was a combination of a platinum-based agent and a taxane, with intravenous (i.v.) cisplatin and paclitaxel being the agents of choice. Subsequently, a combination of paclitaxel and carboplatin showed similar results for response and survival rates, but without the toxicity often related to cisplatin treatment, and with a better quality of life. The standard approach is six cycles of paclitaxel 175mg/m2 administered every three weeks, in combination with carboplatin area under the

In an attempt to improve response rates to chemotherapy, and progression-free and overall survivals, dose intense and dose-dense chemotherapy were introduced. The former refers to the increase of dosages with each drug delivery, whereas the latter implies increasing the frequency of drug administration. It was thought that tumour growth escalated in the initial phase but slowed as the tumour volume increased; hence, delivering higher doses of chemo‐ therapy from the start and at close intervals would increase tumour cell death. This theory was confirmed in a large meta-analysis studying the effects of dose-intense and dose-dense chemotherapy for ovarian cancer [18]. The encouraging results of the JGOG trial, with firstline dose-dense chemotherapy, depict an improvement of median progression-free survival from 17.5 to 28.2 months and an overall survival that was not reached [19].

#### **4. The role of Intraperitoneal (IP) chemotherapy versus Intravenous (IV) chemotherapy**

The route of administration of chemotherapy for ovarian cancer has traditionally been intravenous (i.v.). In the1960s, intraperitoneal (i.p.) chemotherapy was introduced with the aim of controlling malignant ascites. It was found that certain drugs such as cisplatin were cleared from the peritoneal cavity gradually, which meant that a high concentration of the drug could be delivered intraperitoneally without resulting in a systemic overdose of the drug.

Drugs that are particularly suited for i.p. delivery have high molecular weights and are water soluble, leading to a delayed peritoneal but high systemic clearance, and so having a phar‐ macological advantage for treating peritoneal disease.

Ovarian cancer is an ideal cancer for treatment via an i.p. route. The majority of diagnosed cases present with peritoneal disease in the absence of extra-peritoneal metastases [3]. Even in patients who have undergone seemingly curative surgery and adjuvant chemotherapy, up to 70% develop recurrent disease, the majority of which remains confined to the peritoneal cavity. The propensity for peritoneal recurrences as the only site of disease makes this cancer the model candidate for such loco-regional treatment. I.p. chemotherapy has also been used with significant success in mucinous tumours of the appendix and peritoneum [20], colon cancers [21], and has even been shown to provide improved survival in gastric cancers [22].

The underlying principle behind i.p. chemotherapy is the delivery of high concentrations of the appropriate drug to the site that is most likely to develop recurrences, at the opportune moment where tumour burden is at its minimum, i.e., after the performance of complete cytoreduction, with eradication of all macroscopic disease. It is critical that no gross residual disease is present, as penetration of i.p. chemotherapy is up to a depth of 2.5 mm [23–25]; hence, there is an inherent risk that larger volumes of tumour deposits will not be sufficiently treated by the intraperitoneal chemotherapy.

There have been numerous studies examining the results of i.p. chemotherapy in the man‐ agement of ovarian cancer. Amongst the first few randomized controlled trials (RCTs) was that conducted by the Southwest Oncology Group (SWOG) and GOG 104 trial, in which patients with then-defined optimal cytoreduction of less than 2 cm residual disease were administered i.v. cyclophosphamide and either i.v. or i.p. cisplatin. The patients who received the i.p. chemotherapy had significantly increased median overall survival [26]. The GOG 111 trial that combined i.v. paclitaxel with i.v. or i.p. cisplatin reached similar conclusions, in favour of the i.p. treatment. Other RCTs produced progression-free and overall survivals of 28 and 63–66 months, respectively. In the GOG 172 trial, the median overall survival was 65.6 and 49.7 months for the combination i.p./i.v. and cytoreductive surgery (CRS) and i.v. chemother‐ apy alone arms, respectively. There were criticisms of these trials as the i.p. arm in GOG 114 and GOG 172 received two cycles of carboplatin at AUC 9 and i.p. paclitaxel on day 8, respectively; hence, we await the results of additional RCTs that aim to study the effect of i.p. chemotherapy and determine the ideal algorithm for the management of ovarian cancer [27].

#### **5. Recurrent ovarian cancer**

Despite optimal treatment, up to 70% of all patients diagnosed with ovarian cancer suffer from relapse. In the past, early detection of persistent disease by second-look laparotomies was often performed, but as it was found to make no difference in GOG-0158 it is no longer practiced [28]. Currently, the practice of close follow-up of patients by serial CA-125 levels at intervals of one to three months is practiced. In patients who are in clinical complete remission, an increase in CA-125 from initial levels is the most common method to detect disease relapse. However, the MRC-OV05 trial [29], which examined the consequences of early treatment for recurrence versus treatment delayed until clinical symptoms appeared, showed that there was no benefit in the detection of the early presence of disease by CA-125, with only a 1.4 month benefit in survival for the early treatment group.

In patients with clinically evident relapse, treatment options include secondary cytoreduction with or without hyperthermic intraperitoneal chemotherapy (HIPEC), and systemic chemo‐ therapeutic regimes. Systemic treatment is dependent on the platinum sensitivity of the disease. In platinum-sensitive disease, re-treatment with a platinum or platinum-containing combination is advocated, and in platinum-resistance disease, clinical trials involving topote‐ can, docataxel, gemcitabine, paclitaxel, pemetrexed and bevacizumab should be considered.

#### **6. The rationale for Cytoreductive Surgery (CRS) and Hyperthermic Intraperitoneal Chemotherapy (HIPEC)**

Hyperthermic intraperitoneal chemotherapy (HIPEC) was first introduced in the early 1980s for the treatment of peritoneal carcinomatosis. CRS and HIPEC were popularized for the management of peritoneal surface malignancies by Dr. Sugarbaker in the 1990s [21]. The addition of hyperthermia to the i.p. chemotherapy has been shown to boost penetration of the chemotherapy and improve its absorption into the tumour cells, increasing the intracellular accumulation of the drug [30]. The cytotoxic effect appears to be similarly potentiated, secondary to an impairment of the cells' ability to perform DNA repair, and thus has a greater deleterious effect [30, 31]. The drugs selected must be heat stable, with a high molecular weight and a low water solubility to be optimally used in the process of HIPEC. Cisplatin, mitomycin-C, and doxorubicin are the frequently employed drugs.

HIPEC is performed intraoperatively, while the patient is under general anaesthesia, via a pump that maintains the temperature and circulation of the drug solution. In addition to patient comfort, advantages include an ability to ensure that the entire peritoneal surface is bathed in the chemotherapeutic agent, before the formation of obstructing adhesions that may develop in the postoperative period. If HIPEC is administered in an opened-abdomen fashion, the surgeon would be able to manually swirl the chemotherapy to achieve this target. The most important prognostic factor remains the completeness of cytoreduction, with a 5.5% increase in the median overall survival for every 10% of patients undergoing optimal cytoreduction [5], leading to the inevitable conclusion that the changing surgical paradigm for ovarian cancer embracing radical CRS has resulted in meaningfully better survival results [32].

The combination of CRS and HIPEC has shown promising results, with median overall and progression-free survivals of up to 64 and 57 months, respectively [30, 33, 34]. Optimal cytoreduction yields five-year survivals of 12–66% [34].These results are compatible with those of the author's institution [35]. A meta-analysis examining i.p. versus i.v. trials also conclusive‐ lyshowedthe superiorityofthe i.p.overthe i.v. arms,withhazardratiosof 0.79 forbothdiseasefree and overall survivals [36]. Patients with platinum-sensitive disease have better response rates of 20–77% compared with up to 28% in those with platinum-resistant disease [37].

#### **7. CRS and HIPEC: When to do it?**

there is an inherent risk that larger volumes of tumour deposits will not be sufficiently treated

There have been numerous studies examining the results of i.p. chemotherapy in the man‐ agement of ovarian cancer. Amongst the first few randomized controlled trials (RCTs) was that conducted by the Southwest Oncology Group (SWOG) and GOG 104 trial, in which patients with then-defined optimal cytoreduction of less than 2 cm residual disease were administered i.v. cyclophosphamide and either i.v. or i.p. cisplatin. The patients who received the i.p. chemotherapy had significantly increased median overall survival [26]. The GOG 111 trial that combined i.v. paclitaxel with i.v. or i.p. cisplatin reached similar conclusions, in favour of the i.p. treatment. Other RCTs produced progression-free and overall survivals of 28 and 63–66 months, respectively. In the GOG 172 trial, the median overall survival was 65.6 and 49.7 months for the combination i.p./i.v. and cytoreductive surgery (CRS) and i.v. chemother‐ apy alone arms, respectively. There were criticisms of these trials as the i.p. arm in GOG 114 and GOG 172 received two cycles of carboplatin at AUC 9 and i.p. paclitaxel on day 8, respectively; hence, we await the results of additional RCTs that aim to study the effect of i.p. chemotherapy and determine the ideal algorithm for the management of ovarian cancer [27].

Despite optimal treatment, up to 70% of all patients diagnosed with ovarian cancer suffer from relapse. In the past, early detection of persistent disease by second-look laparotomies was often performed, but as it was found to make no difference in GOG-0158 it is no longer practiced [28]. Currently, the practice of close follow-up of patients by serial CA-125 levels at intervals of one to three months is practiced. In patients who are in clinical complete remission, an increase in CA-125 from initial levels is the most common method to detect disease relapse. However, the MRC-OV05 trial [29], which examined the consequences of early treatment for recurrence versus treatment delayed until clinical symptoms appeared, showed that there was no benefit in the detection of the early presence of disease by CA-125, with only a 1.4 month

In patients with clinically evident relapse, treatment options include secondary cytoreduction with or without hyperthermic intraperitoneal chemotherapy (HIPEC), and systemic chemo‐ therapeutic regimes. Systemic treatment is dependent on the platinum sensitivity of the disease. In platinum-sensitive disease, re-treatment with a platinum or platinum-containing combination is advocated, and in platinum-resistance disease, clinical trials involving topote‐ can, docataxel, gemcitabine, paclitaxel, pemetrexed and bevacizumab should be considered.

**6. The rationale for Cytoreductive Surgery (CRS) and Hyperthermic**

Hyperthermic intraperitoneal chemotherapy (HIPEC) was first introduced in the early 1980s for the treatment of peritoneal carcinomatosis. CRS and HIPEC were popularized for the

by the intraperitoneal chemotherapy.

212 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

**5. Recurrent ovarian cancer**

benefit in survival for the early treatment group.

**Intraperitoneal Chemotherapy (HIPEC)**

The time points at which CRS and HIPEC have been used in the management of advanced ovarian cancer include the primary setting, after neoadjuvant chemotherapy, at the point of recurrence, and as a second-line treatment [38]. In the Milan 2006 consensus statement, it was concluded that CRS and HIPEC could be feasible at all of these time points.

The morbidity and mortality for such a procedure range from 0 to 40 and 0 to 10%, respective‐ ly[34,35],andincludenauseaandvomiting,gastrointestinaldisturbancesandileus,anastomot‐ ic leaks, perioperative bleeding, pleural effusions and pneumothoraces, intra-abdominal collections/abscesses, andsepsis.Thekeyis inpatient selection, anditis imperative thatpatients with a good ECOG and an ability to tolerate such a radical procedure be chosen. The best candidates have long disease-free intervals and low volume disease that can be confidently optimallydebulked.Manyofthe studies includedinthe reviewofCRSandHIPECfor advanced ovarian cancer show the usage of this modality of treatment in the recurrent setting. Howev‐ er, evidence supporting the use of i.p. chemotherapy in the initial setting of advanced ovarian cancer [39] suggests that there is sound rationale behind CRS and HIPEC, even in the primary setting. There are more than 40 studies that have reported on the role of HIPEC in the manage‐ ment of ovarian cancer, but many of these studies are small in number and heterogeneous in theirdesign.Furthertrials suchas the ItalianHORSEstudy(available athttp://clinicaltrials.gov/ show/ NCT01539785) that randomizes patients with platinum-sensitive disease to CRS and HIPEC with cisplatin 75mg/m2 and CRS alone, and the French CHIPOR study (available at http://clinicaltrials.gov/show/NCT01376752) that randomizes patients with recurrent plati‐ num-sensitive disease (relapse beyond six months) after they have received platinum-based chemotherapy and optimal cytoreduction (less than 2.5 mm residual disease) to HIPEC with i.p. cisplatin 75mg/m2 and no HIPEC [37] will provide answers about the role of HIPEC in patients with platinum-sensitive disease. There is a need for phase 3 randomized trials to elucidate which timing and cohort of patients would be most beneficial for CRS and HIPEC.

#### **8. Future directions**

#### **8.1. CRS and HIPEC**

It is evident that complete cytoreduction, with no residual disease, yields the best clinical outcome. However, in a significant proportion of patients recurrence in the peritoneal cavity occurs, and CRS and HIPEC are considered. Perhaps the role of CRS and HIPEC as an adjuvant treatment should be considered an upfront treatment option for primary ovarian cancers, especially with improved morbidity results for this treatment modality. A randomized trial examining the overall and disease-free survivals of patients managed with CRS and adjuvant i.v. chemotherapy and those who undergo CRS and HIPEC, with adjuvant i.v. chemotherapy, for ovarian cancer in the primary setting would enable this questions to be addressed.

#### **8.2. Intraperitoneal bevacizumab**

The role of vascular endothelial growth factor (VEGF) in ovarian cancer has received much attention because VEGF increases vascular permeability and enhances angiogenesis [40]. Overexpression of VEGF has been reported in ovarian cancer [41-43] and several studies have indicated that VEGF-regulated angiogenesis is an important component of ovarian cancer growth [44, 45]. Microvessel density and level of VEGF expression in ovarian cancer directly correlate with poor prognosis, suggesting that angiogenesis, possibly mediated at least in part by VEGF, influences disease progression [44, 45]. Currently, vascular endothelial growth factor receptor (VEGFR) antibody, bevacizumab, is given intravenously for select patients with ovarian cancer.

The role of intraperitoneal VEGF inhibition using bevacizumab has been explored for the treatment of malignant ascites [46]. In a mouse peritoneal model of human ovarian cancer, the author demonstrated that the administration of i.p. bevacizumab and rapamycin not only reduced ascites, but was also able to suppress the development of peritoneal carcinomatosis [47]. This is an indication that this therapy may potentially be useful for the treatment of peritoneal carcinomatosis and may also be a novel, efficient strategy for reducing recurrence of ovarian cancers.

#### **9. Conclusion**

cancer [39] suggests that there is sound rationale behind CRS and HIPEC, even in the primary setting. There are more than 40 studies that have reported on the role of HIPEC in the manage‐ ment of ovarian cancer, but many of these studies are small in number and heterogeneous in theirdesign.Furthertrials suchas the ItalianHORSEstudy(available athttp://clinicaltrials.gov/ show/ NCT01539785) that randomizes patients with platinum-sensitive disease to CRS and HIPEC with cisplatin 75mg/m2 and CRS alone, and the French CHIPOR study (available at http://clinicaltrials.gov/show/NCT01376752) that randomizes patients with recurrent plati‐ num-sensitive disease (relapse beyond six months) after they have received platinum-based chemotherapy and optimal cytoreduction (less than 2.5 mm residual disease) to HIPEC with i.p. cisplatin 75mg/m2 and no HIPEC [37] will provide answers about the role of HIPEC in patients with platinum-sensitive disease. There is a need for phase 3 randomized trials to elucidate which timing and cohort of patients would be most beneficial for CRS and HIPEC.

214 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

It is evident that complete cytoreduction, with no residual disease, yields the best clinical outcome. However, in a significant proportion of patients recurrence in the peritoneal cavity occurs, and CRS and HIPEC are considered. Perhaps the role of CRS and HIPEC as an adjuvant treatment should be considered an upfront treatment option for primary ovarian cancers, especially with improved morbidity results for this treatment modality. A randomized trial examining the overall and disease-free survivals of patients managed with CRS and adjuvant i.v. chemotherapy and those who undergo CRS and HIPEC, with adjuvant i.v. chemotherapy, for ovarian cancer in the primary setting would enable this questions to be addressed.

The role of vascular endothelial growth factor (VEGF) in ovarian cancer has received much attention because VEGF increases vascular permeability and enhances angiogenesis [40]. Overexpression of VEGF has been reported in ovarian cancer [41-43] and several studies have indicated that VEGF-regulated angiogenesis is an important component of ovarian cancer growth [44, 45]. Microvessel density and level of VEGF expression in ovarian cancer directly correlate with poor prognosis, suggesting that angiogenesis, possibly mediated at least in part by VEGF, influences disease progression [44, 45]. Currently, vascular endothelial growth factor receptor (VEGFR) antibody, bevacizumab, is given intravenously for select patients with

The role of intraperitoneal VEGF inhibition using bevacizumab has been explored for the treatment of malignant ascites [46]. In a mouse peritoneal model of human ovarian cancer, the author demonstrated that the administration of i.p. bevacizumab and rapamycin not only reduced ascites, but was also able to suppress the development of peritoneal carcinomatosis [47]. This is an indication that this therapy may potentially be useful for the treatment of peritoneal carcinomatosis and may also be a novel, efficient strategy for reducing recurrence

**8. Future directions**

**8.2. Intraperitoneal bevacizumab**

**8.1. CRS and HIPEC**

ovarian cancer.

of ovarian cancers.

Ovarian cancer often presents at an advanced stage, with the involvement of the peritoneal surface either at initial diagnosis or at recurrence. Despite the advances made in surgical techniques and chemotherapeutic options regarding agents, schedule, and route of adminis‐ tration, the majority of the patients relapse and eventually succumb to their disease. A change in the surgical approach, supporting more radical and extensive surgical procedures in a bid to attain optimal cytoreduction with no gross residual disease, has seen an improvement in survival, as has the use of intraperitoneal chemotherapy in combination with i.v. agents. Although peritoneal carcinomatosis has always been a poor prognostic factor, it ceases to be a factor of much importance if complete CRS can be achieved [31, 48]. CRS and HIPEC provide the combined benefits of surgical eradication and effective chemotherapy, and can be per‐ formed with acceptable morbidity and mortality [49]. Further trials are being undertaken to examine its role in the primary as well as recurrent settings of advanced ovarian cancer, and to determine the ideal drug combinations and dosages [50–52].

#### **Author details**

Melissa Ching Ching Teo\* and Grace Hwei Ching Tan

\*Address all correspondence to: melissa.teo.c.c@nccs.com.sg

Department of Surgical Oncology, National Cancer Centre Singapore, Singapore

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## **Anti-Angiogenic and Anti-Cancer Effects by Targeting the Protein Kinase G Type-Iα (PKG-Iα) Signaling Pathway and its Downstream Effects on Expression of Inhibitor of Apoptosis Proteins, C-IAP1, Livin and Survivin**

Janica C. Wong, Priyatham Gorjala, Benjamin Costantino and Ronald R. Fiscus

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60774

#### **Abstract**

Ovarian cancer is often difficult to treat because of the development of resistance to many of the currently-used therapeutic agents (i.e. chemoresistance). The progression and chemoresistance of ovarian cancer can involve tumor angiogenesis, the develop‐ ment of new blood vessels bringing more blood and nutrients to the growing tumor. Tumor angiogenesis also involves the vascular endothelium-induced stimulation of cancer cell growth (1) and the higher expression levels of certain "cell survival proteins", such as the Inhibitor of Apoptosis Proteins (IAPs, including c-IAP1, Livin and Survivin), which are expressed in both the proliferating cancer cells (2, 3) and the vascular endothelial cells involved in tumor angiogenesis (4).

**Keywords:** Anti-angiogenesis, cancer treatment, protein kinase G (PKG), chemore‐ sistance, cIAP-1, Livin, Survivin, Inhibitor of Apoptosis Proteins (IAPs)

#### **1. Introduction**

Ovarian cancer is often difficult to treat because of the development of resistance to many of the currently-used therapeutic agents (i.e. chemoresistance). The progression and chemore‐

© 2016 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.

sistance of ovarian cancer can involve tumor angiogenesis, the development of new blood vessels bringing more blood and nutrients to the growing tumor. Tumor angiogenesis also involves the vascular endothelium-induced stimulation of cancer cell growth [1] and the higher expression levels of certain "cell survival proteins", such as the Inhibitor of Apoptosis Proteins (IAPs, including c-IAP1, Livin and Survivin), which are expressed in both the proliferating cancer cells [2, 3] and the vascular endothelial cells involved in tumor angiogenesis [4].

Although there are a number of cell signaling pathways that are known to promote angio‐ genesis and the higher levels of expression of the IAPs, one pathway that has become recog‐ nized as an important pro-growth and pro-survival mechanism within both ovarian cancer cells and vascular endothelial cells is the nitric oxide (NO)/cyclic GMP (cGMP)/protein kinase G type-1α (PKG-Iα) signaling pathway. Originally, the NO/cGMP/PKG signaling pathway was recognized to be a key cellular mechanism in regulating the cardiovascular system, specifically involved in promoting vasodilation (relaxation of vascular smooth muscle cells) and in preventing the onset of hypertension and other cardiovascular diseases [5-12]. More recent data from our laboratory have shown that the NO/cGMP/PKG signaling pathway, mediated via one of the isoforms of PKG, i.e. the PKG-Iα splice variant of PKG-I, is involved in promoting cell proliferation and enhanced cell survival (inhibiting the onset of apoptosis) in many types of mammalian cells, including neural cells [3, 6, 10, 13-15], uterine epithelial cells [16], OP9 bone marrow-derived mesenchymal (stromal) stem cells [17], and a number of different type of cancer cells, including ovarian cancer cells [18-22], neuroblastoma cells [15] and lung cancer cells [22].

Our studies have shown that the catalytic/kinase activity of PKG-Iα plays a key role in the phosphorylation of four proteins, BAD, CREB, c-Src and VASP, within mammalian cells, promoting DNA synthesis/cell proliferation and inhibiting the onset of apoptotic cell death, thus enhancing cell survival [22]. We have found that PKG-Iα is hyperactivated in several types of cancer cells, including ovarian cancer cells and lung cancer cells, resulting in abnor‐ mally high levels of phosphorylation of BAD, CREB, c-Src and VASP, which contributes to the exaggerated cell proliferation and resistance to certain chemotherapy, such as cisplatin (i.e. platinum resistance) [21]. The key role played by PKG-Iα in promoting DNA synthesis/cell proliferation and chemoresistance has been established by both pharmacological inhibitors and gene knockdown techniques using siRNA and shRNA that target PKG-Iα [21, 22].

Figure 1 shows a cellular model illustrating our findings about the role of NO at low physio‐ logical levels, i.e. 10 picomolar (pM) to 1 nanomolar (nM), and its downstream activation of PKG-Iα in promoting increased tumor growth and chemoresistance in cancer cells of epithelial origin, including human ovarian cancer cells [3, 19-22]. The model highlights the recent finding from our laboratory regarding the substrate proteins that can be directly phosphorylated by PKG-Iα, including the apoptosis-regulating protein BAD at serine-155 [15], the transcription factor CREB at serine-133 [3, 6, 22, 23] and the oncogenic tyrosine kinase c-Src at serine-17 [22, 23]. The enhanced phosphorylation of CREB caused by the hyperactivation of PKG-Iα in nonsmall cell lung cancer (NSCLC) cells was found to be responsible for the maintenance of high levels of expression of several "cell survival proteins", including Mcl-1 and three of the IAPs (c-IAP1, Livin and Survivin) [3]. Gene knockdown of PKG-Iα expression using siRNA targeting this PKG isoform clearly showed that PKG-Iα plays a critical role in promoting the high-level expression of Mcl-1, c-IAP1, Livin and Survivin in lung cancer cells. Ongoing experiments in the Fiscus Lab in the College of Medicine at Roseman University of Health Sciences are currently determining if a similar relationship between PKG-Iα hyperactivation and the high-level expression of "cell survival protein", such as Mcl-1, c-IAP1, Livin and Survivin, also occurs in human ovarian cancer cells, like in lung cancer cells.

sistance of ovarian cancer can involve tumor angiogenesis, the development of new blood vessels bringing more blood and nutrients to the growing tumor. Tumor angiogenesis also involves the vascular endothelium-induced stimulation of cancer cell growth [1] and the higher expression levels of certain "cell survival proteins", such as the Inhibitor of Apoptosis Proteins (IAPs, including c-IAP1, Livin and Survivin), which are expressed in both the proliferating cancer cells [2, 3] and the vascular endothelial cells involved in tumor angiogenesis [4].

222 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

Although there are a number of cell signaling pathways that are known to promote angio‐ genesis and the higher levels of expression of the IAPs, one pathway that has become recog‐ nized as an important pro-growth and pro-survival mechanism within both ovarian cancer cells and vascular endothelial cells is the nitric oxide (NO)/cyclic GMP (cGMP)/protein kinase G type-1α (PKG-Iα) signaling pathway. Originally, the NO/cGMP/PKG signaling pathway was recognized to be a key cellular mechanism in regulating the cardiovascular system, specifically involved in promoting vasodilation (relaxation of vascular smooth muscle cells) and in preventing the onset of hypertension and other cardiovascular diseases [5-12]. More recent data from our laboratory have shown that the NO/cGMP/PKG signaling pathway, mediated via one of the isoforms of PKG, i.e. the PKG-Iα splice variant of PKG-I, is involved in promoting cell proliferation and enhanced cell survival (inhibiting the onset of apoptosis) in many types of mammalian cells, including neural cells [3, 6, 10, 13-15], uterine epithelial cells [16], OP9 bone marrow-derived mesenchymal (stromal) stem cells [17], and a number of different type of cancer cells, including ovarian cancer cells [18-22], neuroblastoma cells [15]

Our studies have shown that the catalytic/kinase activity of PKG-Iα plays a key role in the phosphorylation of four proteins, BAD, CREB, c-Src and VASP, within mammalian cells, promoting DNA synthesis/cell proliferation and inhibiting the onset of apoptotic cell death, thus enhancing cell survival [22]. We have found that PKG-Iα is hyperactivated in several types of cancer cells, including ovarian cancer cells and lung cancer cells, resulting in abnor‐ mally high levels of phosphorylation of BAD, CREB, c-Src and VASP, which contributes to the exaggerated cell proliferation and resistance to certain chemotherapy, such as cisplatin (i.e. platinum resistance) [21]. The key role played by PKG-Iα in promoting DNA synthesis/cell proliferation and chemoresistance has been established by both pharmacological inhibitors and gene knockdown techniques using siRNA and shRNA that target PKG-Iα [21, 22].

Figure 1 shows a cellular model illustrating our findings about the role of NO at low physio‐ logical levels, i.e. 10 picomolar (pM) to 1 nanomolar (nM), and its downstream activation of PKG-Iα in promoting increased tumor growth and chemoresistance in cancer cells of epithelial origin, including human ovarian cancer cells [3, 19-22]. The model highlights the recent finding from our laboratory regarding the substrate proteins that can be directly phosphorylated by PKG-Iα, including the apoptosis-regulating protein BAD at serine-155 [15], the transcription factor CREB at serine-133 [3, 6, 22, 23] and the oncogenic tyrosine kinase c-Src at serine-17 [22, 23]. The enhanced phosphorylation of CREB caused by the hyperactivation of PKG-Iα in nonsmall cell lung cancer (NSCLC) cells was found to be responsible for the maintenance of high levels of expression of several "cell survival proteins", including Mcl-1 and three of the IAPs (c-IAP1, Livin and Survivin) [3]. Gene knockdown of PKG-Iα expression using siRNA

and lung cancer cells [22].

**Figure 1.** Involvement of picomolar levels of NO and downstream activation of PKG-Iα on promoting tumor angio‐ genesis and the proliferation and chemoresistance of cancer cells.

Figure 1 also illustrates the role of physiological-level NO and PKG-Iα in mediating the protumor-angiogenesis effects of Vascular Endothelial Growth Factor (VEGF). VEGF is a proangiogenesis factor known to be released from many types of cancer cells, including human ovarian cancer cells, and is secreted in especially high amount by higher grade malignancies of the ovary [24]. Interestingly, higher VEGF expression by the ovarian cancer cells of patients was shown to be an independent predictor of poor prognosis of the disease [25].

The model of tumor angiogenesis shown in Figure 1 incorporates the early finding of Hood and Granger published in 1998, showing that VEGF stimulates cell proliferation and tube formation in human umbilical vein endothelial cells (HUVECs), a model of tumor angiogen‐ esis, in a manner that was completely dependent on endogenous PKG [26]. Their study did not determined which isoform of PKG was expressed in the HUVECs, however, our laboratory has recently shown that HUVECs express both of the two PKG-I splice variants (i.e. both PKG-Iα and PKG-Iβ) [4], which has been confirmed in the data illustrated below in Figure 3. We have proposed that it is likely that the PKG-Iα isoform is the splice variant that mediates the enhanced cell proliferation of HUVECs, based on our many studies with other cells (e.g. N1E-115 and NG108-15 neural cells) that express exclusively the PKG-Iα splice variant of PKG-I [3, 4, 15, 17, 21-23]. Two key findings by Hood and Granger was that PKG directly interacted with Raf-1 (c-Raf), as assessed by co-immunoprecipitation, and that PKG activity was neces‐ sary for VEGF-induced activation of Raf-1 and the subsequent downstream activations of MEK and ERK1/2, regulating in enhanced endothelial cell proliferation [26].

Figure 2 shows a comparison of the two splice variants of PKG-I, illustrating how two very similar protein kinases, identical in their catalytic and regulatory domains, can have very different biological effects. The key difference between these two splice variants is the Nterminal region, the first 100 amino acids, which is encoded by the unique first exon, Iα in the case of PKG-Iα and Iβ in the case of PKG-Iβ. The first 100 amino acids provide the leucine zipper/protein-protein interaction domain, which determines the subcellular localization of these two isoforms and further determines which downstream target protein is phosphory‐ lated by the two PKG-I isoforms within cells. Because of phosphorylating very different subsets of substrate proteins, PKG-Iα and PKG-Iβ are involved in mediating very different biological effects, in some cases, even opposite effects.

The PKG-Iα splice variant promotes cell proliferation and cell survival in both normal nontransformed cells (e.g. neural cells and OP9 bone marrow-derived mesenchymal stem cells) as well as malignant cells (contributing to exaggerated cell proliferation and chemoresistance in lung cancer and ovarian cancer cells) [3, 4, 15, 17, 20-23]. In contrast, the PKG-Iβ splice variant, at least when there is chemically-induced hyperactivation (e.g. using Exisulind to cause large increases in the intracellular cGMP levels) or forced overexpression (e.g. Deguchi et al., 2004), promotes effects that are just opposite of those mediated by PKG-Iα, i.e. inhibition of cell proliferation and induction of cell death mediated by PKG-Iβ [22, 23, 27]. This has led to confusion over the real function of PKG in regulating cell proliferation and cell survival, where PKG appears to have opposite effects in different experiments and/or different laboratories. Our studies have shown that it is critically important to differentiate between the actions of the different splice variants of PKG-I to avoid this confusion and the misunderstanding regarding the functions of PKG.

Recently, NO has been shown to be a positive regulator of the Warburg effect in ovarian cancer cells, promoting a metabolic switch toward increased glycolysis, with increased glucose consumption, enhanced uptake of glutamine and increased release of lactate [28]. The downstream signaling pathway mediating these metabolic effects of NO in ovarian cancer cells was not reported, but likely involves a mediator role for the different PKG isoforms that are downstream from the NO exposure.

esis, in a manner that was completely dependent on endogenous PKG [26]. Their study did not determined which isoform of PKG was expressed in the HUVECs, however, our laboratory has recently shown that HUVECs express both of the two PKG-I splice variants (i.e. both PKG-Iα and PKG-Iβ) [4], which has been confirmed in the data illustrated below in Figure 3. We have proposed that it is likely that the PKG-Iα isoform is the splice variant that mediates the enhanced cell proliferation of HUVECs, based on our many studies with other cells (e.g. N1E-115 and NG108-15 neural cells) that express exclusively the PKG-Iα splice variant of PKG-I [3, 4, 15, 17, 21-23]. Two key findings by Hood and Granger was that PKG directly interacted with Raf-1 (c-Raf), as assessed by co-immunoprecipitation, and that PKG activity was neces‐ sary for VEGF-induced activation of Raf-1 and the subsequent downstream activations of MEK

Figure 2 shows a comparison of the two splice variants of PKG-I, illustrating how two very similar protein kinases, identical in their catalytic and regulatory domains, can have very different biological effects. The key difference between these two splice variants is the Nterminal region, the first 100 amino acids, which is encoded by the unique first exon, Iα in the case of PKG-Iα and Iβ in the case of PKG-Iβ. The first 100 amino acids provide the leucine zipper/protein-protein interaction domain, which determines the subcellular localization of these two isoforms and further determines which downstream target protein is phosphory‐ lated by the two PKG-I isoforms within cells. Because of phosphorylating very different subsets of substrate proteins, PKG-Iα and PKG-Iβ are involved in mediating very different biological

The PKG-Iα splice variant promotes cell proliferation and cell survival in both normal nontransformed cells (e.g. neural cells and OP9 bone marrow-derived mesenchymal stem cells) as well as malignant cells (contributing to exaggerated cell proliferation and chemoresistance in lung cancer and ovarian cancer cells) [3, 4, 15, 17, 20-23]. In contrast, the PKG-Iβ splice variant, at least when there is chemically-induced hyperactivation (e.g. using Exisulind to cause large increases in the intracellular cGMP levels) or forced overexpression (e.g. Deguchi et al., 2004), promotes effects that are just opposite of those mediated by PKG-Iα, i.e. inhibition of cell proliferation and induction of cell death mediated by PKG-Iβ [22, 23, 27]. This has led to confusion over the real function of PKG in regulating cell proliferation and cell survival, where PKG appears to have opposite effects in different experiments and/or different laboratories. Our studies have shown that it is critically important to differentiate between the actions of the different splice variants of PKG-I to avoid this confusion and the misunderstanding

Recently, NO has been shown to be a positive regulator of the Warburg effect in ovarian cancer cells, promoting a metabolic switch toward increased glycolysis, with increased glucose consumption, enhanced uptake of glutamine and increased release of lactate [28]. The downstream signaling pathway mediating these metabolic effects of NO in ovarian cancer cells was not reported, but likely involves a mediator role for the different PKG isoforms that are

and ERK1/2, regulating in enhanced endothelial cell proliferation [26].

224 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

effects, in some cases, even opposite effects.

regarding the functions of PKG.

downstream from the NO exposure.

#### **Figure 2.** Opposite effects on cell proliferation and cell survival mediated by the two splice variants of PKG-I. The dif‐ ferent biological effects of PKG-Iα and PKG-Iβ likely result from the different subcellular distributions of these to iso‐ form (determined by the unique exon, Iα versus Iβ, of the two splice variants), resulting in the phosphorylation and activation of very different downstream substrates proteins, as depicted in the models. Modified from the original model in R.R. Fiscus, E.L. Leung, J.C. Wong and M.G. Johlfs, 2012 [22].

### **2. Hyeractivation of PKG-Iα kinase activity in cancer cells contributes to the higher expression levels of Mcl-1 and certain Inhibitor of Apoptosis Proteins (IAPs), including c-IAP1, Livin and Survivin**

Downstream from PKG-Iα hyperactivity in cancer cells is the high levels of expression of "cell survival proteins", including Mcl-1 and certain IAPs, such as c-IAP1, Livin and Survivin [22]. These IAPs have been shown to regulate apoptosis and tumorigenesis [29]. Eight human IAPs have been identified [30] and are known to suppress apoptotic cell death, thus promoting cell survival and, in cancer cells, chemoresistance [31]. c-IAP1 and c-IAP2 possess a caspase recruitment domain [32], and c-IAP1, c-IAP2 and XIAP are known to directly inhibit caspase-3 and caspase-7 activity [33, 34]. Elevated expression of IAP proteins has been shown in almost all types of human cancers and has been implicated as therapeu‐ tic targets [35]. Particularly, XIAP was shown to play a predominant role in the inactiva‐ tion of apoptosome in non-small cell lung cancer NCI-H460 cells [36]. Survivin-specific siRNA was shown to increase apoptosis and inhibit cell proliferation in A549 lung cancer cells associated with activation of caspase-9 [37].

Although the NF-κB transcription factor is traditionally thought to regulate the expression of the IAPs, another transcription factor, CREB, has also been implicated in regulating some IAPs. For example, CREB phosphorylation at serine-133 and its subsequent activation are thought to be key events in the induction of c-IAP2 and Livin expression, potentially mediated by multiple protein kinases, including PKA, ERK1/2 and p38 MAPK, in colon cancer cells [38, 39].

Recent data from our laboratory have shown that the expression of three of the IAPs, c-IAP1, Livin and Survivin, are dependent on the NO/cGMP/PKG-Iα signaling pathway in non-small cell lung cancer (NSCLC) cells, which appears to involve the exaggerated phosphorylation of CREB at serine-133 catalyzed by the hyperactivated PKG-Iα [3]. We have also shown that some of these same signaling proteins, especially PKG-Iα and downstream elevation of the protein expression of c-IAP1and Livin as well as two other IAPs (c-IAP2 and XIAP) are involved in angiogenesis, using human endothelial cells (the HUVECs) as a model of tumor angiogenesis [4]. Interestingly, we found that resveratrol, a polyphenol from red wine, grapes, berries and peanuts known for its protection against cancers, when added at anti-angiogenesis and anticancer concentrations, inhibits the intracellular catalytic/kinase activity of PKG-Iα in the HUVECs, dramatically decreasing protein expression levels of c-IAP1, c-IAP2, Livin and XIAP [4]. Our data suggest that certain naturally-occurring anti-cancer agents, such as resveratrol, may prevent cancers by suppressing the PKG-Iα signaling pathway and lowering the expres‐ sion levels of the IAPs in the vascular endothelial cells of tumors, thus suppressing tumor angiogenesis.

### **3. Identification of PKG-I splice variants expressed in human ovarian cancer cells by using a new ultrasensitive advanced nano-proteomics technology, the NanoPro 1000 system**

Recent studies from our laboratory have used an ultrasensitive "advanced nano-proteomics" technology, called NanoPro 1000 (ProteinSimple, San Jose, CA, USA), to determine expression and phosphorylation levels of PKG-Iα as well as other protein kinases (especially Akt and c-Src, which interact with PKG-Iα and co-mediate the enhanced cell survival and resistance to apoptosis), and the various IAPs. This technology uses a robotic system for collecting samples and analyzing the samples, involving capillary isoelectricfocusing (cIEF) for separating proteins based on pI values, rather than molecular weight. This has a clear advantage over Western blot analysis when attempting to separate and identify proteins with similar molec‐ ular weight, such as isoforms of proteins.

This new NanoPro 1000 technology provides a sensitivity that is >500-times better than conventional Western blot analysis, thus allowing discovery of new signaling proteins that can be used for developing new therapeutic agents. This new technology allows for accurate measurements of lower abundance proteins (undetectable by conventional Western blot analysis), often using fewer than 1,000 mammalian cells for the analysis.

Anti-Angiogenic and Anti-Cancer Effects by Targeting the Protein Kinase G Type-Iα (PKG-Iα) Signaling Pathway… http://dx.doi.org/10.5772/60774 227

siRNA was shown to increase apoptosis and inhibit cell proliferation in A549 lung cancer

Although the NF-κB transcription factor is traditionally thought to regulate the expression of the IAPs, another transcription factor, CREB, has also been implicated in regulating some IAPs. For example, CREB phosphorylation at serine-133 and its subsequent activation are thought to be key events in the induction of c-IAP2 and Livin expression, potentially mediated by multiple protein kinases, including PKA, ERK1/2 and p38 MAPK, in colon cancer cells [38, 39]. Recent data from our laboratory have shown that the expression of three of the IAPs, c-IAP1, Livin and Survivin, are dependent on the NO/cGMP/PKG-Iα signaling pathway in non-small cell lung cancer (NSCLC) cells, which appears to involve the exaggerated phosphorylation of CREB at serine-133 catalyzed by the hyperactivated PKG-Iα [3]. We have also shown that some of these same signaling proteins, especially PKG-Iα and downstream elevation of the protein expression of c-IAP1and Livin as well as two other IAPs (c-IAP2 and XIAP) are involved in angiogenesis, using human endothelial cells (the HUVECs) as a model of tumor angiogenesis [4]. Interestingly, we found that resveratrol, a polyphenol from red wine, grapes, berries and peanuts known for its protection against cancers, when added at anti-angiogenesis and anticancer concentrations, inhibits the intracellular catalytic/kinase activity of PKG-Iα in the HUVECs, dramatically decreasing protein expression levels of c-IAP1, c-IAP2, Livin and XIAP [4]. Our data suggest that certain naturally-occurring anti-cancer agents, such as resveratrol, may prevent cancers by suppressing the PKG-Iα signaling pathway and lowering the expres‐ sion levels of the IAPs in the vascular endothelial cells of tumors, thus suppressing tumor

**3. Identification of PKG-I splice variants expressed in human ovarian cancer cells by using a new ultrasensitive advanced nano-proteomics**

Recent studies from our laboratory have used an ultrasensitive "advanced nano-proteomics" technology, called NanoPro 1000 (ProteinSimple, San Jose, CA, USA), to determine expression and phosphorylation levels of PKG-Iα as well as other protein kinases (especially Akt and c-Src, which interact with PKG-Iα and co-mediate the enhanced cell survival and resistance to apoptosis), and the various IAPs. This technology uses a robotic system for collecting samples and analyzing the samples, involving capillary isoelectricfocusing (cIEF) for separating proteins based on pI values, rather than molecular weight. This has a clear advantage over Western blot analysis when attempting to separate and identify proteins with similar molec‐

This new NanoPro 1000 technology provides a sensitivity that is >500-times better than conventional Western blot analysis, thus allowing discovery of new signaling proteins that can be used for developing new therapeutic agents. This new technology allows for accurate measurements of lower abundance proteins (undetectable by conventional Western blot

analysis), often using fewer than 1,000 mammalian cells for the analysis.

cells associated with activation of caspase-9 [37].

226 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

**technology, the NanoPro 1000 system**

ular weight, such as isoforms of proteins.

angiogenesis.

**Figure 3.** Electropherograms generated by the new technology of NanoPro 1000 system, a capillary isoelectrofocusing (cIEF) instrument for quantifying protein expression levels and phosphorylations levels, with a sensitive >500-times greater than traditional Western blot analysis. The HUVECs are used as positive controls for illustrating the expression of both splice variants of PKG-I in the same cell population. All three of the human ovarian cancer cell lines express only the PKG-Iα splice variant.

Figure 3 illustrates recent experiments showing the expression of PKG-I splice variants in three different ovarian cancer cell lines, CAOV3, SKOV3 and OCC-1 cells, determined by using the NanoPro 1000 technology. The top electropherogram shows that the HUVECs, human endothelial cells, express both PKG-I splice variants, confirming our previous studies of HUVECs using the older, related technology, the NanoPro 100 system, recently published by our laboratory in *Anticancer Research* [4].

Note that all three of the ovarian cancer cell lines shown in Figure 3 express exclusively the PKG-Iα splice variant, in contrast to the expression pattern shown in the human endothelial cells expressing both splice variants of PKG-I. The protein expression levels of PKG-Iα in all of these human ovarian cancer cell lines are typically below the detection limits when analyzed by traditional Western blot analysis, as shown in our previous book chapter on ovarian cancer cells [22]. Thus, the advanced nano-proteomic technology of the robotic cIEF-based NanoPro 1000 system provides a valuable new research tool for studying the expression levels of lower abundance proteins that are undetectable by the 30-year-old technique of Western blot analysis (500-times less sensitive compared to the NanoPro 1000 system).

## **4. Human ovarian cancer cells have hyperactivated PKG-Iα, as quantified by a newly- developed ultrasensitive near-infrared-fluorescence (NIRF) based kinase assay for measuring PKG catalytic/kinase activity in tissue samples and cell lysates**

Recently, our laboratory has successfully development a new, ultrasensitive methodology for accurately measuring the catalytic/kinase activity of any protein kinase within biological samples (cell lysates and tissue homogenates) using NIRF-labeled peptide substrates rather than the old technique of using radioactive (32P- or 33P-labeled) ATP. The radioactive protein kinase assays were originally developed in the 1970s and 1980s for measuring the catalytic activity of protein kinase A (PKA) and PKG in freshly-prepared tissue homogenates [5-10, 22, 23]. The new NIRF-based protein kinase assays were developed for improving safety and for lowering the cost of analysis.

Figure 4 illustrates the use of this new methodology, showing that four different ovarian cancer cell lines, CAOV3, OCC1, SKOV3 and A2780cp cells, all possess measureable levels of endogenous PKG-Iα catalytic/kinase activity and that this kinase activity is indeed hyperac‐ tivated in all of the ovarian cancer cell lines. The NIRF-labeled peptide substrate used in this assay can also be phosphorylated by eight of the most common isoforms of protein kinase C (PKC), but not by other related protein kinases, such as Akt1, Akt2, p70S6-kinase and RSK2. Thus, the catalytic activities of both PKG and PKC can be measured simultaneously in the same biological sample. To define the component of kinase activity contributed by endogenous PKC, we used a combination of four PKC inhibitors (AEB071, Gö 6976, Gö 6983 and LY333,531), which selectively inhibit the eight isoforms of PKC capable of phosphorylating the NIRFpeptide substrate. This defines the PKC catalytic activity from PKG catalytic activity in complex mixtures of protein kinases, such as tissue homogenates and cell lysates. The kinase

catalytic activity remaining in the presence of the four PKC inhibitors represents only PKG catalytic activity, show in Figure 4.

Figure 3 illustrates recent experiments showing the expression of PKG-I splice variants in three different ovarian cancer cell lines, CAOV3, SKOV3 and OCC-1 cells, determined by using the NanoPro 1000 technology. The top electropherogram shows that the HUVECs, human endothelial cells, express both PKG-I splice variants, confirming our previous studies of HUVECs using the older, related technology, the NanoPro 100 system, recently published by

Note that all three of the ovarian cancer cell lines shown in Figure 3 express exclusively the PKG-Iα splice variant, in contrast to the expression pattern shown in the human endothelial cells expressing both splice variants of PKG-I. The protein expression levels of PKG-Iα in all of these human ovarian cancer cell lines are typically below the detection limits when analyzed by traditional Western blot analysis, as shown in our previous book chapter on ovarian cancer cells [22]. Thus, the advanced nano-proteomic technology of the robotic cIEF-based NanoPro 1000 system provides a valuable new research tool for studying the expression levels of lower abundance proteins that are undetectable by the 30-year-old technique of Western blot analysis

**4. Human ovarian cancer cells have hyperactivated PKG-Iα, as quantified by a newly- developed ultrasensitive near-infrared-fluorescence (NIRF) based kinase assay for measuring PKG catalytic/kinase activity in tissue**

Recently, our laboratory has successfully development a new, ultrasensitive methodology for accurately measuring the catalytic/kinase activity of any protein kinase within biological samples (cell lysates and tissue homogenates) using NIRF-labeled peptide substrates rather than the old technique of using radioactive (32P- or 33P-labeled) ATP. The radioactive protein kinase assays were originally developed in the 1970s and 1980s for measuring the catalytic activity of protein kinase A (PKA) and PKG in freshly-prepared tissue homogenates [5-10, 22, 23]. The new NIRF-based protein kinase assays were developed for improving safety and for

Figure 4 illustrates the use of this new methodology, showing that four different ovarian cancer cell lines, CAOV3, OCC1, SKOV3 and A2780cp cells, all possess measureable levels of endogenous PKG-Iα catalytic/kinase activity and that this kinase activity is indeed hyperac‐ tivated in all of the ovarian cancer cell lines. The NIRF-labeled peptide substrate used in this assay can also be phosphorylated by eight of the most common isoforms of protein kinase C (PKC), but not by other related protein kinases, such as Akt1, Akt2, p70S6-kinase and RSK2. Thus, the catalytic activities of both PKG and PKC can be measured simultaneously in the same biological sample. To define the component of kinase activity contributed by endogenous PKC, we used a combination of four PKC inhibitors (AEB071, Gö 6976, Gö 6983 and LY333,531), which selectively inhibit the eight isoforms of PKC capable of phosphorylating the NIRFpeptide substrate. This defines the PKC catalytic activity from PKG catalytic activity in complex mixtures of protein kinases, such as tissue homogenates and cell lysates. The kinase

our laboratory in *Anticancer Research* [4].

228 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

**samples and cell lysates**

lowering the cost of analysis.

(500-times less sensitive compared to the NanoPro 1000 system).

The percent activation of PKG-Iα in the four human ovarian cancer cell lines was all above 90%, indicating that PKG-Iα is indeed hyperactivated (Figure 4) in these malignant cells. For comparison, homogenates of two normal tissues, vascular smooth muscle (V.S.M.) from rat aorta and human pancreatic islets, were also analyzed. The % activation of PKG in smooth muscle tissue was 21%, similar to the % activation measured by the 32P-ATP-based method‐ ology developed and used by Dr. R.R. Fiscus in the early and mid-1980s [5, 7-9], and the PKG activation in pancreatic islets was 31%. Thus, compared to normal tissues, all of the ovarian cancer cell lines have highly-activated PKG. This hyperactivation of PKG-Iα in the ovarian cancer cells would result in downstream (hyper)activation of c-Src and CREB, resulting in an exaggerated expression of "cell survival proteins", such as Mcl-1 and certain IAPs, e.g. c-IAP1, Livin and Survivin, likely contributing to the aggressive nature of this form of cancer.

**Figure 4.** Hyperactivation of PKG catalytic/kinase activity in four ovarian cancer cell lines. A, picture representative of the near infrared-fluorescence (NIRF)-based kinase assay, using the CAOV3 ovarian cancer cells as an example. The % of phosphorylation was calculated based on the fluorescence signal measured by LI-COR Odyssey CLx scanner. The activity ratio, activity –cGMP divided by activity +cGMP (i.e. kinase activity with versus without the addition of exoge‐ nous cGMP (10 µM), an allosteric activator of PKG). All measurements were done in the presence of a combination of select PKC inhibitors (to remove kinase activity contributed by PKC isoforms). The remaining kinase activity reflects the PKG catalytic activity in these cell lines. The data in A., i.e. 2.41, 2.50, 2.27 and 2.44, represent the percent phosphor‐ ylation of the NIRF-labeled peptide substrate during a 2-min reaction. B, The % of PKG activation (i.e. 100 X the activi‐ ty ratio) for each sample analyzed is shown in a bar graph. Data was obtained by measuring kinase activity by the new NIRF-based kinase assay method. The four ovarian cancer cell lines, A2780cp, SKOV3, OCC-1 and CAOV3, had % of PKG activation of 93%, 98%, 115% and 94%, respectively, showing that PKG is clearly hyperactivated in all of the ovar‐ ian cancer cells tested. For comparison, tissue samples of freshly-isolated vascular smooth muscle (V.S.M.) from rat aorta and human pancreatic islets (purchased from Lonza) were used to show PKG catalytic/kinase activity in normal non-cancerous tissues. Note that the % activation of PKG is considerably lower in normal tissue, showing 21% activa‐ tion of PKG in vascular smooth muscle cells (V.S.M.) and 31% activation of PKG in human pancreatic islets.

#### **5. Future experiments**

Our future studies will focus on: 1) determining the expression of PKG isoform and its various phospho-forms in human ovarian cancer cell lines and clinical samples of ovarian tumors using the NanoPro 1000 system, 2) determining the PKG kinase activity of other ovarian cancer cell lines and clinical samples of ovarian tumors using our patented NIRF-based kinase assay, and 3) studying the phosphorylation/activation of the transcription factor CREB and the expression profile of the IAPs using our advanced nano-proteomics technology, with special focus on c-IAP1, c-IAP2, Livin, Survivin and XIAP, in ovarian cancer cells and vascular endothelial cells. The goal of our future experiments is to ultimately develop new therapeutic agents that can target these novel signaling pathways in order to effectively treat chemoresistant ovarian cancer and tumor angiogenesis.

The NanoPro 1000 system is especially useful for studying the multiple phospho-forms of proteins, because of its ability to separate proteins based on pI values rather than molecular weight. Each addition of a phosphate molecule to a protein typically causes a measurable (and resolvable) shift in the pI value, which can be used to determine intracellular activation and/or the catalytic function of a protein kinase within cells. Likewise, our patented technology using NIRF-based kinase assays will be used to determine the effectiveness and potency of protein kinase inhibitors that could potentially be used to treat cancer.

#### **6. Conclusions**

Our studies of human ovarian cancer cells as well as other types of cancer cells (e.g. breast cancer, lung cancer, mesothelioma, neuroblastoma and prostate cancer) have identified the NO/cGMP/PKG-Iα signaling pathway as a key cellular mechanism involved in mediating the exaggerated cell proliferation and chemoresistance of these cancers [3, 15, 19-22]. We have shown that the PKG-Iα splice variant of PKG-I, which is expressed in all of these cancer cells, directly phosphorylates important intracellular proteins, including BAD, CREB and c-Src, leading to enahanced cell survival (i.e. chemoresistance) and exaggerated cell proliferation. Specifically, phosphorylation of CREB at serine-133 following activation of PKG-Iα results in increased gene expression of several "cell survival proteins", including Mcl-1 and some of the the IAPs.

In human lung cancer cells, the NO/PKG-Iα signaling pathway represents a major mechanism for the enhanced gene expression of three IAPs, c-IAP1, Livin and Survivin, which corresponds with exaggerated cell proliferation and resistance to cisplatin-induced apoptosis [3]. Blocking the PKG-Iα catalytic activity or knocking-down the gene expression of PKG-Iα using siRNA dramatically sensitizes these chemoresistant cells to the cancer-killing effects of cisplatin. We anticipate that our future studies with ovarian cancer cells will show similar results.

In HUVECs, human endothelial cells used as a model of tumor angiogenesis, the eNOS/NO/ cGMP/PKG signaling pathway is now recognized to mediate the pro-angiogenesis effects of VEGF [4]. VEGF is a factor that is released in large amounts from cancer cells, including human ovarian cancer cells. Our studies with HUVECs show that four IAPs, c-IAP1, c-IAP2, Livin and XIAP, are all downregulated by resveratrol, a polyphenol from grapes, berries, peanuts and red wine, and that this response corresponds to the inhibition of PKG catalytic activity in the HUVECs.

**5. Future experiments**

230 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

cancer and tumor angiogenesis.

**6. Conclusions**

the IAPs.

Our future studies will focus on: 1) determining the expression of PKG isoform and its various phospho-forms in human ovarian cancer cell lines and clinical samples of ovarian tumors using the NanoPro 1000 system, 2) determining the PKG kinase activity of other ovarian cancer cell lines and clinical samples of ovarian tumors using our patented NIRF-based kinase assay, and 3) studying the phosphorylation/activation of the transcription factor CREB and the expression profile of the IAPs using our advanced nano-proteomics technology, with special focus on c-IAP1, c-IAP2, Livin, Survivin and XIAP, in ovarian cancer cells and vascular endothelial cells. The goal of our future experiments is to ultimately develop new therapeutic agents that can target these novel signaling pathways in order to effectively treat chemoresistant ovarian

The NanoPro 1000 system is especially useful for studying the multiple phospho-forms of proteins, because of its ability to separate proteins based on pI values rather than molecular weight. Each addition of a phosphate molecule to a protein typically causes a measurable (and resolvable) shift in the pI value, which can be used to determine intracellular activation and/or the catalytic function of a protein kinase within cells. Likewise, our patented technology using NIRF-based kinase assays will be used to determine the effectiveness and potency of

Our studies of human ovarian cancer cells as well as other types of cancer cells (e.g. breast cancer, lung cancer, mesothelioma, neuroblastoma and prostate cancer) have identified the NO/cGMP/PKG-Iα signaling pathway as a key cellular mechanism involved in mediating the exaggerated cell proliferation and chemoresistance of these cancers [3, 15, 19-22]. We have shown that the PKG-Iα splice variant of PKG-I, which is expressed in all of these cancer cells, directly phosphorylates important intracellular proteins, including BAD, CREB and c-Src, leading to enahanced cell survival (i.e. chemoresistance) and exaggerated cell proliferation. Specifically, phosphorylation of CREB at serine-133 following activation of PKG-Iα results in increased gene expression of several "cell survival proteins", including Mcl-1 and some of the

In human lung cancer cells, the NO/PKG-Iα signaling pathway represents a major mechanism for the enhanced gene expression of three IAPs, c-IAP1, Livin and Survivin, which corresponds with exaggerated cell proliferation and resistance to cisplatin-induced apoptosis [3]. Blocking the PKG-Iα catalytic activity or knocking-down the gene expression of PKG-Iα using siRNA dramatically sensitizes these chemoresistant cells to the cancer-killing effects of cisplatin. We

In HUVECs, human endothelial cells used as a model of tumor angiogenesis, the eNOS/NO/ cGMP/PKG signaling pathway is now recognized to mediate the pro-angiogenesis effects of

anticipate that our future studies with ovarian cancer cells will show similar results.

protein kinase inhibitors that could potentially be used to treat cancer.

Figure 5 shows a model representing the involvement of the NO/cGMP/PKG-Iα signaling pathway in mediating the pro-angiogenesis effects of VEGF within vascular endothelial cells, promoting the increased expression of the IAPs, c-IAP1, c-IAP2, Livin and XIAP, based on our recent publication [4]. Also shown is the inhibitory action of resveratrol, at anti-angiogenic and anti-cancer concentrations, on the PKG-Iα catalytic actions and downstream expression of c-IAP1, c-IAP-2, Livin and XIAP. We have proposed that the ability of resveratrol to prevent cancers may relate to its ability to inhibit PKG-Iα catalytic activity selectively in tumor endothelial cells involved in tumor angiogenesis, thus suppressing the expression of IAPs in the endothelial cells and the tumor angiogenesis.

**Figure 5.** Involvement of eNOS, NO, cGMP and PKG-Iα in the pro-angiogenesis actions of VEGF in vascular endothe‐ lial cells. Enhanced PKG-Iα catalytic activity induced by VEGF can increase CREB phosphorylation and activation, in‐ creasing expression of certain IAPs. The traditional transcription factor thought to be involved in promoting the gene expression of the IAPs, i.e. NF-κB, is also shown, although its role in PKG-Iα-mediated increases in the IAPs and tu‐ mor angiogenesis is not yet known.

#### **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 the University of Southern Nevada and the Nevada College of Pharmacy), Henderson, Nevada, USA, from a grant from the U.S. Department of Defense (DOD), grant # W81XWH-07-1-0543, and from Start-up Funding from the Nevada Cancer Institute, Las Vegas, Nevada, USA, all awarded to Dr. Fiscus.

#### **Author details**

Janica C. Wong1,2,3, Priyatham Gorjala1 , Benjamin Costantino1 and Ronald R. Fiscus1,3,4\*

\*Address all correspondence to: rfiscus@roseman.edu

1 Roseman University of Health Sciences, College of Medicine, Department of Biomedical Sciences, the Diabetes & Obesity Research Center and the Cancer Research Center, Roseman Medical Education & Research Building at Summerlin Campus, Las Vegas, Nevada, USA

2 Stanford University, School of Medicine, Department of Pediatrics, Stanford, California, USA

3 Nevada Cancer Institute, Cancer Molecular Biology Section, Las Vegas, Nevada, USA

4 Roseman University of Health Sciences, College of Pharmacy, Henderson, Nevada, USA

#### **References**


[4] Wong JC, Fiscus RR. Resveratrol at anti-angiogenesis/anticancer concentrations sup‐ presses protein kinase G signaling and decreases IAPs expression in HUVECs. Anti‐ cancer Research. 2015;35:273-82.

**Acknowledgements**

Dr. Fiscus.

USA

**References**

**Author details**

Janica C. Wong1,2,3, Priyatham Gorjala1

Control. 2011 Jan;18(1):31-43.

23(2):256-63.

3587-98.

\*Address all correspondence to: rfiscus@roseman.edu

232 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

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 the University of Southern Nevada and the Nevada College of Pharmacy), Henderson, Nevada, USA, from a grant from the U.S. Department of Defense (DOD), grant # W81XWH-07-1-0543, and from Start-up Funding from the Nevada Cancer Institute, Las Vegas, Nevada, USA, all awarded to

, Benjamin Costantino1

1 Roseman University of Health Sciences, College of Medicine, Department of Biomedical Sciences, the Diabetes & Obesity Research Center and the Cancer Research Center, Roseman Medical Education & Research Building at Summerlin Campus, Las Vegas, Nevada, USA

2 Stanford University, School of Medicine, Department of Pediatrics, Stanford, California,

3 Nevada Cancer Institute, Cancer Molecular Biology Section, Las Vegas, Nevada, USA

4 Roseman University of Health Sciences, College of Pharmacy, Henderson, Nevada, USA

[1] Teoh DG, Secord AA. Antiangiogenic therapies in epithelial ovarian cancer. Cancer

[2] Chen L, Liang L, Yan X, Liu N, Gong L, Pan S, et al. Survivin status affects prognosis and chemosensitivity in epithelial ovarian cancer. Int J Gynecol Cancer. 2013 Feb;

[3] Wong JC, Bathina M, Fiscus RR. Cyclic GMP/protein kinase G type-Iα (PKG-Iα) sig‐ naling pathway promotes CREB phosphorylation and maintains higher c-IAP1, livin, survivin, and Mcl-1 expression and the inhibition of PKG-Iα kinase activity synergiz‐ es with cisplatin in non-small cell lung cancer cells. J Cell Biochem. 2012 Nov;113(11):

and Ronald R. Fiscus1,3,4\*


PKG-Iα autocrine loop in promoting proliferation and cell survival of OP9 bone mar‐ row stromal cells. J Cell Biochem. 2011 Mar;112(3):829-39.


[28] Caneba CA, Yang L, Baddour J, Curtis R, Win J, Hartig S, et al. Nitric oxide is a posi‐ tive regulator of the Warburg effect in ovarian cancer cells. Cell Death Dis. 2014;5:e1302.

PKG-Iα autocrine loop in promoting proliferation and cell survival of OP9 bone mar‐

[18] Fraser M, Chan SL, Chan SS, Fiscus RR, Tsang BK. Regulation of p53 and suppres‐ sion of apoptosis by the soluble guanylyl cyclase/cGMP pathway in human ovarian

[19] Leung EL, Fraser M, Fiscus RR, Tsang BK. Cisplatin alters nitric oxide synthase levels in human ovarian cancer cells: involvement in p53 regulation and cisplatin resist‐

[20] Leung EL, Wong JC, Johlfs MG, Tsang BK, Fiscus RR. Protein kinase G type Ialpha activity in human ovarian cancer cells significantly contributes to enhanced Src acti‐ vation and DNA synthesis/cell proliferation. Mol Cancer Res. 2010 Apr;8(4):578-91.

[21] Wong JC, Fiscus RR. Protein kinase G-Iα hyperactivation and VASP phosphorylation in promoting ovarian cancer cell migration and platinum resistance. In: Diaz-Padilla I, editor. Ovarian Cancer - A Clinical and Translational Update: INTECH; 2013. p.

[22] Fiscus RR, Leung EL, Wong JC, Johlfs MG. Nitric oxide/protein kinase G-Iα promote c-Src activation, proliferation and chemoresistance in ovarian cancer. In: Farghaly S, editor. Ovarian Cancer - Basic Science Perspective: Intech Open Access Publisher;

[23] Fiscus RR, Johlfs MG. Protein kinase G (PKG): Involvement in promoting neural cell survival, proliferation, synaptogenesis and synaptic plasticity and the use of new ul‐ trasensitive capillary-electrophoresis-based methodologies for measuring PKG ex‐ pression and molecular actions. In: Mukai H, editor. Protein Kinase Technologies -

[24] Santin AD, Hermonat PL, Ravaggi A, Cannon MJ, Pecorelli S, Parham GP. Secretion of vascular endothelial growth factor in ovarian cancer. Eur J Gynaecol Oncol.

[25] Duncan TJ, Al-Attar A, Rolland P, Scott IV, Deen S, Liu DT, et al. Vascular endothe‐ lial growth factor expression in ovarian cancer: a model for targeted use of novel

[26] Hood J, Granger HJ. Protein kinase G mediates vascular endothelial growth factorinduced Raf-1 activation and proliferation in human endothelial cells. J Biol Chem.

[27] Deguchi A, Thompson WJ, Weinstein IB. Activation of protein kinase G is sufficient to induce apoptosis and inhibit cell migration in colon cancer cells. Cancer Res. 2004

row stromal cells. J Cell Biochem. 2011 Mar;112(3):829-39.

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## **Cancer of the Vulva — A Review**

Fernando Anschau and Manoel Afonso Guimarães Gonçalves

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61579

#### **Abstract**

Cancer of the Vulva: a review In reporting on cancer of the vulva, we should keep in mind some important aspects of its epidemiology and its early detection. Most of the pa‐ pers on the subject refer to vulvar cancer as a rare disease, accounting for 4 to 5% of all malignant neoplasms of the female genital tract and less than 1% of women's cancers. The incidence varies from 1 to 3.6 cases per 100,000 women, with peak incidence at ages 70-79 years. Even though the incidence increases with age, the proportion of young patients with vulvar cancer has greatly increased due to its association with infection with human papillomavirus (HPV). The risk of developing cancer of the vulva is related to behavioral, reproductive, hormonal and genetic aspects. Factors that increase risk include other geni‐ tal cancers, chronic inflammatory diseases of the vulva, smoking, history of genital warts and vulvar intraepithelial neoplasia (VIN). We can consider that, given the epidemiologi‐ cal evidence, there are two etiologic pathways for vulvar cancer: one related to older pa‐ tients, in the seventh or eighth decades of life, associated with mutations in TP53 and non-neoplastic epithelial disorders such as chronic inflammation or vulvar lichen, shows precursor lesions of differentiated VIN; the other is more common in young patients, ac‐ counts for approximately 43-60% of squamous carcinoma of the vulva, is associated with HPV infection, and is a common precursor lesion of VIN. Eighty-five to ninety percent of vulvar cancers are squamous in origin (squamous cell carcinoma); however, when con‐ sidering the embryological origin of the vulva - the three germ layers - different histolog‐ ic types can compose neoplasms affecting the region.

**Keywords:** Vulvar cancer, clinical presentation, staging, treatment, prognostic factor, re‐ view, signs and symptoms, therapy, innovations

#### **1. Introduction**

In reporting on cancer of the vulva, we should keep in mind some important aspects of its epidemiology and its early detection. Most of the papers on the subject refer to vulvar cancer as a rare disease, accounting for 4–5% of all malignant neoplasms of the female genital tract

© 2016 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.

and less than 1% of women's cancers. The incidence varies from 1 to 3.6 cases per 100,000 women, with peak incidence at ages 70–79 years. [1, 2, 3, 4] Even though the incidence increases with age, the proportion of young patients with vulvar cancer has greatly increased due to its association with infection with human papillomavirus (HPV). 5 The risk of developing cancer of the vulva is related to behavioral, reproductive, hormonal, and genetic aspects. Factors that increase risk include other genital cancers, chronic inflammatory diseases of the vulva, smoking, history of genital warts, and vulvar intraepithelial neoplasia (VIN).

We can consider that, given the epidemiological evidence, there are two etiologic path‐ ways for vulvar cancer: one is related to older patients, in the seventh or eighth decades of life, associated with mutations in TP53 and non-neoplastic epithelial disorders such as chronic inflammation or vulvar lichen, and shows precursor lesions of differentiated VIN; the other is more common in young patients, accounts for approximately 43–60% of squamous carcinoma of the vulva, is associated with HPV infection, and is a common precursor lesion of VIN. [6, 7, 8, 9]

Eighty-five to ninety percent of vulvar cancers are squamous in origin (squamous cell carcinoma); however, when considering the embryological origin of the vulva – the three germ layers – different histologic types can compose neoplasms affecting the region. Melanoma is the second most common and should be discussed separately because of its peculiar charac‐ teristics.

Prognosis is strongly related to lymph node status and the stage of disease, reaching 90% survival for early stages without lymph node involvement.[1, 10]

Various important advances in the treatment of vulvar cancer were made in recent decades toward more conservative surgery without compromising survival and toward reduction of comorbidities, such as: (1) conservation of the vulva in patients with unifocal tumors, and normal vulva in other aspects; (2) omission of inguinal lymphadenectomy in patients with T1 tumors and stromal invasion <1 mm; (3) elimination of routine pelvic lymphadenectomy; (4) use separate incisions for inguinal dissection; (5) use of preoperative radiotherapy in selected cases; and (6) postoperative radiotherapy to reduce inguinal recurrence in patients with multiple compromised inguinal lymph nodes.

#### **2. Clinical presentation**

The diagnosis is often delayed, since vulvar cancer does not show specific signs and symptoms, and older patients do not usually examine their vulva preventively and report their symptoms. Vulvar cancer may be asymptomatic, but the majority of patients present with nodules or vulvar ulcer. Such signs may be accompanied by pain, but it may also be absent. The long‐ standing pruritus is frequent and may be associated with vulvar dystrophy. Secretions and bleeding are symptoms that are occasionally present, as well as dyspareunia and burning sensation. Putrid odor due to tissue necrosis may also be diagnostic. Enlarged lymph nodes, especially in the inguinal region, denote disease in later stages.[1, 10, 11] A study of delayed diagnosis showed that in 88% of patients, symptoms were already present for about 6 months and in 28% for more than 5 years. In the same study, emphasis was placed on clinical suspicion in view of symptoms, since 31% of patients had office visits three or more times, but only 25% of cases had undergone biopsy. [12]

As previously mentioned, vulvar melanoma will be addressed separately, but any pigmenta‐ tion of the vulva deserves attention for diagnosis.

#### **3. Screening**

and less than 1% of women's cancers. The incidence varies from 1 to 3.6 cases per 100,000 women, with peak incidence at ages 70–79 years. [1, 2, 3, 4] Even though the incidence increases with age, the proportion of young patients with vulvar cancer has greatly increased due to its association with infection with human papillomavirus (HPV). 5 The risk of developing cancer of the vulva is related to behavioral, reproductive, hormonal, and genetic aspects. Factors that increase risk include other genital cancers, chronic inflammatory diseases of the vulva,

We can consider that, given the epidemiological evidence, there are two etiologic path‐ ways for vulvar cancer: one is related to older patients, in the seventh or eighth decades of life, associated with mutations in TP53 and non-neoplastic epithelial disorders such as chronic inflammation or vulvar lichen, and shows precursor lesions of differentiated VIN; the other is more common in young patients, accounts for approximately 43–60% of squamous carcinoma of the vulva, is associated with HPV infection, and is a common

Eighty-five to ninety percent of vulvar cancers are squamous in origin (squamous cell carcinoma); however, when considering the embryological origin of the vulva – the three germ layers – different histologic types can compose neoplasms affecting the region. Melanoma is the second most common and should be discussed separately because of its peculiar charac‐

Prognosis is strongly related to lymph node status and the stage of disease, reaching 90%

Various important advances in the treatment of vulvar cancer were made in recent decades toward more conservative surgery without compromising survival and toward reduction of comorbidities, such as: (1) conservation of the vulva in patients with unifocal tumors, and normal vulva in other aspects; (2) omission of inguinal lymphadenectomy in patients with T1 tumors and stromal invasion <1 mm; (3) elimination of routine pelvic lymphadenectomy; (4) use separate incisions for inguinal dissection; (5) use of preoperative radiotherapy in selected cases; and (6) postoperative radiotherapy to reduce inguinal recurrence in patients with

The diagnosis is often delayed, since vulvar cancer does not show specific signs and symptoms, and older patients do not usually examine their vulva preventively and report their symptoms. Vulvar cancer may be asymptomatic, but the majority of patients present with nodules or vulvar ulcer. Such signs may be accompanied by pain, but it may also be absent. The long‐ standing pruritus is frequent and may be associated with vulvar dystrophy. Secretions and bleeding are symptoms that are occasionally present, as well as dyspareunia and burning sensation. Putrid odor due to tissue necrosis may also be diagnostic. Enlarged lymph nodes, especially in the inguinal region, denote disease in later stages.[1, 10, 11] A study of delayed diagnosis showed that in 88% of patients, symptoms were already present for about 6 months

survival for early stages without lymph node involvement.[1, 10]

multiple compromised inguinal lymph nodes.

**2. Clinical presentation**

smoking, history of genital warts, and vulvar intraepithelial neoplasia (VIN).

238 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

precursor lesion of VIN. [6, 7, 8, 9]

teristics.

There is no standard procedure for screening for vulvar cancer. However, we must maintain a level of suspicion when seeing patients with signs and symptoms related to the vulvar region and to be attentive to the examination of the female genitalia at check-ups. Patients with history of vaginal and cervical cancer should have the vulva inspected, with or without colposcopy (vulvoscopy), as part of a regular follow-up. Similarly, those with lichen sclerosus or VIN history deserve regular monitoring.

#### **4. Gynecological examination**

The vulvar cancer can arise in any region of the vulva, but the most common locations are labia (80%), clitoris (10%), and frenulum region (10%). Most tumors are unilateral, but may present as bilateral or multicentric (Figures 1, 2, and 3). Any malignant neoplastic lesion involving the vagina and the vulva should be classified as vulvar cancer.

**Figure 1.** Squamous cell carcinoma of the vulva – clitoral region with involvement of labia minora and majora.

**Figure 2.** Adenocarcinoma of the vulva – frenulum region extending to the top of the right thigh.

**Figure 3.** (a) Squamous cell carcinoma of the vulva with destruction of vulvar anatomy. (b) Carcinoma of the vulva associated with lichen sclerosus Presence of white areas, cracks, macules, plaques, thin and hypochromic skin, and tu‐ mor infiltrating next to the clitoris.

On the basis of this knowledge, a careful inspection of the vulva and vagina are important points in the gynecological examination and may include vulvoscopy and colposcopy. Suspicious areas with color changes, redness, ulcers, papules, macules, erosions, thickening of skin, bumps, and cracks must be observed carefully. The lesions may grow and form infiltrating endophytic or exophytic tissue with the formation of visible tumors. When identifying suspicious areas, a histological examination should be performed by biopsy that includes not only the skin but also the subepithelial stroma. Multifocal lesions require multiple biopsies for the investigation of histological changes (Figure 3.1).

Considering that a significant proportion of vulvar cancers are related to HPV infection, especially in younger patients, the vagina (again) and the cervix should be examined. The cytological examination of the cervix should be carried out according to the current screening parameters. Cytology of the vulva – depending on its collection and proper laboratory analysis – achieves a sensitivity of 95% and specificity of 64% 13; although there is no clear reason to recommend it, it would make the biopsy much more effective. In addition to the inspection, palpation of the genitals, anal/rectal region, pelvic walls, and inguinal regions should be performed. 14 Collins' test (based on toluidine blue staining) was used to demonstrate nuclear changes reacting with toluidine blue; however, the method is non-specific and has not been used in the diagnosis of cancer. The use of acetic acid can highlight lesions and their dimen‐ sions, but does not adequately differentiate benign from malignant lesions (Figures 4 and 5).

**Figure 4.** Macula next to right labium majora

**Figure 5.** After application of 2% acetic acid – acetowhite lesion with more defined edges (biopsy-confirmed VIN)

#### **5. Staging**

**Figure 2.** Adenocarcinoma of the vulva – frenulum region extending to the top of the right thigh.

240 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

**Figure 3.** (a) Squamous cell carcinoma of the vulva with destruction of vulvar anatomy. (b) Carcinoma of the vulva associated with lichen sclerosus Presence of white areas, cracks, macules, plaques, thin and hypochromic skin, and tu‐

On the basis of this knowledge, a careful inspection of the vulva and vagina are important points in the gynecological examination and may include vulvoscopy and colposcopy. Suspicious areas with color changes, redness, ulcers, papules, macules, erosions, thickening of skin, bumps, and cracks must be observed carefully. The lesions may grow and form infiltrating endophytic or exophytic tissue with the formation of visible tumors. When identifying suspicious areas, a histological examination should be performed by biopsy that includes not only the skin but also the subepithelial stroma. Multifocal lesions require multiple

Considering that a significant proportion of vulvar cancers are related to HPV infection, especially in younger patients, the vagina (again) and the cervix should be examined. The

biopsies for the investigation of histological changes (Figure 3.1).

mor infiltrating next to the clitoris.

The staging system of cancer of the vulva has changed in recent years and since 1988 has become surgical. The final diagnosis and therefore the stage classification depends on the histopathological evaluation of the surgical specimen (vulva and lymph nodes). The classifi‐ cation of the International Federation of Gynecology and Obstetrics (FIGO) was last changed in 2009 by the FIGO Committee on Gynecologic Oncology and provides a good discrimination between prognosis and stages (Table 1). [15] The International Union for Cancer Control (UICC) provides a classification for tumor (T), lymph nodes (N), and metastasis (M) (TNM classification), which is shown in the following text compared to the FIGO classification (Table 2). [16]


<sup>a</sup> The depth of invasion is defined as the measurement of the tumor from the epithelial-stromal junction of the adjacent most superficial dermal papilla to the deepest point of invasion.

**Table 1.** FIGO classification of cancer of the vulva



**Table 2.** Staging of cancer of the vulva (FIGO and UICC (TNM))

#### **6. Principles of staging**

cation of the International Federation of Gynecology and Obstetrics (FIGO) was last changed in 2009 by the FIGO Committee on Gynecologic Oncology and provides a good discrimination between prognosis and stages (Table 1). [15] The International Union for Cancer Control (UICC) provides a classification for tumor (T), lymph nodes (N), and metastasis (M) (TNM classification), which is shown in the following text compared to the FIGO classification (Table

IA Lesions ≤2 cm in size, confined the vulva or perineum and with stromal invasion ≤1.0mm<sup>a</sup>

II Tumor of any size with extension to adjacent perineal structures (lower third of urethra, lower third of

III Tumor of any size with or without extension to adjacent perineal structures (lower third of urethra, lower

IIIA (i) with 1 lymph node metastasis (≥5 mm), or (ii) with 1–2 lymph node metastasis(es) (<5 mm) IIIB (i) with 2 lymph node metastasis (≥5 mm), or (ii) with 3 lymph node metastasis(es) (<5 mm)

<sup>a</sup> The depth of invasion is defined as the measurement of the tumor from the epithelial-stromal junction of the adjacent

T3 Invasion of the upper part of urethra/vagina, bladder, rectal mucosa, bone, fixation in pelvic

(i) upper urethral and/or vaginal mucosa, bladder mucosa, rectal mucosa, or fixed to pelvic bone

, no

, confined to the vulva or perineum, with

2). [16]

**FIGO Stage Description**

I Tumor confined to the vulva

nodal metastasis

vagina, anus) with negative nodes

negative lymph nodes

242 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

IB Lesions >2 cm in size or with stromal invasion >1.0mm<sup>a</sup>

third of vagina, anus) with positive inguinofemoral lymph nodes

IV Tumor invades other regional (upper 2/3 urethra, upper 2/3 vagina), or distant structures

(ii) fixed or ulcerated inguinofemoral lymph nodes

IVB Any distant metastasis including pelvic lymph nodes

T2 Tumor with invasion of the lower part of urethra/vagina/anus

IIIC With positive nodes with extracapsular spread

IVA Tumor invades any of the following:

most superficial dermal papilla to the deepest point of invasion.

T1 Tumor confined to vulva and/or perineum T1a <2 cm with stromal invasion <1.0 mm T1b >2 cm with stromal invasion >1.0 mm

**Table 1.** FIGO classification of cancer of the vulva

bones

**TNM Description**

Cancer ofthe vulva can spread from the original site by the following: local invasion of adjacent tissues; embolizationto regionallymphnodes,usually to the superficial anddeepinguinal ones and eventually to the pelvic ones (Figure 6); and via blood, rarely reaching the lungs, liver, and bones. Lymph node involvement is the most important prognostic factor, and lymphatic embolization is the major route of spread.[14] The evaluation of patients with vulvar cancer begins with the physical examination, palpation of inguinal and supraclavicular lymph nodes, vaginal examination, and digital rectal examination. Oncologic cervical cytology, colposcopy of the cervix and vagina (because of the association with squamous intraepithelial lesions), hematological/biochemical tests, and chest X-ray are routine. Cystoscopy and sigmoidoscopy are indicated in suspected cases of bladder or rectal invasion. Pelvic computed tomography (CT), magnetic resonance imaging (MRI), and intravenous urography can be used to evaluate the possibility of metastatic disease in pelvic lymph nodes or surgical planning.[1, 17]

**Figure 6.** Lymphatic dissemination of vulva

#### **7. Histopathological classification**

As mentioned, squamous cell carcinoma is the most common cancer of the vulva, and it is associated with HPV infection especially in younger patients. Melanoma is the second most common vulvar cancer, with approximately 4.8% of these patients; this will be reviewed separately. The other histopathological types of vulvar cancer are verrucous carcinoma, Paget's disease, adenocarcinoma not otherwise specified (NOS), basal cell carcinoma NOS, Bartholin gland carcinoma, and sarcoma.[1]

The three-grade system (G1, well differentiated, G2, moderately differentiated, G3, poorly differentiated or undifferentiated, Gx, grade cannot be accessed) can be used to grade the tumor pathology. In the same aspect, from knowledge of the histopathology, it is important to determine the depth of stromal invasion. The depth of invasion is defined as the measure‐ ment of the tumor from the epithelial-stromal junction of the adjacent most superficial dermal papilla to the deepest point of invasion.[11, 15, 18]

#### **8. Prognostic and recurrence factors**

As already discussed, prognosis is strongly related to lymph node status and stage of disease. Positive lymph nodes show a direct correlation with the depth and extent of the invasion. FIGO staging indicates 5-year survival rates of 90.4% for stage I, 77.1% for stage II, 51.3% for stage III, and 18% for stage IV. Another study of the Gynecologic Oncology Group (GOG) pointed to a 5-year survival rate of 97.9% for tumors with a diameter <2 cm with negative lymph nodes. In this paper, the authors classified patients with vulvar cancer into 4 groups according to histopathological surgical findings (tumor size and extent of lymph node metastases) (Table 3).[19] Other factors with the histological type and the disease-free interval, although they have been considered possible prognostic factors, do not have great clinical relevance. The free surgical margins are the most important predictive factor for local recurrence. Some studies have indicated recurrence rates of 22.5–50% when the disease-free margins are ≤8 mm.[20, 21] In these same studies, the authors demonstrated that in 50% of cases with margins ≤8 mm in histological specimens, margins on macroscopic examination were 1 cm. Thus, a macro‐ scopic tumor-free margin of 1 cm increased to 2 cm for a positive prognosis.[22, 23]


**Table 3.** Risk groups and survival (GOG)

Women over 50 years of age have a higher risk of vulvar cancer mortality and this risk increases with age. Likewise, a racial disparity in survival has been shown for vulvar cancer, with a poorer prognosis among white patients.

#### **9. Treatment**

(CT), magnetic resonance imaging (MRI), and intravenous urography can be used to evaluate

As mentioned, squamous cell carcinoma is the most common cancer of the vulva, and it is associated with HPV infection especially in younger patients. Melanoma is the second most common vulvar cancer, with approximately 4.8% of these patients; this will be reviewed separately. The other histopathological types of vulvar cancer are verrucous carcinoma, Paget's disease, adenocarcinoma not otherwise specified (NOS), basal cell carcinoma NOS,

The three-grade system (G1, well differentiated, G2, moderately differentiated, G3, poorly differentiated or undifferentiated, Gx, grade cannot be accessed) can be used to grade the tumor pathology. In the same aspect, from knowledge of the histopathology, it is important to determine the depth of stromal invasion. The depth of invasion is defined as the measure‐ ment of the tumor from the epithelial-stromal junction of the adjacent most superficial dermal

As already discussed, prognosis is strongly related to lymph node status and stage of disease. Positive lymph nodes show a direct correlation with the depth and extent of the invasion. FIGO

the possibility of metastatic disease in pelvic lymph nodes or surgical planning.[1, 17]

244 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

**Figure 6.** Lymphatic dissemination of vulva

**7. Histopathological classification**

Bartholin gland carcinoma, and sarcoma.[1]

papilla to the deepest point of invasion.[11, 15, 18]

**8. Prognostic and recurrence factors**

Surgery is the treatment of choice for patients with vulvar cancer; however, treatment needs to be individualized. Currently, there is no standard surgery, and the emphasis is on finding the most conservative treatment associated with possible cure of the disease. Aimed at decreasing psychosexual morbidity, where possible, a more conservative surgery is sought, such as local excision of the tumor, with tumor-free margin, rather than radical vulvectomy. Surgical removal, to be effective in controlling the disease locally, needs to have lateral margins of at least 1 cm (histologically) and the deep margin should be inferior fascia of the urogenital diaphragm.[1, 24, 25]

With the introduction of radical vulvectomy with en bloc bilateral inguinofemoral lympha‐ denectomy (butterfly incision), overall survival for vulva cancer went from 20% to 60%, when compared with the simple excision of the tumor. Thus, for a long period, it was the default operation for the treatment of vulvar cancer.[26, 27] Even in the early stages, all patients underwent inguinofemoral lymphadenectomy; although only 20–30% of these showed lymph node metastases. In case of metastasis in inguinofemoral lymph nodes, the best treatment option was pelvic radiotherapy instead of pelvic lymphadenectomy.[28]

En bloc resection (radical vulvectomy with bilateral inguinofemoral lymphadenectomy) is no longer done these days, except in tumors located in the upper regions of the vulva, near the inguinal incisions. This butterfly incision was replaced by triple incision (Figures 7 and 8), which involves the complete excision of the tumor by radical vulvectomy or local excision (with safety margin) and removal of the lymph nodes by two separate inguinal incisions but without the additional skin removal.[1, 23, 25, 29] The triple incision surgery involves less morbidity, with less risk of seroma and lymphedema, as well as lower rate of dehiscence and pain, without increasing the risk of recurrence or mortality compared with en bloc resection.

**Figure 7.** Triple incision (immediate postoperative period) with preservation of the upper part of vulva and clitoris

Patients in stage IA with microinvasive vulvar cancer can be managed with a wide local excision, without the need for inguinal dissection.[30, 31] There is indication of at least ipsilateral inguinal lymphadenectomy in patients with stage IB, II, or any tumor with more than 1 mm stromal invasion.[1] Patients with lateral tumors (in labia majora or minora) without involvement of the midline can be subjected to radical hemivulvectomy, instead of radical vulvectomy, with inguinofemoral lymphadenectomy.

Bilateral inguinal dissection must be performed in patients with tumors in medial regions for those involving the anterior portion of the labia minora and for those with large lateral tumors (>2 cm in diameter, >5 mm of invasion), as well as for patients with positive ipsilateral lymph nodes.[32]

**Figure 8.** Triple incision (immediate post-operative period) with preservation of clitoris

Patients with FIGO stage III or IV or with extensive involvement of inguinal lymph nodes are considered having advanced disease, for which a multimodal treatment plan should be proposed. Radical vulvectomy combined with partial or total pelvic exenteration is an option for patients with locally advanced disease with clinically resectable lesions.[33, 34]

#### **10. Sentinel lymph nodes**

With the introduction of radical vulvectomy with en bloc bilateral inguinofemoral lympha‐ denectomy (butterfly incision), overall survival for vulva cancer went from 20% to 60%, when compared with the simple excision of the tumor. Thus, for a long period, it was the default operation for the treatment of vulvar cancer.[26, 27] Even in the early stages, all patients underwent inguinofemoral lymphadenectomy; although only 20–30% of these showed lymph node metastases. In case of metastasis in inguinofemoral lymph nodes, the best treatment

En bloc resection (radical vulvectomy with bilateral inguinofemoral lymphadenectomy) is no longer done these days, except in tumors located in the upper regions of the vulva, near the inguinal incisions. This butterfly incision was replaced by triple incision (Figures 7 and 8), which involves the complete excision of the tumor by radical vulvectomy or local excision (with safety margin) and removal of the lymph nodes by two separate inguinal incisions but without the additional skin removal.[1, 23, 25, 29] The triple incision surgery involves less morbidity, with less risk of seroma and lymphedema, as well as lower rate of dehiscence and pain, without increasing the risk of recurrence or mortality compared with en bloc resection.

**Figure 7.** Triple incision (immediate postoperative period) with preservation of the upper part of vulva and clitoris

vulvectomy, with inguinofemoral lymphadenectomy.

nodes.[32]

Patients in stage IA with microinvasive vulvar cancer can be managed with a wide local excision, without the need for inguinal dissection.[30, 31] There is indication of at least ipsilateral inguinal lymphadenectomy in patients with stage IB, II, or any tumor with more than 1 mm stromal invasion.[1] Patients with lateral tumors (in labia majora or minora) without involvement of the midline can be subjected to radical hemivulvectomy, instead of radical

Bilateral inguinal dissection must be performed in patients with tumors in medial regions for those involving the anterior portion of the labia minora and for those with large lateral tumors (>2 cm in diameter, >5 mm of invasion), as well as for patients with positive ipsilateral lymph

option was pelvic radiotherapy instead of pelvic lymphadenectomy.[28]

246 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

Since only 25–35% of patients with vulvar cancer present with metastasis to lymph nodes, only a small number of patients show a real benefit from inguinofemoral lymphadenectomy. It is therefore evident that alternatives to lymphadenectomy are needed. A sentinel lymph node biopsy has been shown to be a reasonable alternative to complete inguinal and femoral lymphadenectomy in selected patients. In a study of patients with stage I and II with tumor <4 cm, stromal invasion less than 1 mm and clinically negative lymph nodes, sentinel lymph nodes have been shown to have a sensitivity of 94.1% and a negative predictive value of 97.1%. [35] Other studies also demonstrated a sensitivity of 92% and negative predictive value of 97– 98%, making sentinel lymph node evaluation an accurate way to stage vulvar cancer.[36, 37] When disease is found in sentinel lymph nodes, or when sentinel lymph node assessment is not possible, bilateral inguinofemoral lymphadenectomy must be performed.

#### **11. Radiotherapy/chemotherapy**

Patients undergoing inguinal lymphadenectomy with subsequent identification of a macro‐ metastasis (>5 mm in diameter), extracapsular metastatic spread, or ≥2 micrometastases (<5 mm) should receive bilateral inguinal and pelvic radiotherapy. If the lymph nodes are clinically positive, one should not proceed with full lymphadenectomy, since the inguinal dissection with postoperative irradiation has the potential to cause severe lymphedema. In these cases, where possible, only the largest lymph nodes should be surgically removed before the patient is subjected to postoperative radiotherapy.[28, 38]

Radiotherapy has also been used preoperatively in patients with advanced disease aimed at providing a more complete surgery. Adjuvant radiotherapy has been added in some studies to decrease local recurrence in patients with positive or slim surgical margins; however, other authors are not of the same opinion.[39, 40]

The use of chemotherapy combined with radiotherapy in the primary treatment of locally advanced carcinoma of the vulva shows better results (regarding clinical response and recurrence) compared to radiotherapy alone. If the primary surgery has the potential to result in an intestinal or urinary tract stoma, it is preferable to employ primary chemoradiotherapy, followed by a more limited resection of the tumor bed, or any residual tumor lesion.[41, 42]

Chemotherapy alone is not a common treatment in primary cancer of the vulva. However, studies have already pointed to the characterization of vulvar cancer as chemosensitive, making chemotherapy a valid alternative for the management of these tumors; but data are still insufficient.[43, 44]

#### **12. Melanoma**

Vulvar melanomas occur more frequently in postmenopausal white women. Most patients are asymptomatic except for the pigmented lesion. Most lesions of vulvar melanoma occur on the clitoris or labia minora; it is not unusual for them to extend to the urethra and vagina. FIGO staging for melanoma does not apply, since the lesions are smaller and prognosis is related to the depth of tumor invasion. The system of levels created by Clark or that defined by Breslow may be used to stage melanoma. These systems measure the depth of invasion of the skin. A detailed histological analysis of the surgical specimen is required to carry out these micro‐ stagings. The staging of melanoma is defined by the system of the American Joint Committee on Cancer (AJCC), which includes other prognostic factors such as primary ulceration of the tumor, number of metastatic lymph nodes, micrometastatic disease based on sentinel lymph node biopsy, sites of distant metastatic disease, and serum levels of lactate dehydrogenase (LDH). Any pigmented vulvar lesions should be biopsied, except when known that there has been no change for several years.[45, 46, 47]

There is a tendency for more conservative treatment of vulvar melanoma. Lesions with less than 1 mm invasion can be simply treated with radical local excision. More invasive lesions require resection of the primary tumor and inguinofemoral lymph nodes. Currently, there is controversy as to the benefit of inguinofemoral lymphadenectomy. [1]

#### **Author details**

positive, one should not proceed with full lymphadenectomy, since the inguinal dissection with postoperative irradiation has the potential to cause severe lymphedema. In these cases, where possible, only the largest lymph nodes should be surgically removed before the patient

Radiotherapy has also been used preoperatively in patients with advanced disease aimed at providing a more complete surgery. Adjuvant radiotherapy has been added in some studies to decrease local recurrence in patients with positive or slim surgical margins; however, other

The use of chemotherapy combined with radiotherapy in the primary treatment of locally advanced carcinoma of the vulva shows better results (regarding clinical response and recurrence) compared to radiotherapy alone. If the primary surgery has the potential to result in an intestinal or urinary tract stoma, it is preferable to employ primary chemoradiotherapy, followed by a more limited resection of the tumor bed, or any residual tumor lesion.[41, 42]

Chemotherapy alone is not a common treatment in primary cancer of the vulva. However, studies have already pointed to the characterization of vulvar cancer as chemosensitive, making chemotherapy a valid alternative for the management of these tumors; but data are

Vulvar melanomas occur more frequently in postmenopausal white women. Most patients are asymptomatic except for the pigmented lesion. Most lesions of vulvar melanoma occur on the clitoris or labia minora; it is not unusual for them to extend to the urethra and vagina. FIGO staging for melanoma does not apply, since the lesions are smaller and prognosis is related to the depth of tumor invasion. The system of levels created by Clark or that defined by Breslow may be used to stage melanoma. These systems measure the depth of invasion of the skin. A detailed histological analysis of the surgical specimen is required to carry out these micro‐ stagings. The staging of melanoma is defined by the system of the American Joint Committee on Cancer (AJCC), which includes other prognostic factors such as primary ulceration of the tumor, number of metastatic lymph nodes, micrometastatic disease based on sentinel lymph node biopsy, sites of distant metastatic disease, and serum levels of lactate dehydrogenase (LDH). Any pigmented vulvar lesions should be biopsied, except when known that there has

There is a tendency for more conservative treatment of vulvar melanoma. Lesions with less than 1 mm invasion can be simply treated with radical local excision. More invasive lesions require resection of the primary tumor and inguinofemoral lymph nodes. Currently, there is

controversy as to the benefit of inguinofemoral lymphadenectomy. [1]

is subjected to postoperative radiotherapy.[28, 38]

248 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

authors are not of the same opinion.[39, 40]

been no change for several years.[45, 46, 47]

still insufficient.[43, 44]

**12. Melanoma**

Fernando Anschau1,2,3,4 and Manoel Afonso Guimarães Gonçalves2,3,5\*

\*Address all correspondence to: mafonsog@terra.com.br

1 Pontifícia Universidade Católica do Rio Grande do Sul, PUCRS, Brazil

2 Faculty of Medicine at the Pontifical Catholic University of Rio Grande do Sul, Brazil

3 Gynecologic Oncology Sector of Gynecology Service at São Lucas Hospital, Rio Grande do Sul, Brazil

4 Conceição Hospital Group - board member, Brazil

5 Universidade Federal de São Paulo, UNIFESP, Brazil

#### **References**


[9] Forman D, De Martel C, Larsey CJ, et al. Global burden of human papillomavirus

[10] Duong TH, Flowers LC. Vulvo-vaginal cancers: risks, evaluation, prevention and ear‐

[11] Deppe G, Mert I, WinerIS.Management of squamous cell vulvar cancer: A review. J

[12] Jones RW, Joura EA. Analysing prior clinical events at presentation in 102 women with vulvar carcinoma: Evidence of diagnostic delays. J Reprod Med 1999; 44:766-8.

[13] Nauth HF. Vulva-Zytologie. Makroskopie, Histologie und Zytologie der normalen

[14] Dittmer C, Fischer D, Diedrich K, Thill M. Diagnosis and treatment options of vulvar

[15] FIGO Committee on Gynecologic Oncologic. Revised FIGO staging for carcinoma of the vulva, cervix, and endometrium. Int J GynecolObstet 2009; 105(2):103-4.

[16] Sobin LH, Gospodarowicz MK, Wittekind CH. UICC: TNM classification of malig‐ nant tumors, 7th ed. Wiley-Black-well, Oxford, ISBN 978-1-4443-3241-4. 2009.

[17] Hawnaur JM, Reynolds K, Wilson G, Hillier V, Kitchener HC. Identification of ingui‐ nal lymph node metastases from vulvar carcinoma by magnetic resonance imaging:

[18] Edge SB, Compton CC. The American Joint Committe on Cancer: The 7th edition of the AJCC câncer staging manual and the future of TNM. Ann SurgOncol 2010;

[19] Landrum LM, Lanneau GS, Skaggs VJ, Gould N, Walker JL, McMeekin DS, Gold MA. Gynecologic oncology group risk groups for vulvar carcinoma: improvement in

[20] Heaps JM, Fu YS, Montz FJ, Hacker NF, Berek JS. Surgical-pathologic variables pre‐ dictive of local recurrence in squamous cell carcinoma of the vulva. GynecolOncol

[21] De Hullu JA, Hollema H, Lolkema S, et al. Vulvar carcinoma. The price of less radical

[22] Heaps JM, Fu YS, Montz FJ, Hacker NF, Berek JS. Surgical-pathologic variables pre‐ dictive of local recurrence in squamous cell carcinoma of the vulva. GynecolOncol

[23] De Hullu JA, Hollema H, Lolkema S, et al. Vulvar carcinoma. The price of less radical

survival in the modern era. GynecolOncol 2007; 106(3):521-5.

und erkranten Vulva. Lehrbuch und Atlas. Thieme, Stuttgart. 1986.

cancer: a review. Arch GynecolObstet 2012; 285:183-193.

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surgery. Cancer 2002; 95:2331-8.

surgery. Cancer 2002; 95:2331-8.

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ly detection. ObstetGynecolClin North Am 2007; 34:783-802.


## **Robotic Surgery in the Management of Endometrial Cancer**

[39] Faul CM, Mirmow D, Huang Q, Gerszten K, Day R, Jones MW. Adjuvant radiation for vulvar carcinoma: Improved local control. Int J RadiatOncolBiolPhys 1997;

[40] Groenen SM, Timmers PJ, Burger CW. Recurrence rate in vulvar carcinoma in rela‐ tion to pathological margin distance. Int J Gynecol Cancer 2010; 20: 869-73.

[41] Hacker NF, Berek JS, Juillard GJF, Lagasse LD. Preopeative radiation therapy for lo‐

[42] Boronow RC, Hickman BT, Reagan MT, Smith RA, Steadham RE. Combined therapy as an alternative to exenteration for locally advanced vulvovaginal cancer. Results, complications and dosimetric and surgical considerations. Am J ClinOncol

[43] Tomao F, Di Tucci C, Marchetti C, Perniola G, Bellati F, Panici PB. Role of chemother‐ apy in the management of vulvar carcinoma. Crit Rev OncolHematol 2012; 82:25-39.

[44] Deppe G, Mert I, Belotte J, Winer IS. Chemotherapy of vulvar cancer: a review. Wien

[45] Clark WH, From L, Bernardino EA, et al. The histogenesis and biologic behavior of primary human malignant melanomas of the skin. Cancer Res 1969; 29:705-27. [46] Breslow A. Thickness, cross-sectional área and depth of invasion in the prognosis of

[47] Kim CJ, Reintgen DS, Balch CM for the AJCC Melanoma Staging Committee. The

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new melanoma staging system. Cancer Control 2002; 9:9-15.

 Mark Williams, John Villeneuve and Bernice Robinson-Bennett

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60835

#### **Abstract**

Endometrial cancer remains the most common gynecologic malignancy in the Unites States and Western Europe. It has been estimated that in 2014, about 52, 630 new cases of endometrial cancer was diagnosed and about 8,590 died from the disease. Prior to 1988, endometrial cancer was staged clinically. Since that time surgical staging has been adopted. With the advancement in technology, the vast majority of cases are being staged and treated surgically via minimally invasive approaches. Conventional laparoscopy has been a major advancement in staging and treatment of uterine cancers. However, technical challenges such as 2–D imaging, rigid instruments, and lack of precision and surgeon fatigue did not translate into widespread adoption of this technique. With the advent of computer–enhanced robotic telesurgery, this has dramatically changed the surgical management and staging of endometrial cancer.

**Keywords:** Endometrial cancer, robotic surgery, oncology, surgical techniques, out‐ comes

#### **1. Introduction**

Endometrial cancer is a general definition that often includes all cancers that develop in the uterus, the most common being endometrioid cancer, or cancers arising in the lining of the uterus or endometrium.[1-3] In the United States, endometrial cancer is the fourth most common cancer among women and also the most common gynecological cancer, with on

© 2016 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.

average about 6% of all female cancers.[4] In the United States, the number of new cases estimated to be diagnosed in 2015 is about 55,000 based on data from the American Cancer Society. Of note, the number of deaths based on these data is expected to be roughly 10,000, with death rates rising between 0.8% and 1.9% per year within the last 5 years.[2] However, there is quite a notable variation in worldwide incidence. For example, the highest incidence is observed in Western Europe, United States, and Canada, while in comparison, Africa and Asia are shown to have the lowest rates of incidence.[5] The overall increase in the incidence of this disease during the last decades is mainly related to higher life expectancy within the developed world.[5]

Endometrial cancer is often described as principally a menopausal state disease, since the majority of the cases occur in advanced age.[4, 5, 6, 8] The risk of endometrial cancer increases with the age of the woman. The median age of diagnosis is around 61 years, with the peak incidence happening between 55 and 70 years old. Data from the research community show that most cases, about 95%, occur in patients over 40 years of age with only up to 5% of disease development occurring in women younger than 40.[9-11] Interestingly, the median age of death is around 73 years.[11] The majority of cases are postulated to be of a sporadic etiology, although there is a minority with evidence of a hereditary basis. A number of research articles have been published detailing the correlation of increased risk of endometrial cancer occur‐ rence in women from families with the autosomal dominant hereditary non-polyposis colorectal cancer (HNPCC) gene. Importantly, endometrial carcinoma is the most common extra-colonic cancer seen in this condition referred to as Lynch syndrome.[6, 12] The lifetime risk in these women is about 40% to 60%, and they have a risk of about 40% of developing endometrial cancer by 70 years old.[6, 11, 13]

A unifying theme among the risk factors is that of increased estrogen exposure.[92] The gradual growth in incidence especially within the last decade also has some correlation with dietary and hormonal factors.[14] Obesity, along with increased abdominal girth, is a known risk factor.[2, 15, 72-73] There has been a notable worldwide increased prevalence of obesity. It appears from some data that developed countries are more disproportionately affected by this phenomenon. Increased body mass index (BMI) is also suggested to result in a higher all–cause mortality and endometrial cancer–specific mortality in endometrial cancer survivors.[16-17] By contrast, Park et al looked at the relation of pre-treatment BMI on known prognostic factors, the impact of disease–free survival, and the cause–specific survival in a recent Korean study of women with endometrial cancer. The study population results, however, found that BMI was not a significant factor for both disease-free and cause-specific survival.[18] Of note, endometrial cancer is the third most common gynecological cancer in Korea. A Swedish cohort study of 11659 women evaluated various lifestyle factors including diet and physical activity and possible association of risk of endometrial cancer. Overall, 133 cases of endometrial cancer were observed. The data suggested that an increased risk was noted with very low intake of fruits and vegetables and statistically significant decreased risk (*p* < 0.01) with increased physical activity.[19] Increased weight in early adult life as well as middle age also increased the risk. The management of peri– and postmenopausal symptoms by unopposed exogenous estrogen is yet another risk factor.[20-21]

Risk factors often associated with decreased risk of endometrial cancer are those that help to decrease the amount of circulating estrogen. These include oral contraceptive (OCP) use, intrauterine device (IUD) use, and cigarette smoking. OCPs have been shown to lower the risk by up to 40%, with protection still noted up to 15 years after last use. A proportional correlation is seen with protection and length of use.

Although the exact etiology still remains elusive, it is known that most cases arise from atypical hyperplasia of the lining of the uterus.[72-76] Epithelial cancers of the uterus are generally divided into two groups. Type I endometrial cancers make up 75% – 90% of endometrial cancers and are usually low grade, diagnosed in early stages with good prognosis and of endometrioid histology. They are estrogen–dependent and tend to develop in an environment of hyperplasia or unopposed estrogen exposure, whether physiological or pharmacological. In addition, this subset may have phosphatase and tensin homologue mutations. Type II endometrial cancers, however, are estrogen–independent, usually high grade, have a poor prognosis, often diagnosed at later stages and are usually of papillary serous, clear cell, or even high-grade endometrioid pathology. Type II cancers may have a link with P53 tumor sup‐ pression mutation and the endometrial milieu of Type II cancers is often associated with polyps or simply atrophic in nature.

At diagnosis, the malignancy is frequently found to be localized or within the borders of the uterine corpus in 72-75% of instances, especially since they present early with abnormal uterine bleeding.[11, 15] As mentioned earlier, the chance that postmenopausal bleeding is a result of cancer substantially escalates with a woman's age. Endometrial cancer is usually diagnosed in early stages, although up to 20% of patients with clinical stage I may have indications of extrauterine spread at time of surgical intervention.[22] The relative estimated survival rates at the 5- and 10-year mark are approximately 82% and 79%, respectively.[2]

#### **2. Surgical management**

average about 6% of all female cancers.[4] In the United States, the number of new cases estimated to be diagnosed in 2015 is about 55,000 based on data from the American Cancer Society. Of note, the number of deaths based on these data is expected to be roughly 10,000, with death rates rising between 0.8% and 1.9% per year within the last 5 years.[2] However, there is quite a notable variation in worldwide incidence. For example, the highest incidence is observed in Western Europe, United States, and Canada, while in comparison, Africa and Asia are shown to have the lowest rates of incidence.[5] The overall increase in the incidence of this disease during the last decades is mainly related to higher life expectancy within the

Endometrial cancer is often described as principally a menopausal state disease, since the majority of the cases occur in advanced age.[4, 5, 6, 8] The risk of endometrial cancer increases with the age of the woman. The median age of diagnosis is around 61 years, with the peak incidence happening between 55 and 70 years old. Data from the research community show that most cases, about 95%, occur in patients over 40 years of age with only up to 5% of disease development occurring in women younger than 40.[9-11] Interestingly, the median age of death is around 73 years.[11] The majority of cases are postulated to be of a sporadic etiology, although there is a minority with evidence of a hereditary basis. A number of research articles have been published detailing the correlation of increased risk of endometrial cancer occur‐ rence in women from families with the autosomal dominant hereditary non-polyposis colorectal cancer (HNPCC) gene. Importantly, endometrial carcinoma is the most common extra-colonic cancer seen in this condition referred to as Lynch syndrome.[6, 12] The lifetime risk in these women is about 40% to 60%, and they have a risk of about 40% of developing

A unifying theme among the risk factors is that of increased estrogen exposure.[92] The gradual growth in incidence especially within the last decade also has some correlation with dietary and hormonal factors.[14] Obesity, along with increased abdominal girth, is a known risk factor.[2, 15, 72-73] There has been a notable worldwide increased prevalence of obesity. It appears from some data that developed countries are more disproportionately affected by this phenomenon. Increased body mass index (BMI) is also suggested to result in a higher all–cause mortality and endometrial cancer–specific mortality in endometrial cancer survivors.[16-17] By contrast, Park et al looked at the relation of pre-treatment BMI on known prognostic factors, the impact of disease–free survival, and the cause–specific survival in a recent Korean study of women with endometrial cancer. The study population results, however, found that BMI was not a significant factor for both disease-free and cause-specific survival.[18] Of note, endometrial cancer is the third most common gynecological cancer in Korea. A Swedish cohort study of 11659 women evaluated various lifestyle factors including diet and physical activity and possible association of risk of endometrial cancer. Overall, 133 cases of endometrial cancer were observed. The data suggested that an increased risk was noted with very low intake of fruits and vegetables and statistically significant decreased risk (*p* < 0.01) with increased physical activity.[19] Increased weight in early adult life as well as middle age also increased the risk. The management of peri– and postmenopausal symptoms by unopposed exogenous

developed world.[5]

endometrial cancer by 70 years old.[6, 11, 13]

254 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

estrogen is yet another risk factor.[20-21]

Although not the focus of this chapter, initial evaluation and workup is usually achieved via endometrial biopsy and ultrasound. Abnormal uterine bleeding is often the most common presentation that is seen. Current recommendations from the American College of Obstetri‐ cians and Gynecologists and other governing bodies still recommend the evaluation of all patients with postmenopausal bleeding for likelihood of endometrial cancer. In addition, any female over the age of 40 years with abnormal bleeding should also be evaluated. This evaluation consists of obtaining tissue either by an endometrial biopsy or dilatation and curettage.

After histologically confirmed diagnosis, additional evaluation to rule out metastasis may be considered. A chest radiograph may be helpful to note any simultaneous pulmonary disease or involvement and to rule out possible metastases to the lung.[13] In some cases, the meas‐ urement of CA-125 is also obtained because in some women with advanced stage disease at time of diagnosis, CA-125 usually may be elevated. These elevated levels can help in deter‐ mining adequate response to treatment or recurrence of disease during post–treatment surveillance.[23-24] However, for the typical histology of Type I, endometrioid Grade 1 and clinical stage 1 patient, a physical examination and chest X-ray is usually only required.

In the majority of cases, the subsequent step involves surgery for definitive treatment, staging to determine the extent of the disease, and to reduce tumor burden in advanced stages with extrauterinedisease.In 1971,the International Federation ofGynecology andObstetrics (FIGO) put forth a comprehensive clinical staging system, which was used worldwide.[25] This initial step helpedto standardize to somedegree thediagnosing andrelevanttreatment ofthedisease. However, since 1988, clinical staging has mainly been replaced by surgical staging especially since itdoesnotfully evaluate significanthistopathologicalfeatures.[22, 76-78]TheGynecolog‐ ic Oncology Group (GOG) carried out two large–scale prospective trials looking at surgical staging in 1984 and 1987.[22, 26] The results from these studies aided in determining the important prognostic factors along with indicated treatment goal. Along with age, race, and endocrine status, it was shown that prognosis is related to the presence or absence of certain uterine and extrauterine risk factors. Uterine factors include histologic type, grade, depth of invasion into the myometrium, isthmus-cervix extension, and lymphovascular space inva‐ sion. Extrauterine factors include adnexal metastasis, intraperitoneal spread, positive perito‐ neal cytology, pelvic and aortic node metastasis, and estrogen/progesterone receptor activity. [27] FIGO stage is often considered to be the single strongest predictor regarding outcome in endometrial cancer based on results from various multivariate analyses.[28]

Current staging is based on the FIGO 2009 staging criteria.[25-26, 29-31] The procedure of surgical staging includes an adequate evaluation of the peritoneal contents, peritoneal cytologic washings, hysterectomy, bilateral salpingo-oophorectomy, cytoreduction of all visible disease, and bilateral pelvic and para-aortic lymph node dissection.[11, 13] Ideally, the procedure should entail the same components whether done via a laparotomy or by minimally invasive surgery (MIS). In the instance where a patient is unable to undergo surgery, whole pelvic and intracavitary radiation may be used as definitive treatment. However, some data have shown a notable decrease in 5-year survival times for clinical stage 1 disease treated in this maner (67%) compared to surgery alone (87%).[11]

#### **3. History of laparoscopy in management and staging of endometrial cancer**

The introduction and involvement of laparoscopy has become truly integral and beneficial in management of endometrial cancer. For more than 30 years, gynecologic surgeons have used laparoscopy for many procedures, including oophorectomies, ovarian cystectomies, and bilateral tubal ligations. Earlier research studies published information on both the feasibility and technique of radical hysterectomy with pelvic and para-aortic lymphadenectomy.[32-35] These helped set the foundation for the possibility of full and comprehensive surgical staging using an MIS approach. Of note, laparoscopic intervention would not get its introduction into the field of gynecologic endometrial cancer until the earlier part of the 1990s. A 1992 publication by Childers et al.[36] was the first to report on laparoscopically–assisted vaginal hysterectomy (LAVH) for management of endometrial cancer based on two cases. The case report also mentioned techniques such as port placement, insufflation methods, and lymphadenectomy involving pelvic and para-aortic nodes. In a subsequent study series, Childers et al. published data involving the first use of laparoscopy in surgical staging of endometrial cancer.[37] The data showed a total of two conversions to laparotomy in their population (*n*= 59), with most common indications being complications such as transected ureter and incidental cystotomy. Interestingly, additional deductions from this study were that this new technique appeared to have similar operating times to conventional laparotomy, however, with an increased degree and length of learning curve for surgeons. Another study looked at the utility of LAVH and laparoscopic lymph node dissection with supporting results of association of decreased postoperative pain and blood loss with increased lymph node yield. Yet this study did show increased operating time compared to open surgery.

Since the initial case reports and other similar retrospective studies done around that time, the development and advancement of minimally invasive laparoscopic methodologies to the surgical staging of endometrial cancer has continued. Later studies have included multiple variables such as description of feasibility reports of the standard laparoscopic method, outcome analysis of open surgical versus laparoscopic techniques, analysis of cost-effective‐ ness, and even development of laparoendoscopic single-site surgery (LESS).[37-46]

#### **4. History of robotics in gynecologic surgery**

surveillance.[23-24] However, for the typical histology of Type I, endometrioid Grade 1 and clinical stage 1 patient, a physical examination and chest X-ray is usually only required.

In the majority of cases, the subsequent step involves surgery for definitive treatment, staging to determine the extent of the disease, and to reduce tumor burden in advanced stages with extrauterinedisease.In 1971,the International Federation ofGynecology andObstetrics (FIGO) put forth a comprehensive clinical staging system, which was used worldwide.[25] This initial step helpedto standardize to somedegree thediagnosing andrelevanttreatment ofthedisease. However, since 1988, clinical staging has mainly been replaced by surgical staging especially since itdoesnotfully evaluate significanthistopathologicalfeatures.[22, 76-78]TheGynecolog‐ ic Oncology Group (GOG) carried out two large–scale prospective trials looking at surgical staging in 1984 and 1987.[22, 26] The results from these studies aided in determining the important prognostic factors along with indicated treatment goal. Along with age, race, and endocrine status, it was shown that prognosis is related to the presence or absence of certain uterine and extrauterine risk factors. Uterine factors include histologic type, grade, depth of invasion into the myometrium, isthmus-cervix extension, and lymphovascular space inva‐ sion. Extrauterine factors include adnexal metastasis, intraperitoneal spread, positive perito‐ neal cytology, pelvic and aortic node metastasis, and estrogen/progesterone receptor activity. [27] FIGO stage is often considered to be the single strongest predictor regarding outcome in

endometrial cancer based on results from various multivariate analyses.[28]

this maner (67%) compared to surgery alone (87%).[11]

256 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

Current staging is based on the FIGO 2009 staging criteria.[25-26, 29-31] The procedure of surgical staging includes an adequate evaluation of the peritoneal contents, peritoneal cytologic washings, hysterectomy, bilateral salpingo-oophorectomy, cytoreduction of all visible disease, and bilateral pelvic and para-aortic lymph node dissection.[11, 13] Ideally, the procedure should entail the same components whether done via a laparotomy or by minimally invasive surgery (MIS). In the instance where a patient is unable to undergo surgery, whole pelvic and intracavitary radiation may be used as definitive treatment. However, some data have shown a notable decrease in 5-year survival times for clinical stage 1 disease treated in

**3. History of laparoscopy in management and staging of endometrial cancer**

The introduction and involvement of laparoscopy has become truly integral and beneficial in management of endometrial cancer. For more than 30 years, gynecologic surgeons have used laparoscopy for many procedures, including oophorectomies, ovarian cystectomies, and bilateral tubal ligations. Earlier research studies published information on both the feasibility and technique of radical hysterectomy with pelvic and para-aortic lymphadenectomy.[32-35] These helped set the foundation for the possibility of full and comprehensive surgical staging using an MIS approach. Of note, laparoscopic intervention would not get its introduction into the field of gynecologic endometrial cancer until the earlier part of the 1990s. A 1992 publication by Childers et al.[36] was the first to report on laparoscopically–assisted vaginal hysterectomy (LAVH) for management of endometrial cancer based on two cases. The case report also The natural progression in medicine, and science and technology, tends to show that with new research comes novel breakthroughs. This is evidenced in a wide variety of procedures, algorithms, medications and in this case equipment. More often than not, these tend to be helpful in the advancement of the art of medicine. This effect is seen directly with the estab‐ lishment of robotic surgery in the field of gynecologic surgery. We have clearly seen a revolution in the armada of gynecological interventions with MIS over the last three decades. [47] The field of robotic surgery has undergone rapid advancement, especially in gynecology, [7-8], since it was originally developed for medical and surgical use in battle zones. The goal was that robotic surgery could be used by surgeons in a remote location to perform simple or complex procedures with similar skill, technique and outcomes as if done in a regular operating room.

There are earlier models that helped to pave the way for the advanced systems currently in use and lead to development in the field and technique. One such model and the first robot to assist in a surgery was a single robotic arm known as Automated Endoscopic System for Optimal Positioning (AESOP) developed by Computer Motion Inc. (Computer Motion, Inc., Santa Barbara, CA, USA).[48-49] Cleared by the Food and Drug Administration (FDA) in 1994, this device was made to hold and manipulate an endoscope and remove the need for an assistant. In addition, it was designed to give a surgeon improved control over visualization and also allow command over the laparoscope using voice-activation.[50] The first commer‐ cially available robotic system, ROBODOC, described in 1992 was a robotic arm designed for use in orthopedic hip prosthesis surgery[49], and allowed for accurate incisions in the femur bone for implant insertion. In 1998, Computer Motion Inc., marketed another model that had been in development called ZEUS Surgical System (ZSS), which had a 2-D imaging system and robotic arms made to mimic the surgeon's arms. Two arms were added to the AESOP to create the ZEUS. The arms also allowed for downscaling of movement from the surgeon's hands and elimination of tremors. The surgeon sat at a console, which helped to decrease fatigue especially in longer operations and expanded on the possibility of telesurgery or remote location surgery.

**Figure 1.** Da Vinci Surgical System with additional training console

Another company, Intuitive Surgical, Inc. (Intuitive Surgical, Mountain View, CA, USA) also developed a robotic surgery assist model called the Da Vinci Surgical System (DVSS) shown in Image 1.[91] Unlike the ZSS, the DVSS used a 3–D vision system produced from two endoscopes, which results in a perceptual 3–D image. It also was designed with the EndoWrist system, which offered seven degrees of freedom. This resulted in the recreation of the dexterity and range of motion of a surgeon's hand, therefore allowing a high degree of accuracy and flexibility. Instruments could thus also be rotated a full 3600 .[51] The first successful surgery was done in 1997 in Belgium.[52] The DVSS was eventually cleared by the FDA in 2005 for use in gynecologic procedures and has full regulatory clearance with the coveted Conformité Européenne (CE) mark in Europe.[53]

#### **5. Advantages of robotic surgery**

Even with the initially high cost of acquisition (estimated between 1– and 2 million U.S. dollars) of a DVSS for an institution, there are many advantages of robotic surgery that make it worthwhile. More than 7,000 peer–reviewed publications have been published on

computer–enhanced robotic surgery. For example, the amount of clinical evidence and data on the robotic surgery system and technique is increasing at a rate of 100 publications per month on average.[54]

#### **5.1. Dexterity**

bone for implant insertion. In 1998, Computer Motion Inc., marketed another model that had been in development called ZEUS Surgical System (ZSS), which had a 2-D imaging system and robotic arms made to mimic the surgeon's arms. Two arms were added to the AESOP to create the ZEUS. The arms also allowed for downscaling of movement from the surgeon's hands and elimination of tremors. The surgeon sat at a console, which helped to decrease fatigue especially in longer operations and expanded on the possibility of telesurgery or remote

Another company, Intuitive Surgical, Inc. (Intuitive Surgical, Mountain View, CA, USA) also developed a robotic surgery assist model called the Da Vinci Surgical System (DVSS) shown in Image 1.[91] Unlike the ZSS, the DVSS used a 3–D vision system produced from two endoscopes, which results in a perceptual 3–D image. It also was designed with the EndoWrist system, which offered seven degrees of freedom. This resulted in the recreation of the dexterity and range of motion of a surgeon's hand, therefore allowing a high degree of accuracy and

was done in 1997 in Belgium.[52] The DVSS was eventually cleared by the FDA in 2005 for use in gynecologic procedures and has full regulatory clearance with the coveted Conformité

Even with the initially high cost of acquisition (estimated between 1– and 2 million U.S. dollars) of a DVSS for an institution, there are many advantages of robotic surgery that make it worthwhile. More than 7,000 peer–reviewed publications have been published on

.[51] The first successful surgery

location surgery.

**Figure 1.** Da Vinci Surgical System with additional training console

258 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

flexibility. Instruments could thus also be rotated a full 3600

Européenne (CE) mark in Europe.[53]

**5. Advantages of robotic surgery**

The invention of the ergonomic wrist instruments allowed for more accurate replication of the movement at the wrist, including rotation.

#### **5.2. Precision**

The robotic surgical system provides the ability to improve the precision with which the surgical procedure is carried out. This is due in part to different factors such as the EndoWrist concept, tremor reduction (explained below), and improved field of view.

#### **5.3. Field and depth of view**

The ability to see in a 3–D image as mentioned earlier is truly a remarkable feature. As an old adage of excellent surgical techniques, the need for adequate exposure and visualization is vital. This is also one improved quality over the laparoscopic technique, which has a limited 2–D view. In addition, robotic surgery does have the benefit of greatly increased magnification which adds ability for more precise fine microsurgery techniques. The robotic setup that includes a viewing station for the assistants and other staff in the room provides both a great interactive learning and teaching viewing option unique to this system.

#### **5.4. Control and motion dampening**

This technology was also seen in the earlier Zeus system. It gives the ability to reduce the tremors created naturally by the extension of the fingers or the resultant tension due to fatigue as operating times increase. This is somewhat similar to being on a cruise ship and not feeling the rocking of huge waves but just that gentle back and forth enough to give the calm feeling of being at sea. However, the robot is able to filter out unnecessary hand and finger motions which results in safer, more accurate intracorporeal movements.

#### **5.5. Decreased blood loss**

This can be seen as a direct result of the factors above, especially greater instrument control, viewing ability, and small entry sites.

#### **5.6. Learning curve**

Studies have looked at the required learning to be proficient in both laparoscopic and robotic techniques, with the latter often noted to be less difficult to acquire. This is due to the ergonomic setup and more counter-intuitive hand movements needed compared to a laparoscopic style. It is suggested that 20 robotic procedures are needed for proficiency.[55-56] Operative times tend to decrease and the nodal counts increase with increased surgeon's experience.

#### **5.7. Decreased surgeon fatigue**

The surgeon operator console provided on the robotic surgical system is a great enhancement, especially in longer operations. [57]

#### **6. Techniques of robotic surgery**

The use of port sites and the selection of these ports are important in MIS. As mentioned earlier, the actual technique of staging should ideally be the same regardless of type of abdominal incision.

**Figure 2.** Schematic demonstration of the port placement in robotic assisted operation for endometrial carcinoma

Techniques include either single-site port or multiple port use and other factors play a role in determining which the best approach is used.

**Figure 3.** Pelvic lymph node retrieval

Our patients undergo a bowel prep regimen. Patients are positioned securely on a foam memory pad with Velcro straps placed across the breasts. This is critical for the 290 Trende‐ lenburg position for the entire case. Patients are placed in lithotomy in Allen stirrups. A uterine manipulator is used in the majority of cases. Typically, 5 ports are placed as shown in the diagram, Figure 1.[58] The camera is placed approximately 25 cm above the pubic symphysis. The robot is then docked from the patient's left side. A 00 scope is used. The fenestrated bipolar and the monopolar hot shears are used as the operating instruments.

**Figure 4.** Para-aortic lymph node retrieval.

**5.7. Decreased surgeon fatigue**

incision.

especially in longer operations. [57]

**6. Techniques of robotic surgery**

260 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

determining which the best approach is used.

**Figure 3.** Pelvic lymph node retrieval

The surgeon operator console provided on the robotic surgical system is a great enhancement,

The use of port sites and the selection of these ports are important in MIS. As mentioned earlier, the actual technique of staging should ideally be the same regardless of type of abdominal

**Figure 2.** Schematic demonstration of the port placement in robotic assisted operation for endometrial carcinoma

Techniques include either single-site port or multiple port use and other factors play a role in

During indicated lymphadenectomy, pelvic and/or para–aortic, adequate dissection is essential. The robotically assisted system and technique help with improving node yield. The following images shown are during lymph node harvesting for both the pelvic and the paraaortic lymph nodes during cases at our institution. Image 2 shows the intraoperative dissection and removal of nodes in the pelvic cavity, while Image 3 shows the dissection and removal of para-aortic nodes. In Image 3, the aorta is seen on the left of the image (top-down view) branching into the right and left common iliac arteries.

#### **7. Comparison of outcomes between open surgery and MIS**

Total laparoscopic hysterectomy was compared to total abdominal hysterectomy in the LACE trial.[59] In this trial, there was notable improvement in quality of life for up to 6 months for the participants. Also, on that trial, there were more frequent serious adverse outcomes in the laparotomy versus the laparoscopy group.

The largest randomized prospective multicenter study to evaluate outcomes between open surgery and MIS is known as the Gynecologic Oncology Group (GOG) LAP2 trial, a 10-year data accrual study.[60] It compares outcomes between incidence of surgical complications, perioperative morbidity and mortality in stage I or IIa, grades I to III endometrial/uterine cancer in patients being staged with either traditional laparotomy approach versus compre‐ hensive laparoscopic staging. The GOG in this trial aimed to evaluate the feasible role of a laparoscopic method in the primary surgical treatment of endometrial cancer in terms of staging rates, safety, recurrence, and survival. In that study, the rate of conversion to open surgery was 26%. The reasons for conversion were poor visualization, extrauterine spread and bleeding. It was noted that as the BMI increased, so did the rate of conversion. With a BMI of less than 20, the success rate was 90% compared to 34% with a BMI of 50. The median number of lymph nodes harvested was not significantly different. The complications were also evaluated. In the LAP2 study, the hospital time was 2 days in laparoscopic group compared to 4 days in the laparotomy group. The oncologic outcomes of the comparison were reassuring. The estimated 5–year survival rate was 11.6% and 13.7% for laparoscopy and laparotomy, respectively. The overall survival rate was essentially equivalent between the two groups at 89.8% at 5 years.

There have been many retrospective and other studies comparing outcomes and complications between laparotomy and robotic/laparoscopic surgery.[79-81] The key differences between the two are outlined below.

#### **7.1. Estimated blood loss**

The use of smaller multiple incisions compared to a larger incision portends to an expected decreased blood loss. In addition, the magnification and ability to control small blood vessels contributes significantly to the decreased blood loss noted. In a study by Gaia et al. comparing outcomes in laparotomy versus laparoscopic techniques, outcomes were similar except for a statistically significant reduction in blood loss favoring the laparoscopy group. However, there was no difference in transfusion rates.[61] In other studies, however, robotic surgery was associated with reduced blood loss and transfusion when compared to conventional laparo‐ scopy.[53]

#### **7.2. Length of operating time**

Operating time has been shown in studies to be shorter in open surgery compared to MIS. Some studies suggest that operating time was on average 30 min longer for laparoscopic procedures.[59] In the LAP2 trial, operative was longer for laparoscopy by about a median of 70 min.[60] To some degree, this also is based on surgeon expertise with MIS and potential limitations with instrumentation. Some of these limitations are overcome by the robotic platform.

#### **7.3. Increased exposure/visualization**

Laparotomy may seem to have increased exposure simply due to the large abdominal incision created during the procedure. However, with the magnification obtained during laparoscopy visualization is superior, especially in the deep pelvis.

#### **7.4. Length of Hospital Stay (LOHS)**

Due to smaller incisions with minimally invasive techniques, faster expected healing and recovery time are seen in MIS compared to open surgery. This results in decreased need for prolonged admission after operation. As an example, in the LAP2 trial, hospital time was shorter, 2 days with laparoscopy compared to 4 days with laparotomy.[60] He et al. [62] in a meta–analysis noted shorter length of hospital stay (mean difference [MD], -3.42; 95% confidence interval [CI], -3.81 to -3.03; *p* < 0.01) with laparoscopy compared to laparotomy. Other studies have also demonstrated similar results regarding decreased LOHS with laparoscopy.[60, 63-65]

#### **7.5. Lymph node yield**

laparoscopic method in the primary surgical treatment of endometrial cancer in terms of staging rates, safety, recurrence, and survival. In that study, the rate of conversion to open surgery was 26%. The reasons for conversion were poor visualization, extrauterine spread and bleeding. It was noted that as the BMI increased, so did the rate of conversion. With a BMI of less than 20, the success rate was 90% compared to 34% with a BMI of 50. The median number of lymph nodes harvested was not significantly different. The complications were also evaluated. In the LAP2 study, the hospital time was 2 days in laparoscopic group compared to 4 days in the laparotomy group. The oncologic outcomes of the comparison were reassuring. The estimated 5–year survival rate was 11.6% and 13.7% for laparoscopy and laparotomy, respectively. The overall survival rate was essentially equivalent between the two groups at

262 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

There have been many retrospective and other studies comparing outcomes and complications between laparotomy and robotic/laparoscopic surgery.[79-81] The key differences between the

The use of smaller multiple incisions compared to a larger incision portends to an expected decreased blood loss. In addition, the magnification and ability to control small blood vessels contributes significantly to the decreased blood loss noted. In a study by Gaia et al. comparing outcomes in laparotomy versus laparoscopic techniques, outcomes were similar except for a statistically significant reduction in blood loss favoring the laparoscopy group. However, there was no difference in transfusion rates.[61] In other studies, however, robotic surgery was associated with reduced blood loss and transfusion when compared to conventional laparo‐

Operating time has been shown in studies to be shorter in open surgery compared to MIS. Some studies suggest that operating time was on average 30 min longer for laparoscopic procedures.[59] In the LAP2 trial, operative was longer for laparoscopy by about a median of 70 min.[60] To some degree, this also is based on surgeon expertise with MIS and potential limitations with instrumentation. Some of these limitations are overcome by the robotic

Laparotomy may seem to have increased exposure simply due to the large abdominal incision created during the procedure. However, with the magnification obtained during laparoscopy

Due to smaller incisions with minimally invasive techniques, faster expected healing and recovery time are seen in MIS compared to open surgery. This results in decreased need

89.8% at 5 years.

scopy.[53]

platform.

two are outlined below.

**7.1. Estimated blood loss**

**7.2. Length of operating time**

**7.3. Increased exposure/visualization**

**7.4. Length of Hospital Stay (LOHS)**

visualization is superior, especially in the deep pelvis.

Various studies including the previously mentioned GOG LAP2 trial have focused on lymph node yield in either approach. Some studies have shown a similar amount of nodes sampled or retrieved while others have shown increased on either side. Also, in the 2013 meta–analysis of nine randomized controlled trials by He et al., the data showed no statistically significant difference between either approach pelvic node yield (MD, 0.45; 95% CI, -0.41 to 1.32; *p* = 0.30).[62]

#### **7.6. Complications**

In the GOG LAP2 trial, which remains the largest prospective randomized trial comparing laparoscopy to laparotomy in the management of endometrial cancer, the combined compli‐ cation rate inclusive of vascular, urinary, bowel, and nerve was higher in the laparoscopy patients (10%) in comparison to the laparotomy group (8%).[60] DeNardis et al. have however shown reduced complications in robotic cohorts. In that study, major peri–operative compli‐ cation was found to be 3.6% in the robotically-assisted cohort compared to 20.8% in the laparotomy group.[66]

#### **8. Comparison of robotic surgery versus conventional laparoscopy in endometrial cancer**

These two techniques are both types of MIS, although one can often think of robotic surgery as being the younger, more advanced sibling. In this way, robotic surgery in many ways has helped to enhance the techniques and outcomes involved in laparoscopic surgery. Two areas of note where this unique edge is definitely appreciated is in the treatment of both the elderly and the obese with endometrial cancer. These two conditions require additional concern due to possibility of co–morbidities, access, and post–operative survival and outcomes. Cho and Nezhat in their review of 754 case identified complication rates of 10.5%, 12.2%, and 44.6% in robotic hysterectomy, laparoscopic hysterectomy, and abdominal hysterectomy, respectively. [67] In the open cohort, the majority of complications were related to wound infections and bowel dysmotility. There was also a lower rate of conversion in the robotic group when compared to the laparoscopic group. The above–mentioned advancement in the robotic system is no doubt responsible for this observation.

#### **8.1. Decreased Estimated Blood Loss (EBL)**

Decreased blood loss is seen in many studies comparing the two techniques with robotic being associated the lesser amount. In Seamon's study, estimated blood loss was 100 and 250 mL, respectively, for robotic versus conventional laparoscopy.[68]

#### **8.2. Decreased Length of Hospital Stays (LOHS)**

Especially in the current medical climate with current societal economic situations, the trend is to improve the proper utilization of resources while, at the same time, decreasing costs wherever possible. Although robotic surgery comes with a substantial investment cost, having shorter hospital admissions especially postoperatively can help to reduce operating costs from a different angle. Studies have shown either similar in some cases or usually a slightly decreased LOHS in robotic surgery.[68]

#### **8.3. Decreased Operating Room (OR) time**

This is related to surgeon skill, experience and expertise. Since robotic surgery has been shown to have an improved learning curve, this may play a role in overall operating time. Also, coupled with the other benefits robotic surgery offers, this may result in faster times from incision to incision or from docking to incision in some studies. However, some studies have shown similar operating times between the two methods. Seamon et al. in their comparison of robotic to conventional laparoscopy reported reduced mean operative times in the robot group, 242 versus 287 min.[68]

#### **8.4. Decreased chance of conversion to laparotomy**

Some studies, both observational and retrospective comparison, have noted less chance for conversion with decreased visualization, body habitus, patient weight, and comorbidities often being reasons cited for having to do so. This occurs less in robotic than laparoscopic surgery. Gaia et al. demonstrated a 9.9% conversion rate in laparoscopy compared to 4.9% for the robotic approach.[61]

#### **8.5. Patients with increased BMI**

The prevalence of obesity is increasing. Obesity is associated with increased surgical morbid‐ ity. There is an associated increase in blood loss, operative times, wound complications, and venous thromboembolism. Hence, the development of newer techniques that will provide a comparable surgical staging with reduced morbidities is very attractive. The obesity factor affects both techniques but the qualities of the robotic surgery tend to lend toward decreased morbidity compared to laparoscopic surgery. Recall in the LAP2 trial that the success of surgical staging was decreased with increasing BMI. The robot seems to overcome this limitation associated with conventional laparoscopy. In a retrospective study by Gehrig,[69] complete surgical staging was accomplished in 92% of robotic patients in contrast to 84% in the laparoscopic group. Also notable was the shorter operative times (189 vs 215 min, *p* = 0.004, less blood loss (50 v 150 mL, *p* < 0.001) and a statistically significantly shorter hospital stay. Another retrospective study done by Mendivil et al. comparing robotic, laparotomy, and conventional laparoscopic cohorts of morbidly obese patients (BMI > 40 kg/m2 ) compared the outcomes of each procedure.[82] Robotic surgery had the longest operating time compared to laparoscopy and laparotomy (2.78 vs 1.82 and 1.35 h, *p* < 0.001) but had the least estimated blood loss respectively (100 vs 175 and 250 mL, *p* = 0.002). The length of hospital stay was significantly shorter with both minimally invasive methods compared to laparotomy (2 vs 4 days, *p* = 0.002)

#### **8.6. Patients of advanced age**

**8.1. Decreased Estimated Blood Loss (EBL)**

264 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

**8.2. Decreased Length of Hospital Stays (LOHS)**

decreased LOHS in robotic surgery.[68]

group, 242 versus 287 min.[68]

the robotic approach.[61]

**8.5. Patients with increased BMI**

**8.3. Decreased Operating Room (OR) time**

**8.4. Decreased chance of conversion to laparotomy**

respectively, for robotic versus conventional laparoscopy.[68]

Decreased blood loss is seen in many studies comparing the two techniques with robotic being associated the lesser amount. In Seamon's study, estimated blood loss was 100 and 250 mL,

Especially in the current medical climate with current societal economic situations, the trend is to improve the proper utilization of resources while, at the same time, decreasing costs wherever possible. Although robotic surgery comes with a substantial investment cost, having shorter hospital admissions especially postoperatively can help to reduce operating costs from a different angle. Studies have shown either similar in some cases or usually a slightly

This is related to surgeon skill, experience and expertise. Since robotic surgery has been shown to have an improved learning curve, this may play a role in overall operating time. Also, coupled with the other benefits robotic surgery offers, this may result in faster times from incision to incision or from docking to incision in some studies. However, some studies have shown similar operating times between the two methods. Seamon et al. in their comparison of robotic to conventional laparoscopy reported reduced mean operative times in the robot

Some studies, both observational and retrospective comparison, have noted less chance for conversion with decreased visualization, body habitus, patient weight, and comorbidities often being reasons cited for having to do so. This occurs less in robotic than laparoscopic surgery. Gaia et al. demonstrated a 9.9% conversion rate in laparoscopy compared to 4.9% for

The prevalence of obesity is increasing. Obesity is associated with increased surgical morbid‐ ity. There is an associated increase in blood loss, operative times, wound complications, and venous thromboembolism. Hence, the development of newer techniques that will provide a comparable surgical staging with reduced morbidities is very attractive. The obesity factor affects both techniques but the qualities of the robotic surgery tend to lend toward decreased morbidity compared to laparoscopic surgery. Recall in the LAP2 trial that the success of surgical staging was decreased with increasing BMI. The robot seems to overcome this limitation associated with conventional laparoscopy. In a retrospective study by Gehrig,[69] complete surgical staging was accomplished in 92% of robotic patients in contrast to 84% in the laparoscopic group. Also notable was the shorter operative times (189 vs 215 min, *p* = 0.004, less blood loss (50 v 150 mL, *p* < 0.001) and a statistically significantly shorter hospital stay.

Elderly patients usually have more co–morbidities and are generally poorer surgical candi‐ dates with concomitant more advanced disease, which may require more surgical intervention. There have been studies looking at the utility of robotic surgery in this scenario, as well as laparoscopic surgeries. In a retrospective analysis by Scribner et al., laparoscopic staging was completed in 77.6% of patients. The operative time was increased for the laparoscopic group, however, there was no increased morbidity from longer anesthetic times.[70] Another study by Lavoue et al.[87] compared a population (*n* = 113) of advanced age patients (greater than or equal to 70 years) with endometrial cancer undergoing surgical staging by either robotic or traditional open surgery. The robotic group had longer operating times (244 vs 217 min, *p* = 0.009) but less estimated mean EBL (75 vs 334 mL, *p* < 0.0001), less minor adverse events (17 vs 60%, *p* < 0.001) and decreased mean LOHS (3 vs 6 days, *p* < 0.0001). However, no statistical difference (*p* = 0.61) was noted in the 2-year disease–free survival during follow-up.

A single institution retrospective chart review looked at the safety of robotic surgery in a cohort of patients with endometrial cancer (*n* = 228) compared to laparotomy.[88] The cohort was subdivided by method of surgery (robotic vs laparotomy) and age (<65 vs 65 years and older). Older patients undergoing robotic surgery had decreased estimated blood loss (131 vs 235 mL, *p* = 0.03), decreased rate of postoperative ileus (0 vs 15%, *p* = 0.04), decreased perioperative surgical complication rate (4 vs 30%, *p* = 0.01), and decreased LOHS (2.2 vs 4.4 days, *p* < 0.01) compared with laparotomy. The rate of discharge home was similar with compared to laparotomy (96 vs 91%, *p* = 0.45).

Robotic surgery with the associated advantages such as decreased EBL, decreased LOHS, and potentially decreased postoperative morbidity may show potential for improved outcomes compared to laparoscopic surgery and laparotomy. Further studies may be needed to evaluate this comparison.

#### **8.7. Single-Port Access (SPA)**

The progression from traditional open surgery toward minimally invasive methods, both robotic and conventional laparoscopic, has resulted in further innovation such as attempts at using fewer port entry sites, less trocars, and smaller abdominal incisions.

Laparoendoscopic single–site surgery (LESS), a novel technique, may lead to an additional decrease in the overall invasiveness of conventional laparoscopy. Fanfani et al.[83] in a 2012 publication of a single institution cohort trial looked at laparoendoscopic single–site surgery (LESS) in surgical management of early–stage endometrial cancer. The results showed median age of 57 years (42–68), median BMI of 24 kg/m2 (21–30) with median operating time of 105 min, and median EBL of 20 mL (10–180). The skin and fascial incision needed for this singleport access approach was 2.5 cm (median 2.2 cm, range of 2.0–2.5) with all patients reported being satisfied with both pain control postoperatively and the cosmetic results.

Some difficulties inherent with LESS include instrument crowding as well as clashing, decreased and/or poor visualization, loss of triangulation, and ergonomic issues.[85] The combination of robotic surgery with LESS may help to overcome some of the technical limitations noted with LESS. A retrospective case–control study by Fagotti et al. looked at the comparison outcomes between robotic and laparoendoscopic single–site hysterectomy for treatment of early endometrial cancer.[86] Although the median OR time was less in the robotic versus laparoendoscopic group (90 min vs 107 min), the data did not produce any seemingly clinically relevant differences.

**Figure 5.** Single site port system

The robotic single–site port system[89] which enables operating through a small umbilical incision in common procedures such as benign hysterectomies, cholecystectomies, or salpin‐ go–oopherectomies. The recommended size of incision needed for this five–lumen port (see Image 4[93]) is typically about 1.5 cm. As seen the port has five channels. There is a channel for the 8.5 mm scope, two robotic arms, a surgical assistant port, and an insufflation port. The instruments are semiflexible and capable of triangulation. Currently, the instruments lack the EndoWrist articulation, which might be disadvantageous. The advantages of this technique include the promise of potentially virtually scarless surgery due to the small incision, in addition to the known ones of robotic surgery such as decreased EBL and LOHS.[90] More prospective studies with larger numbers are needed to compare robotic single–site surgery with standard robotic multi-site surgery for procedures commonly done.

A pilot study published in 2013 by Vizza et al.[88] looked at the feasibility and safety of using robotic single-site hysterectomy in patients with low–risk early stage endometrial cancer. The five–lumen port described above was used with the size of the umbilical incision ranging from 2 to 2.5 cm. The median age was 64 years with a median BMI of 26.6 kg/m2 . The results showed median OR time to be 90 min, median blood loss of 75 mL with no reported conversion to laparoscopy or laparotomy, and median LOHS of 2 days. No reported complications occurred neither intra– nor postoperatively. The study concluded that robotic single–site hysterectomy was a safe and technically possible option in this patient group. Future studies with other gynecologic oncologic procedures and cases need to be carried out to evaluate the feasibility and advantages of this technique.

#### **8.8. Cost**

(LESS) in surgical management of early–stage endometrial cancer. The results showed median

min, and median EBL of 20 mL (10–180). The skin and fascial incision needed for this singleport access approach was 2.5 cm (median 2.2 cm, range of 2.0–2.5) with all patients reported

Some difficulties inherent with LESS include instrument crowding as well as clashing, decreased and/or poor visualization, loss of triangulation, and ergonomic issues.[85] The combination of robotic surgery with LESS may help to overcome some of the technical limitations noted with LESS. A retrospective case–control study by Fagotti et al. looked at the comparison outcomes between robotic and laparoendoscopic single–site hysterectomy for treatment of early endometrial cancer.[86] Although the median OR time was less in the robotic versus laparoendoscopic group (90 min vs 107 min), the data did not produce any seemingly

The robotic single–site port system[89] which enables operating through a small umbilical incision in common procedures such as benign hysterectomies, cholecystectomies, or salpin‐ go–oopherectomies. The recommended size of incision needed for this five–lumen port (see Image 4[93]) is typically about 1.5 cm. As seen the port has five channels. There is a channel for the 8.5 mm scope, two robotic arms, a surgical assistant port, and an insufflation port. The instruments are semiflexible and capable of triangulation. Currently, the instruments lack the EndoWrist articulation, which might be disadvantageous. The advantages of this technique include the promise of potentially virtually scarless surgery due to the small incision, in addition to the known ones of robotic surgery such as decreased EBL and LOHS.[90] More prospective studies with larger numbers are needed to compare robotic single–site surgery

A pilot study published in 2013 by Vizza et al.[88] looked at the feasibility and safety of using robotic single-site hysterectomy in patients with low–risk early stage endometrial cancer. The five–lumen port described above was used with the size of the umbilical incision ranging from

with standard robotic multi-site surgery for procedures commonly done.

2 to 2.5 cm. The median age was 64 years with a median BMI of 26.6 kg/m2

being satisfied with both pain control postoperatively and the cosmetic results.

(21–30) with median operating time of 105

. The results showed

age of 57 years (42–68), median BMI of 24 kg/m2

266 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

clinically relevant differences.

**Figure 5.** Single site port system

Any cost analysis of the different modalities of surgery and staging in endometrial cancer cannot overlook the impact of hospital stay on overall cost. Since the robot has been effective in shifting hysterectomy and staging to an essentially an outpatient procedure, there will be an anticipated decrease in overall hospital cost.

A cost effective analysis of robotically assisted management of new endometrial cancer was performed by Leitao et al.[71] The costs were inclusive of all surgical aspects of care provided up to 6 months following discharge. In that study, the total mean amortized cost per case was \$20,487 for laparoscopy, 20,467 for robotically assisted, and \$24, 642 for laparotomy. It was concluded that when laparotomy rates are reduced by virtue of the robot, then there is notable cost neutralization. A similar finding of laparoscopy being the least expensive approach was noted. Interestingly, if the cost of the robotic disposable instruments did not exceed \$1,046, then from a societal perspective the robotic approach would be the least expensive.[46] In other studies, the utilization of the robot was deemed to be approximately 1.5 times higher than conventional laparoscopy. However, the mentioned reduction in completion of case and decreased conversion to laparotomy cannot be ignored.

Future studies will probably examine the use of the robot in debulking advanced cases of uterine cancers. Single–site surgeries will probably become more popular. Advancements in the actual technology are only expected to sky rocket. One can only imagine what the next step involves or what direction robotic computer-enhanced telesurgery would take. The important factor overall is being able to find that balance of effective patient care and management with the proper utilization of resources based on overall cost as well as reimbursement.

#### **Author details**

Mark Williams1,2, John Villeneuve1,2 and Bernice Robinson-Bennett1,2\*

\*Address all correspondence to: bernice.robinson-bennett@readinghealth.org

1 Department of Gynecologic Oncology, McGlinn Cancer Institute, Reading Hospital, Reading Health System, Reading, Pennsylvania, USA

2 Department of Obstetrics and Gynecology, Reading Hospital, Reading Health System, Reading, Pennsylvania, USA

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## **Cervical Cancer in Human Immunodeficiency Virus (HIV) Positive Patients**

#### Atara Ntekim

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60664

#### **Abstract**

The clinical management of cervical cancer in human immunodeficiency virus (HIV) positive patients is associated with challenges mainly due to the state of their immunity. They are managed like their HIV seronegative counterparts with surgery or chemoradiotherapy. HIV, cervical cancer, radiotherapy and chemotherapy lower immunity through reduction in CD4 cell counts.

A perspective on the management of HIV positive patients with cervical cancer is hereby provided.

Available studies were reviewed and peculiar characteristics of HIV patients with cervical cancer were examined. Strategies for managing such patients were identified.

HIV positive patients are younger and have more aggressive disease. They have more treatment related toxicities, poorer disease control with higher rates of incomplete and treatment delays than their HIV negative counterparts. Highly active anti-retroviral therapy (HAART) improves treatment outcome in such patients.

HIV positive patients with cervical cancer should be commenced on HAART at diagnosis. There should be closer monitoring of CD4 cell counts and viral load while on oncology treatment towards early recognition of need for prophylaxes against opportunistic infections. The dosage of the treatment modalities should also be adjusted according to CD4 cells count status. Possible interactions between antiretroviral therapy (ART) with chemotherapy and radiotherapy should not be overlooked.

**Keywords:** HIV, cervical cancer, radiotherapy, chemotherapy

© 2016 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.

#### **1. Introduction**

Cancer of the uterine cervix is the most common gynecological malignancy and occurs worldwide [1]. Close to eighty percent of cervical cancers occur in the developing countries [2]. Chronic persistent infections with high-risk HPV subtypes play an important role in the carcinogenesis of cervical cancer. Human immunodeficiency virus (HIV) infection lowers immunity and is epidemic in some developing countries especially in sub-Sahara Africa. Cervical cancer is very common in HIV seropositive patients and is associated with an aggressive course and poor treatment outcome [3]. The associated compromise in immunity caused by HIV infection poses serious challenges to the clinical management of HIV positive patients diagnosed with cervical cancer. The main modalities of managing cervical cancer are surgery, chemotherapy and radiotherapy with most patients requiring combination therapy. These treatment modalities lead to reduced immunity in patients which is further reduced if one or two modalities are combined. In a patient with immunological challenges due to HIV infection, these treatment modalities can therefore worsen the immunological competence of the individual leading to poorer treatment tolerance, undue treatment toxicity and poor treatment outcome. At present, HIV positive patients diagnosed with cervical cancer are being managed using guidelines for managing HIV seronegative patients diagnosed with cervical cancer. The outcome of treatment in HIV positive patients are worse compared with HIV negative patients and HIV positive patients present late and are less likely to complete oncology treatment [4]. Infection with HIV has also been noted to increase mortality among cancer patients generally [5]. There is therefore need to consider additional therapeutic measures applicable to cervical cancer patients who are HIV positive.

The aim of this chapter is to highlight special features associated with HIV positive patients diagnosed with cervical cancer and provide a perspective on management strategies for these patients. This was done through a review of the evidence from basic, epidemiological and clinical studies which formed the basis for the recommendations for the management of HIV positive patients diagnosed with cervical cancer.

#### **2. Peculiarities of cervical cancer in HIV positive patients**

#### **2.1. Epidemiology of cervical cancer**

Cervical cancer occurs worldwide. The incidence of cervical cancer is still high in developing countries whereas it has decreased significantly in the developed countries over the last several decades. Close to eighty percent of cervical cancer occur in the developing countries [2]. The highest incidence is in sub-Sahara Africa especially in Eastern African countries [6]. Further‐ more, the mortality due to cervical cancer is about ten times higher in the developing countries where screening and treatment modalities are neither common nor easily accessible. In developed countries, screening is the main factor responsible for the decrease in the incidence and mortality rates of cervical cancer.

#### **2.2. Risk factors for cervical cancer**

**1. Introduction**

278 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

Cancer of the uterine cervix is the most common gynecological malignancy and occurs worldwide [1]. Close to eighty percent of cervical cancers occur in the developing countries [2]. Chronic persistent infections with high-risk HPV subtypes play an important role in the carcinogenesis of cervical cancer. Human immunodeficiency virus (HIV) infection lowers immunity and is epidemic in some developing countries especially in sub-Sahara Africa. Cervical cancer is very common in HIV seropositive patients and is associated with an aggressive course and poor treatment outcome [3]. The associated compromise in immunity caused by HIV infection poses serious challenges to the clinical management of HIV positive patients diagnosed with cervical cancer. The main modalities of managing cervical cancer are surgery, chemotherapy and radiotherapy with most patients requiring combination therapy. These treatment modalities lead to reduced immunity in patients which is further reduced if one or two modalities are combined. In a patient with immunological challenges due to HIV infection, these treatment modalities can therefore worsen the immunological competence of the individual leading to poorer treatment tolerance, undue treatment toxicity and poor treatment outcome. At present, HIV positive patients diagnosed with cervical cancer are being managed using guidelines for managing HIV seronegative patients diagnosed with cervical cancer. The outcome of treatment in HIV positive patients are worse compared with HIV negative patients and HIV positive patients present late and are less likely to complete oncology treatment [4]. Infection with HIV has also been noted to increase mortality among cancer patients generally [5]. There is therefore need to consider additional therapeutic

measures applicable to cervical cancer patients who are HIV positive.

**2. Peculiarities of cervical cancer in HIV positive patients**

positive patients diagnosed with cervical cancer.

**2.1. Epidemiology of cervical cancer**

and mortality rates of cervical cancer.

The aim of this chapter is to highlight special features associated with HIV positive patients diagnosed with cervical cancer and provide a perspective on management strategies for these patients. This was done through a review of the evidence from basic, epidemiological and clinical studies which formed the basis for the recommendations for the management of HIV

Cervical cancer occurs worldwide. The incidence of cervical cancer is still high in developing countries whereas it has decreased significantly in the developed countries over the last several decades. Close to eighty percent of cervical cancer occur in the developing countries [2]. The highest incidence is in sub-Sahara Africa especially in Eastern African countries [6]. Further‐ more, the mortality due to cervical cancer is about ten times higher in the developing countries where screening and treatment modalities are neither common nor easily accessible. In developed countries, screening is the main factor responsible for the decrease in the incidence The most important risk factor for the development of cervical cancer is infection with human papilloma virus [HPV] [7]. In particular, chronic persisting infections with high-risk HPV subtypes play an important role in the carcinogenesis of cervical cancer. The subtype mostly implicated in cervical cancer aetiology is types 16 followed by 18. Other subtypes are also implicated but to a lesser extent. Human Immunodeficiency Virus (HIV) infection causes low immunity in those infected. In HIV positive patients, some less carcinogenic subtypes of HPV have been reported to play an important role in the aetiology of cervical cancer (Table 1).


**Table 1.** Most frequent HPV types among women with invasive cervical cancer by any histology. Sources: HIV negative: ICO HPV Information Centre (2014); HIV-1 positive: [8]. Subtypes 52 and 35 that are less carcinogenic in HIV negative patients are more important in HIV positive patients.

#### **2.3. Human Immunodeficiency Virus (HIV) infection**

Immunodeficiency is an important cofactor for persisting infections with HPV. It increases the virulence and aggressiveness of HPV thereby accelerating the progression to malignant transformation of the endo-cervical epithelial cells.

Human immunodeficiency virus (HIV) lowers immunity and is epidemic in most developing countries. Cervical cancer is very common in HIV seropositive patients and has an aggressive course with poor treatment outcome [3]. Regions of high prevalence of cervical cancer corresponds with regions of high prevalence of HIV infection (Figures 1& 2).

**Figure 1.** Adult HIV prevalence by WHO region (WHO 2013) http://www.who.int/gho/hiv/en/. Sub-Sahara Africa has the highest prevalence rate of 4.5% while Western Pacific and Eastern Mediterranean have the least with 0.1% preva‐ lence.

**Figure 2.** Estimated Cervical Cancer Incidence Worldwide in 2012 (GLOBOCAN 2012)

#### **2.4. HIV infection and cervical cancer**

Cervical cancer is one of the malignancies commonly diagnosed in people living with HIV. Other commonly associated malignancies in HIV setting include lymphomas, Kaposis' sarcoma, anal carcinoma and other HPV associated malignancies like vulval and penile cancers. Worldwide, cervical cancer incidence is higher among HIV positive women compared with HIV negative women. In a North American multi-cohort collaboration prospective study involving 13,690 HIV positive and 12,021 HIV negative women, it was found that HIV positive women had 7 times more incidence of cervical cancer than their HIV negative counterparts [9]. In a study in West Africa to assess the relationship be‐ tween HIV infection and cervical cancer, HIV infected women had higher rates of 22/132 (16.7%) than controls 10/120 (8.3%) (p = 0.048) [10].

Cervical cancer has been observed to occur in younger age ranges among HIV positive patients than with HIV negative women (about 10 years younger) and the disease is also noted to have a more aggressive course with metastasis to unusual sites like the skin and brain. Recurrences are much earlier and frequent than in HIV negative women [11, 12]. HIV positive women are also noted to have cervical cancer at higher CD4 counts compared with the low CD4 counts associated with other AID associated malignancies like Kaposis' sarcoma and lymphomas [13].

#### **2.5. Screening for cervical cancer**

**Figure 1.** Adult HIV prevalence by WHO region (WHO 2013) http://www.who.int/gho/hiv/en/. Sub-Sahara Africa has the highest prevalence rate of 4.5% while Western Pacific and Eastern Mediterranean have the least with 0.1% preva‐

280 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

**Figure 2.** Estimated Cervical Cancer Incidence Worldwide in 2012 (GLOBOCAN 2012)

lence.

Screening for cervical cancer is an effective strategy for reducing the incidence and mortality of cervical cancer. The availability of effective screening corresponds with reduced incidence and mortality of the disease. It is the main reason why the incidence of cervical cancer in developed world is less compared with developing (poor resource countries) where screening programs are not available. The recommended schedule of screening of sexually active female populations by The American Cancer Society is summarized in Table 2.


In HIV infected individuals the progression of HPV infection to carcinogenesis is accelerated and there is need to shorten the period of screening in women living with HIV so as to diagnose cervical squamous epithelial changes early. In a report of long term follow up of participants using cervical cytology, Massad and colleagues (2008) reported high grade squamous epithe‐ lial lesion of 4.4 in 1000 person-years in HIV positive patients against 1.3 in 1000-person years among HIV negative women. At ten years observation period, the cumulative risk of abnormal cytology was 77% in HIV positive individuals as against 50% in HIV negative individuals [15]. In another report of a cervical cytology follow up of 409 HIV positive women, progression of cervical lesions occurred in 39 cases. In 24 [61.5%) cases, the first diagnosis was benign cellular changes (BCC) and 21 out of the 24 cases had low-grade squamous intraepithelial lesion (LSIL) after one year. In 11 (28.2%) out of the 204 cases, the first diagnosis was BCC, and 9 cases had high-grade intraepithelial lesion (HSIL) after I year. In 2 (5.0%) out of the 204 cases, the first diagnosis was LSIL and the second was HSIL at one year interval. Two (5.0%) had the first diagnosis as HSIL, and the second as invasive carcinoma at 2-yr interval [16]. Cervical intraepithelial neoplasia (CIN) has also been reported to be more common in HIV positive women with CD4 cell count < 200 cells /ul [17]. Cervical cancer has also been noted to occur in younger women with HIV infection than in those without, and the peak incidence has been reported to be a decade earlier [18]. These results point to the need for shorter screening intervals for HIV positive women. In addition, the diagnosis of abnormal cervical cytology has been shown to be unrelated with current intake of highly active anti-retroviral therapy (HAART) [19]. It could therefore be beneficial to commence cervical cancer screening at an earlier age possibly at age 19 years with a screening interval of 2 years for those with CD4 count ≥ 200 cells/ul and yearly for those with CD4 count < 200 cells/ul irrespective of HAART status.

#### **2.6. Pathophysiology of HIV infection**

HIV infection lowers immunity through the destruction of CD4 lymphocytes. The first target of HIV in the host system is the CD4 T cells. The HIV cell envelope binds to the CD4 cell receptor causing further activation of co-receptors that will eventually lead to the fusion of the host and viral cell membranes. The virus then gets totally into the host cell. This process leads to the destruction of CD4 cells through various mechanisms as the virus multiplies in the host system [20, 21].

The level of destruction is related to the level of HIV viral load in the patients system. CD4 cell count and viral load are the recommended tests to measure HIV positive patients' immune status which can also indicate the rate of destruction of immune cells [22]. Progressive reduction in CD4 cell population reduces the ability of the body to ward off infective agents leading to occurrence of opportunistic infections in HIV infected individuals. Dormant infections such as Herpes zoster can also be reactivated under conditions of depressed immunity. These opportunistic infections add to the deterioration of the clinical states of HIV infected patients leading to poor treatment outcome. Opportunistic infections are common if CD4 cells count is below 200 cells/ul [23]. The list of common infections associated with depressed immunity is presented in Table 3.


**Table 3.** Common opportunistic infections associated with depressed T cell immunity Adapted from Mackall (2000) [23].

#### **2.7. Clinical aspects of HIV infection and cervical cancer**

The higher the HIV viral load, the more likely the compromise in the immune status. Cervical cancer patients with HIV have been reported as having lower levels of CD4 cells count than HIV sero positive patients without cervical cancer. In a report by Leitao and colleagues (2008) comparing the CD4 cells count and viral load in 15 HIV positive cases with cervical cancer with 60 HIV positive patients without cervical cancer controls, the median CD4 count for cases was 208 cells/lL (range, 18-1102 cells/lL) while that for controls was 445 cells/lL (range, 20-1201 cells/lL) (p = 0.03). The median viral load was 16,918 copies/mL (range, 50-214,915 copies/mL) for cases while that for control was 1430 copies/mL (range, 50-571,000 copies/mL) for controls (p= 0.15 [24]). In the WHO staging of HIV, the association of HIV with cervical cancer is classified under stage IV as with other AIDS defining malignancies indicating severity and warrants the commencement of anti-retroviral therapy [25]. HIV infection is also noted to be associated with high grade cervical cancer which leads to rapid progression of the disease.

#### **2.8. Management of cervical cancer**

In HIV infected individuals the progression of HPV infection to carcinogenesis is accelerated and there is need to shorten the period of screening in women living with HIV so as to diagnose cervical squamous epithelial changes early. In a report of long term follow up of participants using cervical cytology, Massad and colleagues (2008) reported high grade squamous epithe‐ lial lesion of 4.4 in 1000 person-years in HIV positive patients against 1.3 in 1000-person years among HIV negative women. At ten years observation period, the cumulative risk of abnormal cytology was 77% in HIV positive individuals as against 50% in HIV negative individuals [15]. In another report of a cervical cytology follow up of 409 HIV positive women, progression of cervical lesions occurred in 39 cases. In 24 [61.5%) cases, the first diagnosis was benign cellular changes (BCC) and 21 out of the 24 cases had low-grade squamous intraepithelial lesion (LSIL) after one year. In 11 (28.2%) out of the 204 cases, the first diagnosis was BCC, and 9 cases had high-grade intraepithelial lesion (HSIL) after I year. In 2 (5.0%) out of the 204 cases, the first diagnosis was LSIL and the second was HSIL at one year interval. Two (5.0%) had the first diagnosis as HSIL, and the second as invasive carcinoma at 2-yr interval [16]. Cervical intraepithelial neoplasia (CIN) has also been reported to be more common in HIV positive women with CD4 cell count < 200 cells /ul [17]. Cervical cancer has also been noted to occur in younger women with HIV infection than in those without, and the peak incidence has been reported to be a decade earlier [18]. These results point to the need for shorter screening intervals for HIV positive women. In addition, the diagnosis of abnormal cervical cytology has been shown to be unrelated with current intake of highly active anti-retroviral therapy (HAART) [19]. It could therefore be beneficial to commence cervical cancer screening at an earlier age possibly at age 19 years with a screening interval of 2 years for those with CD4 count ≥ 200 cells/ul and yearly for those with CD4 count < 200 cells/ul irrespective of HAART

282 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

HIV infection lowers immunity through the destruction of CD4 lymphocytes. The first target of HIV in the host system is the CD4 T cells. The HIV cell envelope binds to the CD4 cell receptor causing further activation of co-receptors that will eventually lead to the fusion of the host and viral cell membranes. The virus then gets totally into the host cell. This process leads to the destruction of CD4 cells through various mechanisms as the virus multiplies

The level of destruction is related to the level of HIV viral load in the patients system. CD4 cell count and viral load are the recommended tests to measure HIV positive patients' immune status which can also indicate the rate of destruction of immune cells [22]. Progressive reduction in CD4 cell population reduces the ability of the body to ward off infective agents leading to occurrence of opportunistic infections in HIV infected individuals. Dormant infections such as Herpes zoster can also be reactivated under conditions of depressed immunity. These opportunistic infections add to the deterioration of the clinical states of HIV infected patients leading to poor treatment outcome. Opportunistic infections are common if CD4 cells count is below 200 cells/ul [23]. The list of common infections associated with

status.

**2.6. Pathophysiology of HIV infection**

depressed immunity is presented in Table 3.

in the host system [20, 21].

The management of cervical cancer follows a multimodality approach. Relevant clinical examinations and investigations to assess the stage of the disease and the suitability of the patient for the modes of therapy have to be done. The choice of treatment depends on the stage of the disease and the performance status of the patient. The treatment choice usually involves surgery, radiotherapy and chemotherapy either alone or in combinations.

The treatment is usually chosen based on the stage of the disease. Treatment follows guidelines that operate in various countries and regions. The European Society for Medical Oncology (ESMO) guidelines (2012) is outlined in Table 4. [26]

#### **2.9. CD4 cell count and cervical cancer treatment**

Chemotherapy leads to suppressed immunity especially through the reduction of CD4 and CD8 cell counts in HIV positive patients. The effect is more marked on CD4 cells. The recovery is slow and better with CD8 than CD4 cells. The recovery of CD4 cells depends on the state of the thymus gland as they are thymus dependent. The thymus gland undergoes involution in the adults and hence recovery of CD4 cells count is usually very slow in those with involute thymus gland. The effect of chemotherapy is more marked with


**Table 4.** Cervical cancer treatment according to Stage (ESMO guideline 2012).

alkylating agents, purine nucleoside analogues and steroids [23]. In a study to assess the activity of the thymus gland after chemotherapy, it was reported that in younger patients aged between 18-49 years, the thymus function was evident in 63% of the participants compared with 0% of their counterparts aged 70-91years three months post treatment [27]. Pelvic radiotherapy has been reported to lower immune cells significantly in HIV sero negative patients. These cells include CD4 T- lymphocytes, B cells and Helper T cells. The reduction could be up to 50% in some instances [28, 29].

The advent of highly active anti-retroviral therapy (HAART) has improved the immunological status of HIV positive patients and control the increase in viral load [30]. HAART leads to rapid reduction in HIV viral load and sometimes to clinically undetectable level [31]. In a study to assess the effects of combination chemotherapy on immune status of HIV associated lympho‐ ma patients, Powles (2002) reported that there was a significant drop in CD4 T cells. Following completion of treatment, the recovery of CD4 T-cells was faster in patients receiving HAART than in those without HAART [32]. The treatment with HAART does not however prevent the development of cervical cancer in HIV positive patients [33].

Patients with compromised immunity usually suffer more treatment toxicities as well. Chemoradiotherapy used in the treatment of cervical cancer affect the immune status of patients. Chemotherapy leads to immune cells suppression and the toxicity following radiotherapy is increased in patients with compromised immunity [18]. HIV positive patients not on HAART are therefore more likely to experience compromised immunity than those on HAART. With decreasing immune status among HIV positive patients, the rate of decrease of CD4 cells can be unpredictable and such patients can suffer from opportunistic infections that will further complicate their conditions.

#### **2.10. Management of cervical cancer in HIV positive patients**

alkylating agents, purine nucleoside analogues and steroids [23]. In a study to assess the activity of the thymus gland after chemotherapy, it was reported that in younger patients aged between 18-49 years, the thymus function was evident in 63% of the participants compared with 0% of their counterparts aged 70-91years three months post treatment [27]. Pelvic radiotherapy has been reported to lower immune cells significantly in HIV sero negative patients. These cells include CD4 T- lymphocytes, B cells and Helper T cells. The

IB1, IIA Radical hysterectomy and PLND Adjuvant CT/RT if risk factors (LVSI, G3,

IB2, IIB–IV Combination CT/RT with cisplatin NACT to large bulky tumors prior to

Conservative surgery

nodes)

nodes)

CT/RT

Adjuvant CT/RT if risk factors (LVSI, G3, positive resection margins, multiple

positive resection margins, multiple

The advent of highly active anti-retroviral therapy (HAART) has improved the immunological status of HIV positive patients and control the increase in viral load [30]. HAART leads to rapid reduction in HIV viral load and sometimes to clinically undetectable level [31]. In a study to assess the effects of combination chemotherapy on immune status of HIV associated lympho‐ ma patients, Powles (2002) reported that there was a significant drop in CD4 T cells. Following completion of treatment, the recovery of CD4 T-cells was faster in patients receiving HAART than in those without HAART [32]. The treatment with HAART does not however prevent the

Patients with compromised immunity usually suffer more treatment toxicities as well. Chemoradiotherapy used in the treatment of cervical cancer affect the immune status of patients. Chemotherapy leads to immune cells suppression and the toxicity following radiotherapy is increased in patients with compromised immunity [18]. HIV positive patients not on HAART are therefore more likely to experience compromised immunity than those on HAART. With decreasing immune status among HIV positive patients, the rate of decrease of CD4 cells can be unpredictable and such patients can suffer from opportunistic infections that will further

reduction could be up to 50% in some instances [28, 29].

**Stage Treatment Issue**

PLND- pelvic lymphadenectomy; LVSI- lymphovascular space invasion; CT- computed

tomography; NACT- neoadjuvant chemotherapy; RT- radiation therapy

**Table 4.** Cervical cancer treatment according to Stage (ESMO guideline 2012).

IA1 Conization or simple hysterectomy ± salpingo-ophorectomy

IA2 Conization/radical trachelectomy or modified radical

284 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

and PLND if LVSI

hysterectomy and PLND

development of cervical cancer in HIV positive patients [33].

complicate their conditions.

Decisions on the management of cervical cancer in HIV positive patients are not straight forward. This is because of the immune concerns about such patients. Contributory factors to immune compromise in such patients include the cancer, the HIV infection and the modalities of treatment – chemotherapy and radiotherapy. There is the fear that oncology treatment will worsen the immune status of treatment. Standard treatment as with HIV seronegative patients are recommended. Radiation treatment of HIV positive patients with cervical cancer however, has been reported to be associated with a seven fold increase in multi-systemic toxicities compared with HIV sero-negative patients [18]. HIV patients with malignancy have also been reported to have impaired ability of the mucosa to repair radiation damage. In treating oropharyngeal tumour with radiation, it was reported that HIV positive patients had mucosal reaction with lesser doses of radiation than HIV negative patients [34]. With regards to cervical cancer, it is likely that the tissues with mucosal lining close to the treatment fields like urinary bladder and gastro intestinal tract (GIT) might be affected in a similar way. This pattern of mucosal reaction is attributable to low immune status of the patients.

Close to about 10% of HIV positive patients are also reported to be co-infected with hepatitis B virus (HBV) or hepatitis C virus (HCV) or with both [35]. HIV positive patients diagnosed with cervical cancer should therefore be routinely screened for HBV and HCV. A study in sub-Sahara Africa reported that among HIV-infected individuals, mean HBV and HCV prevalence rates were 15% and 7%, respectively [36]. Such co-infections have been reported to accelerate the progression of HIV infection [37]. Such infections also warrant extra caution in using HAART drugs that are metabolised in the liver to minimise liver toxicity that may occur [38]. Chemotherapy and radiotherapy may also activate viral hepatitis in such patients leading to complications of therapy [39].

Essential measures to improve treatment tolerance and optimal outcome in HIV positive patients with cervical cancer include early commencement of HAART (at diagnosis) based on WHO recommendation of commencement of anti- retroviral therapy (ART) in WHO HIV stage IV patients. Other studies have also recommended the commencement (at diagnosis) of ART in all patients diagnosed with cancer based on the study results that show better outcome of treatment in such patients. Such patients are noted to have better CD4 cell count and viral load responses compared with those not on ART and are more likely to complete oncology treatment on schedule [40, 41, 42]. Generally, early commencement of ART in HIV positive individuals has been recently shown to be of benefit. In a randomized trial in Cote d'Ivore West Africa, in which 2,056 participants with HIV-1 infection were included in the analysis (Temprano Trial), it was reported that ART reduced the possibility of severe illness by 44% in people starting treatment immediately at diagnosis, as compared to those starting ART only when their CD4 levels drop to below 500/mm3 . The study also reported that prophylaxis against tuberculosis with isoniazid initiated among people living with HIV with a CD4 count greater than 500/mm3 reduced the risk of severe illness by 35%, compared with those without such treatment. Early initiation of isoniazid was also not associated with increase in the development of resistance to isoniazid [43].

Regular monitoring of CD4 cell count and viral load assay is needed towards early intervention in case of derangements below critical levels. Msadabwe (2009) reported the CD4 cells count trend during treatment and up to three months after treatment of HIV positive patients with cervical cancer treated with chemoradiotherapy. The average initial CD4 cells count was 321.06 cells /mm2 at commencement of treatment. This gradually dropped to 62.56 cells/mm2 at the end of treatment giving a mean difference of 258.2 cells /mm2 . There was however, gradual rise after treatment but by 3 months which was the end of the follow up period of the study, the pre-treatment level was not reached. The average count at the end of three months was one third of the pre-treatment value [44]. Significant drop in CD4 cells following radiotherapy applies to both HIV negative and HIV positive patients especially if the radiation fields are around areas with large lymphoid tissues such as the chest and pelvis. In HIV seronegative patients treated for early stage breast cancer and stage I seminoma with radiotherapy, it was reported that the CD4 cells count dropped by about 200 cells/ul and that pre-treatment levels could not be attained after six years follow up [45]. Monthly CD4 cells count assay is therefore needed to monitor the trend in CD4 cells count during treatment and at three monthly intervals after treatment to ensure adequate CD4 count levels. This practice will enable the early commencement of prophylaxis against opportunistic infections if CD4 cells count is below critical levels so as to reduce morbidity in the patients. The recommended CD4 levels for commencement of appropriate prophylaxis are presented in Table 5.


**Table 5.** Criteria for initiating and discontinuing prophylaxis for opportunistic infections in HIV positive patients [adapted from NIH- AIDS Information 2015]. [46]

Other steps include testing for viral load every six months. This will ensure early diagnosis of drug resistances as increasing viral load while patient is on ART may indicate onset of drug resistance which should be promptly investigated and appropriate ART changes made.

External beam radiation therapy should be delivered at a daily dose of 1.8Gy per fraction to HIV positive patients to minimize toxicity [47]. Patients with CD4 cells count less than 200 cells/ul should be treated with 1.5Gy per fraction while the dose of weekly cisplatin should be given at a reduced dose of 30-35mg/m2 . These modifications have been reported to result in treatment tolerance similar to HIV negative patients [45]. Patients with CD4 cells count less than 150 cells /ul may however, not be able to withstand long course of radiation therapy and should be given short course treatments depending on performance status. The rate of completion of chemotherapy has been reported to be 30-45% among HIV positive patients compared with 64-89% among HIV negative patients[48, 49].

The above measures could help in improving the rate of completion of treatment in HIV positive patients. Renal dysfunction not myelo- suppression or gastrointestinal toxicity has been reported in a retrospective study, to be the main cause of chemotherapy suspension in HIV positive patients treated for cervical cancer and that chemotherapy was the most difficult section to be completed in HIV positive patients [4]. Carboplatin chemotherapy may be preferred to cisplatin in order to improve chemotherapy completion rate in HIV positive patients. Patients with CD4 cells count less than 200 cells/ul should however, not receive chemotherapy.

#### **2.11. Drug interactions between chemotherapy and anti-retroviral agents**

Regular monitoring of CD4 cell count and viral load assay is needed towards early intervention in case of derangements below critical levels. Msadabwe (2009) reported the CD4 cells count trend during treatment and up to three months after treatment of HIV positive patients with cervical cancer treated with chemoradiotherapy. The average initial CD4 cells count was 321.06 cells /mm2 at commencement of treatment. This gradually dropped to 62.56 cells/mm2

rise after treatment but by 3 months which was the end of the follow up period of the study, the pre-treatment level was not reached. The average count at the end of three months was one third of the pre-treatment value [44]. Significant drop in CD4 cells following radiotherapy applies to both HIV negative and HIV positive patients especially if the radiation fields are around areas with large lymphoid tissues such as the chest and pelvis. In HIV seronegative patients treated for early stage breast cancer and stage I seminoma with radiotherapy, it was reported that the CD4 cells count dropped by about 200 cells/ul and that pre-treatment levels could not be attained after six years follow up [45]. Monthly CD4 cells count assay is therefore needed to monitor the trend in CD4 cells count during treatment and at three monthly intervals after treatment to ensure adequate CD4 count levels. This practice will enable the early commencement of prophylaxis against opportunistic infections if CD4 cells count is below critical levels so as to reduce morbidity in the patients. The recommended CD4 levels for

**Pathogen Initiate Prophylaxis Preferred agent Discontinuation of Prophylaxis**

once weekly or

twice weekly

daily

**Table 5.** Criteria for initiating and discontinuing prophylaxis for opportunistic infections in HIV positive patients

Other steps include testing for viral load every six months. This will ensure early diagnosis of drug resistances as increasing viral load while patient is on ART may indicate onset of drug resistance which should be promptly investigated and appropriate ART changes made.

External beam radiation therapy should be delivered at a daily dose of 1.8Gy per fraction to HIV positive patients to minimize toxicity [47]. Patients with CD4 cells count less than 200

Trimethoprim/ Sulfamethoxazole

Trimethoprim/ Sulfamethoxazole (TMP/SMX)

Clarithromycin 500mg orally

(TMP/SMX) double strength

Single strength daily or double strength three times weekly.

CD4 <50 cells/mm3 Azithromycin 1200mg orally

end of treatment giving a mean difference of 258.2 cells /mm2

286 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

commencement of appropriate prophylaxis are presented in Table 5.

CD4 <100 cells/mm3 and Positive serology for Toxoplasma (IgG+)

CD4 <200 cells/mm3 or a history of oropharyngeal

candidiasis

[adapted from NIH- AIDS Information 2015]. [46]

Mycobacterium avium complex (MAC)

Toxoplasma gondii encephalitis (TE)

(PCP)

Pneumocystis pneumonia

at the

. There was however, gradual

CD4 count increase to >100 cells/mm3 for ≥3 months in

Patient receiving ART with increase in CD4 count to >200 cells/mm3 for ≥3 months

CD4 count >200 cells/mm3 for >3 months in response to ART ⋅ Adequate viral suppression ⋅ If PCP occurred with CD4 >200 cells/mm3, prophylaxis should be maintained

response to ART

Platinum compounds commonly used in the chemotherapy of cervical cancer are cisplatin and carboplatin. Patients with persistent or recurrent and metastatic disease can have paclitaxel added to their treatment regimen [50]. On the other hand, anti-retroviral therapy in HIV treatment consists of combinations of three different drugs from at least two different drug classes (Table 6).


\*Drugs commonly used for first line treatment of HIV infections.

**Table 6.** Major HIV drug classes. At least three drugs from two drug classes are selected for the treatment of HIV when ART is indicated Sources: NIH-NIAID, 2015; hiv-druginteractions.org 2015). [51]

Commonly used first line drugs are stavudine or tenofovir, lamivudine [Nucleoside Reverse Transcriptase Inhibitors (NRTIs) and efavirenz [Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) [4]. Various other second line combinations are used in cases of drug resistance development or intolerable side effects of the first line combination drug regimen. Pharmacokinetic enhancers like Cobicistat [COBI] can be included with some of the combina‐ tions to increase the effectiveness of the treatment. Most HIV positive patients diagnosed with cervical cancer who are on HAART are likely to be placed on regimen containing the above drugs [52, 53].

Possibilities of drug interactions and potentiation of toxicities exist among these chemotherapy and ART drugs and these can affect treatment outcome. Paclitaxel is metabolized mainly by CYP 2C8 enzyme of the cytochrome P450 system to 6 alpha-hydroxypaclitaxel. Paclitaxel metabolites are inactive in comparison with the parent drug [54]. CYP 2C8 enzyme can be inhibited by some ART drugs such as Delavirdine, Ritonavir, Fosamprenavir, Atazanavir, Indinavir, Lopinavir, Nelfinavir and Saquinavir. Concomitant intake of any of these agents can lead to increased toxicity of paclitaxel. On the other hand, Nevirapine is CYP 2C8 enzyme inducer and on concomitant intake of this agent with paclitaxel can lead to accelerated clearance of the active parent drug leading to ineffectiveness of paclitaxel [55].


a Might increase risk of neuropathy as both drugs could cause neuropathy.

b Cisplatin is eliminated through renal route via organic cation transporter 2 (OCT2) and human multidrug and toxin extrusion 1 (MATE1) enzymes. Cisplatin and lamivudine could compete for OCT2 which could slow their elimination. Lamivudine dose could be adjusted.

c Carboplatin and stavudine administered together can increase the risk of peripheral neuropathy due to additive toxicity.

d. Both have nephrotoxic potential. Dose of tenofovir may need to be adjusted appropriately.

e. Lamivudine may affect renal function hence dose may need to be adjusted.

<sup>f</sup> Efavirenz is a strong inhibitor of CYP2C8 enzyme mostly involved in the metabolism of paclitaxel. Co administration of these agents may increase the toxicity of paclitaxel.

NB. The above interactions are supported by very low levels of evidence.

**Table 7.** Cytotoxic and HIV drugs interactions relevant to HIV and cervical cancer (Source: HIV drugsinteraction.org. Accessed 2015 March 11).

There can be overlapping side effects between chemotherapy and ART drugs. Myelo-sup‐ pression is associated with most chemotherapeutic agents including paclitaxel and platinum compounds and this can also be induced by the ART drug zidovudine. Paclitaxel can also cause neuropathy likewise didanosine and stavudine. Care therefore has to be exercised in patients that take these drugs concomitantly or other alternatives should be given. Cisplatin and carboplatin can cause nephrotoxicity likewise the ART drug tenofovir while nausea and vomiting which is common with most chemotherapy drugs can also be induced by ART drugs in the classes of protease inhibitors, nucleoside and nonnucleoside reverse transcriptase inhibitors [50]. Patients on these agents should have effective management of nausea and vomiting with potent anti-emetics.

The possible interactions of cytotoxic drugs commonly used in cervical cancer chemotherapy with first line drugs used in the treatment of HIV infection are presented in Table 7 above.

Interactions between cytotoxic drugs used in the treatment of cervical cancer and first line ART drugs are quite favorable as contained in Table 7 with the associated levels of evidence. Combining the treatment modalities in HIV positive patients should therefore be tolerated by most patients.

#### **3. Conclusion**

Commonly used first line drugs are stavudine or tenofovir, lamivudine [Nucleoside Reverse Transcriptase Inhibitors (NRTIs) and efavirenz [Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) [4]. Various other second line combinations are used in cases of drug resistance development or intolerable side effects of the first line combination drug regimen. Pharmacokinetic enhancers like Cobicistat [COBI] can be included with some of the combina‐ tions to increase the effectiveness of the treatment. Most HIV positive patients diagnosed with cervical cancer who are on HAART are likely to be placed on regimen containing the above

Possibilities of drug interactions and potentiation of toxicities exist among these chemotherapy and ART drugs and these can affect treatment outcome. Paclitaxel is metabolized mainly by CYP 2C8 enzyme of the cytochrome P450 system to 6 alpha-hydroxypaclitaxel. Paclitaxel metabolites are inactive in comparison with the parent drug [54]. CYP 2C8 enzyme can be inhibited by some ART drugs such as Delavirdine, Ritonavir, Fosamprenavir, Atazanavir, Indinavir, Lopinavir, Nelfinavir and Saquinavir. Concomitant intake of any of these agents can lead to increased toxicity of paclitaxel. On the other hand, Nevirapine is CYP 2C8 enzyme inducer and on concomitant intake of this agent with paclitaxel can lead to accelerated

clearance of the active parent drug leading to ineffectiveness of paclitaxel [55].

Potential interaction d Potential interaction <sup>e</sup>

**Paclitaxel** No interaction No interaction No interaction <sup>f</sup>

d. Both have nephrotoxic potential. Dose of tenofovir may need to be adjusted appropriately.

**Cisplatin** a Potential interaction a Potential interaction <sup>b</sup>

288 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

Might increase risk of neuropathy as both drugs could cause neuropathy.

e. Lamivudine may affect renal function hence dose may need to be adjusted.

NB. The above interactions are supported by very low levels of evidence.

of these agents may increase the toxicity of paclitaxel.

**Drugs Stavudine Tenofovir Lamivudine Efavirenz**

 Cisplatin is eliminated through renal route via organic cation transporter 2 (OCT2) and human multidrug and toxin extrusion 1 (MATE1) enzymes. Cisplatin and lamivudine could compete for OCT2 which could slow their elimination.

Carboplatin and stavudine administered together can increase the risk of peripheral neuropathy due to additive toxicity.

<sup>f</sup> Efavirenz is a strong inhibitor of CYP2C8 enzyme mostly involved in the metabolism of paclitaxel. Co administration

**Table 7.** Cytotoxic and HIV drugs interactions relevant to HIV and cervical cancer (Source: HIV drugsinteraction.org.

Potential interaction No interaction

Potential interaction No interaction

Potential interaction

drugs [52, 53].

**Carboplatin** <sup>c</sup>

Lamivudine dose could be adjusted.

Accessed 2015 March 11).

a

b

c

The outcome of treatment in HIV positive patients diagnosed with cervical cancer is still poor especially in regions with high prevalence of HIV and cervical cancer. This could be improved through prompt commencement of such patients on ART at diagnosis. Close monitoring of the immune status (CD4 cell) and viral load is needed to ensure early diagnosis of depressed immune status and HAART treatment resistance. This could give early indication for com‐ mencement of appropriate prophylaxis against opportunistic infections and review of ART drug combinations. There is need to continue further search for other modes of treatment such as targeted therapies and radio sensitizers that can improve the effectiveness of managing HIV positive patients diagnosed with cervical cancer. Prospective studies are also needed to establish optimal radiation and chemotherapy doses in HIV positive patients diagnosed with cervical cancer.

#### **Author details**

Atara Ntekim\*

Address all correspondence to: tarantekim@yahoo.com

Department of Radiation Oncology, College of Medicine/ University College Hospital, Uni‐ versity of Ibadan, Ibadan, Nigeria

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[30] April MD, Wood R, Berkowitz BK, Paltiel AD, Anglaret X, Losina E, et al. The surviv‐ al benefits of antiretroviral therapy in South Africa. J Infect Dis [Internet]. 2014 Feb 15 [cited 2015 Mar 14];209(4):491–9. Available from: http://www.pubmedcen‐ tral.nih.gov/articlerender.fcgi?artid=3903379&tool=pmcentrez&rendertype=abstract

[31] Sedaghat AR, Siliciano RF, Wilke CO. Low-level HIV-1 replication and the dynamics of the resting CD4+ T cell reservoir for HIV-1 in the setting of HAART. BMC Infect Dis [Internet]. 2008 Jan [cited 2015 Mar 14];8:2. Available from: http://www.pubmed‐ central.nih.gov/articlerender.fcgi?artid=2254415&tool=pmcentrez&rendertype=ab‐

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#### **Chapter 14**

## **Sentinel Lymph Node Detection in Early Stage Cervical Cancer**

Elisa Moreno-Palacios, Elsa Delgado, Javier De Santiago and Ignacio Zapardiel

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61099

#### **Abstract**

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fda-approved-hiv-medicines#

Worldwide, cervical cancer is the fourth most common malignancy among women. Radical hysterectomy and pelvic lymphadenectomy is the standard treatment for early stage cervical cancer. If lymph node metastasis is present at the time of diagnosis, 5-year survival rate drops from 90% to 57%. The risk of lymph node metastases in women with early stage cervical cancer is approximately 15%, and determines the use of adjuvant treatment. Over 80% of patients do not benefit from pelvic lymphadenec‐ tomy, but may suffer from adverse complications or sequelae such as lymphedema, lymphocyst formation, and neurovascular and ureteral injury. The sentinel lymph node is the first node to which metastatic disease will spread from a primary tumor. The clinical benefits of biopsy of only the sentinel lymph node includes a significant reduction in the adverse effects of complete lymphadenectomy. The specific benefits of sentinel lymph node detection in early stage cervical cancer includes improved identification of metastatic lymph nodes through ultrastaging and identification of alternate lymph node drainage sites, as well as the possibility of intraoperative frozen section analysis, which may be used to guide surgical management. Sentinel lymph node detection in early stage cervical cancer could become the standard of care in the near future.

**Keywords:** Cervical cancer, sentinel lymph node, lymphadenectomy, ultrastaging, micrometastasis

© 2016 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.

#### **1. Introduction**

Worldwide, cervical cancer is the fourth most common cancer among women, after breast, colorectal, and lung cancers. Almost 70% of the global burden occurs in developing countries, where it accounts for almost 12% of all female malignancies, being a major public health problem in many developing countries [1]. It is well known that the most important cause of cervical cancer is the presence of a persistent papillomavirus infection [2]. The risk factors for developing cervical cancer are the same as those for acquiring the human papillomavirus (HPV) infection, such as early age intercourse, multiple sexual partners, and sexual contact with high-risk men. HPV type 16 and 18 are responsible for approximately 70–75% of all cervical tumors [3].

However, long-term (1992-2010) cancer incidence trends for all racial and ethnic groups show that cervical cancer has experienced the largest decrease in incidence among women [4]. This decrease in incidence is related mostly to cervical cancer screening programs with Papanico‐ laou smears and HPV DNA cervical detection. Moreover, cervical cancer screening programs are associated with a potentially significant reduction in the diagnosis of advanced cervical cancers and death. Cervical cancer screening is well established in developed countries, but it is still taken of in developing countries.

Most developed countries have introduced HPV vaccination in their vaccination calendar, expecting to lower the incidence of cervical cancer. However, cervical cancer still represents a health problem in developed countries with 54,517 new cases diagnosed and 24,874 deaths from this disease every year in Europe [5].

#### **2. Diagnosis**

Early stage cervical cancer is commonly asymptomatic, diagnosed by pathological Papanico‐ laou smears. Advanced cervical cancer can present with symptoms such as abnormal vaginal bleeding, intercourse bleeding, dyspareunia, or pelvic plain.

The diagnosis of cervical cancer requires histological confirmation in all cases. If the patient presents with a macroscopic cervical lesion, a direct biopsy ought to be performed. If changes are shown in the cytological study but there is no macroscopic lesion in the cervix a colposcopy should be carried out. If the colposcopy findings are suspicious of malignancy directed biopsies ought to be taken, on the other hand if the colposcopy doesn't present any alterations, an endocervical curettage is indicated. If microscopic cervical invasive lesions are present a conization is required for tumor staging.

There are three categories of epithelial tumor of the cervix recognized by the WHO: squamous, glandular (adenocarcinoma), and other epithelial tumors including neuroendocrine tumors and undifferentiated tumors. Squamous cell carcinoma accounts for approximately 70–80% of all cervical cancers, and adenocarcinoma for 10–15%. Neuroendocrine tumors of the cervix are highly aggressive, rare tumors with a prognosis worse than stage-comparable undifferentiated squamous cell carcinoma of the cervix, and have a different therapeutic management [6]. In this chapter we will be referring to squamous and adenocarcinomas of the cervix exclusively.

The histological report of the biopsy and/or conization of the cervix should include the following information: histological type, differentiation grade, tumor size, length of stromal invasion, and the presence or absence of lymph-vascular space invasion (LVSI). Pathological information is very important for the tumor risk assessment.

#### **3. Tumor staging**

**1. Introduction**

296 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

cervical tumors [3].

**2. Diagnosis**

is still taken of in developing countries.

from this disease every year in Europe [5].

conization is required for tumor staging.

bleeding, intercourse bleeding, dyspareunia, or pelvic plain.

Worldwide, cervical cancer is the fourth most common cancer among women, after breast, colorectal, and lung cancers. Almost 70% of the global burden occurs in developing countries, where it accounts for almost 12% of all female malignancies, being a major public health problem in many developing countries [1]. It is well known that the most important cause of cervical cancer is the presence of a persistent papillomavirus infection [2]. The risk factors for developing cervical cancer are the same as those for acquiring the human papillomavirus (HPV) infection, such as early age intercourse, multiple sexual partners, and sexual contact with high-risk men. HPV type 16 and 18 are responsible for approximately 70–75% of all

However, long-term (1992-2010) cancer incidence trends for all racial and ethnic groups show that cervical cancer has experienced the largest decrease in incidence among women [4]. This decrease in incidence is related mostly to cervical cancer screening programs with Papanico‐ laou smears and HPV DNA cervical detection. Moreover, cervical cancer screening programs are associated with a potentially significant reduction in the diagnosis of advanced cervical cancers and death. Cervical cancer screening is well established in developed countries, but it

Most developed countries have introduced HPV vaccination in their vaccination calendar, expecting to lower the incidence of cervical cancer. However, cervical cancer still represents a health problem in developed countries with 54,517 new cases diagnosed and 24,874 deaths

Early stage cervical cancer is commonly asymptomatic, diagnosed by pathological Papanico‐ laou smears. Advanced cervical cancer can present with symptoms such as abnormal vaginal

The diagnosis of cervical cancer requires histological confirmation in all cases. If the patient presents with a macroscopic cervical lesion, a direct biopsy ought to be performed. If changes are shown in the cytological study but there is no macroscopic lesion in the cervix a colposcopy should be carried out. If the colposcopy findings are suspicious of malignancy directed biopsies ought to be taken, on the other hand if the colposcopy doesn't present any alterations, an endocervical curettage is indicated. If microscopic cervical invasive lesions are present a

There are three categories of epithelial tumor of the cervix recognized by the WHO: squamous, glandular (adenocarcinoma), and other epithelial tumors including neuroendocrine tumors and undifferentiated tumors. Squamous cell carcinoma accounts for approximately 70–80% of all cervical cancers, and adenocarcinoma for 10–15%. Neuroendocrine tumors of the cervix are highly aggressive, rare tumors with a prognosis worse than stage-comparable undifferentiated Cervical cancer FIGO classification is based on clinical examination, considering the tumors size, vaginal and parametrial involvement, bladder/rectum extension and distant metastasis. If the clinical examination is difficult or uncertain considering vaginal and/or parametrial involvement, it should be performed under anesthesia.

To determine the tumor's extension, various imaging tests are helpful, such as computed tomography (CT) scan, magnetic resonance imaging (MRI), and positron emission tomogra‐ phy (PET-CT). CT scan, to detect pathological loco-regional lymph nodes. While MR imaging is suited for examining soft tissue alterations, helping to determine the size, degree of stromal invasion, possible parametrial involvement, possible vaginal infiltration, and pelvic extension of the tumor. PET-CT imaging is known to determine accurately the extent of the disease, mainly by detecting possible metastatic lymph nodes and distant metastatic disease.

Cervical cancer FIGO stages IA, IB, and IIA are considered early stage tumors (Table 1). Approximately 44% of all cervical cancers are diagnosed in the early stages. Stage IA tumors are defined as invasive carcinomas that present with a stromal invasion of less than 5mm, and a horizontal extension of less than 7mm. Stage IB tumors are defined as invasive carcinomas limited to the cervix that present with a stromal invasion and a horizontal extension greater than 5mm and 7mm, respectively. Stage IIA tumors are defined as invasive carcinomas that invade beyond the uterus but do not involve the parametrium or the lower third of the vagina.

The lymph node status is not included in the FIGO staging system (Table 1), although it is the most important independent prognostic factor in early stage cervical cancer. If lymph node metastases are present at the time of diagnosis, the 5-year survival rate drops substantially. In stages IB-IIA, the 5-year survival rate drops from 88%–95% without lymph node metastasis to 51–78% with lymph node metastasis [7].

To determine the lymph node status, several imaging tests have been used, including CT and MRI. The major problem of the CT scan and the MRI is that these imaging tests only detect changes in the size and form of the lymph nodes and are not able to distinguish between metastasis and inflammation of the lymph nodes, presenting both low sensitivity and specif‐ icity. More recently, PET-CT has been seen to accurately determine the extent of the disease, particularly determining the lymph node status, with a sensitivity of 53–73% and a specificity as high as 90–97% [8,9]. Although, PET-TC presents higher sensitivity and specificity than CT and MRI, it is known to detect only lymph node metastases larger than 6mm, possibly not


**Table 1.** FIGO stage classification.

accurately detecting lymph node metastases in a high percentage of patients. Until the present moment, pelvic lymphadenectomy has been the standard surgical procedure for the assess‐ ment of the lymph node status in early stage cervical cancer, being an integral component of the definitive surgical management.

#### **4. Treatment**

There are several treatment options for cervical cancer, depending on the stage, the prognostic factors, and the wish to preserve fertility of the patient.

In early stage cervical cancer, surgery is considered the standard treatment, although radio‐ therapy is equally effective, only differing in terms of morbidity and complications. Surgery offers benefits over radiotherapy in early stage cervical cancer, such as ovarian function preservation, maintenance of a more functional vagina, and facilitation of the knowledge of

**Figure 5.** Lymphatic channels with fluorescein.

accurately detecting lymph node metastases in a high percentage of patients. Until the present moment, pelvic lymphadenectomy has been the standard surgical procedure for the assess‐ ment of the lymph node status in early stage cervical cancer, being an integral component of

There are several treatment options for cervical cancer, depending on the stage, the prognostic

In early stage cervical cancer, surgery is considered the standard treatment, although radio‐ therapy is equally effective, only differing in terms of morbidity and complications. Surgery offers benefits over radiotherapy in early stage cervical cancer, such as ovarian function preservation, maintenance of a more functional vagina, and facilitation of the knowledge of

the definitive surgical management.

**Table 1.** FIGO stage classification.

factors, and the wish to preserve fertility of the patient.

298 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

**4. Treatment**

pathological prognostic factors. In locally advanced stages, a combination of radiotherapy and chemotherapy is the standard treatment.

Fertility-preserving surgery, consisting of radical or simple trachelectomy can be offered to young patients with early stage cervical cancer with a strong wish to preserve their fertility [10].

The goal of radical hysterectomy or radical trachelectomy is to remove the tumor with free margins by excising the uterus, cervix, and parametrium. Pelvic lymphadenectomy is per‐ formed to determine the presence or absence of lymph node metastasis, for both prognostic and therapeutic planning. Pelvic lymphadenectomy is a mere staging surgical procedure. In the absence of lymph node metastasis, pelvic lymphadenectomy has no therapeutic effect, with potential complications and associated sequelae.

There are different possible surgical approaches such as laparotomy, laparoscopy, or vaginal surgery. All surgical approaches are considered comparable in terms of oncological results when carried out by experienced surgeons. Minimally invasive surgery (laparoscopy) shows the same efficiency as conventional laparotomy, with lesser blood loss, shorter hospital stay, and lower perioperative morbidity [11].

The risk of lymph node metastasis in early stage cervical cancer is approximately 15%. Consequently, 85% of patients with early stage cervical cancer not only do not benefit from the pelvic lymphadenectomy, but can also suffer complications and morbidity. Pelvic lym‐ phadenectomy is associated with a 4% risk of intraoperative complications such as vascular and neurological lesions, as well as long-term complications, especially lymphocyst formation and lymphedema [12]. Lymphocyst formation occurs in up to 30% of the patients subjected to a pelvic lymphadenectomy. Lymphedema of the lower abdomen, pubis, and lower extremities occurs in 25% of the patients, more frequently in those patients that receive adjuvant radio‐ therapy after surgery [13]. These complications are very hard to treat and can produce an important impact in the patient's quality of life [14].

Surgery is not recommended in patients with early stage cervical cancer who present with poor prognostic factors. There are pathological factors associated with high risk of relapse such as positive or close margins, metastatic lymph nodes, or microscopic parametrial involvement. If one or more of these poor prognostic factors are present at the time of diagnosis, chemoradiotherapy is indicated. Chemo-radiation therapy in high-risk patients is associated with better 4-year overall survival and progression-free survival [15].

Patients with metastatic pelvic lymph nodes are at risk of having para-aortic metastatic lymph nodes. In order to determine the fields of radiation, a para-aortic lymphadenectomy ought to be performed [16]. Radiotherapy administration after radical hysterectomy increases the risk of radiotherapy-related complications, especially intestinal complications by adhesion formation.

A correct pre-therapeutic evaluation is needed to select patients who will benefit from receiving radio-chemotherapy. Radical hysterectomy is not recommended in early stage cervical cancer that presents poor prognostic factors, such as lymph node metastasis, due to the fact that adjuvant radiotherapy and chemotherapy are required in an attempt to improve survival. The objective of avoiding surgery is to prevent the addition of morbidity caused by the association of radiotherapy and surgery.

#### **5. Sentinel lymph node biopsy**

Sentinel lymph node (SLN) is defined as the first node to which metastatic disease will spread from a primary tumor. Consequently, in the absence of metastasis in the sentinel lymph node, all other lymph nodes will also be free of disease. Therefore, if the sentinel lymph node has no trace of disease, lymphadenectomy can be avoided, reducing the morbidity associated with a complete lymphadenectomy.

Sentinel lymph node detection was first described by Cabanas in penile cancer [17]. Since then it has been described in multiples tumors, being the standard of care in melanoma, breast, and vulvar cancer, reducing significantly the morbidity associated with the performance of a complete lymphadenectomy in these patients.

Sentinel lymph nodes are identified by the injection of dye and/or a radioactive tracer around the tumor site. In cervical cancer, the sentinel lymph node is detected by injecting Technetium (Tc-99), blue dye, or both into the cervix. Protocols of detection vary in different studies, reporting that the highest detection rate is found when the combination of Tc-99 and blue dye is used [18]. The cervix point of injection varies in different studies. In some studies, the tracer is injected submucosally into the four quadrants of the cervix, and in others the tracer is injected submucosally into the 3 and 9 o'clock of the cervix; no significant differences have been found between these two techniques [9, 18].

After the radiotracer is injected, a lymphoscintigraphic localization imaging can be conducted. Lymphoscintigraphy is an imaging technique used to identify the lymph drainage basin, the sentinel lymph node, the location of the sentinel lymph node, the number of sentinel lymph nodes, and possible secondary drainage. Lymphoscintigraphy helps the surgeon to identify and localize the sentinel lymph node during the surgical procedure [19].

Different protocols of radiotracer injection and subsequent lymphoscintigraphic imaging have been described in the literature. Protocols differ in the time frame from which the radiotracer is injected till the surgery is performed, defining long and short protocols. In the long protocols the radiotracer is injected the day before surgery and lymphoscintigraphy is performed 1h after the injection [20]. In the short protocol the radiotracer is injected between 2 and 4 hours before surgery, and the lymphoscintigraphy is performed 20 minutes after the injection [9].

radiotherapy is indicated. Chemo-radiation therapy in high-risk patients is associated with

Patients with metastatic pelvic lymph nodes are at risk of having para-aortic metastatic lymph nodes. In order to determine the fields of radiation, a para-aortic lymphadenectomy ought to be performed [16]. Radiotherapy administration after radical hysterectomy increases the risk of radiotherapy-related complications, especially intestinal complications by adhesion

A correct pre-therapeutic evaluation is needed to select patients who will benefit from receiving radio-chemotherapy. Radical hysterectomy is not recommended in early stage cervical cancer that presents poor prognostic factors, such as lymph node metastasis, due to the fact that adjuvant radiotherapy and chemotherapy are required in an attempt to improve survival. The objective of avoiding surgery is to prevent the addition of morbidity caused by

Sentinel lymph node (SLN) is defined as the first node to which metastatic disease will spread from a primary tumor. Consequently, in the absence of metastasis in the sentinel lymph node, all other lymph nodes will also be free of disease. Therefore, if the sentinel lymph node has no trace of disease, lymphadenectomy can be avoided, reducing the morbidity associated with a

Sentinel lymph node detection was first described by Cabanas in penile cancer [17]. Since then it has been described in multiples tumors, being the standard of care in melanoma, breast, and vulvar cancer, reducing significantly the morbidity associated with the performance of a

Sentinel lymph nodes are identified by the injection of dye and/or a radioactive tracer around the tumor site. In cervical cancer, the sentinel lymph node is detected by injecting Technetium (Tc-99), blue dye, or both into the cervix. Protocols of detection vary in different studies, reporting that the highest detection rate is found when the combination of Tc-99 and blue dye is used [18]. The cervix point of injection varies in different studies. In some studies, the tracer is injected submucosally into the four quadrants of the cervix, and in others the tracer is injected submucosally into the 3 and 9 o'clock of the cervix; no significant differences have been found

After the radiotracer is injected, a lymphoscintigraphic localization imaging can be conducted. Lymphoscintigraphy is an imaging technique used to identify the lymph drainage basin, the sentinel lymph node, the location of the sentinel lymph node, the number of sentinel lymph nodes, and possible secondary drainage. Lymphoscintigraphy helps the surgeon to identify

Different protocols of radiotracer injection and subsequent lymphoscintigraphic imaging have been described in the literature. Protocols differ in the time frame from which the radiotracer

and localize the sentinel lymph node during the surgical procedure [19].

better 4-year overall survival and progression-free survival [15].

300 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

the association of radiotherapy and surgery.

complete lymphadenectomy in these patients.

between these two techniques [9, 18].

**5. Sentinel lymph node biopsy**

complete lymphadenectomy.

formation.

The blue dye is injected to the cervix in the surgery room after the anesthetic induction is performed, with the same technique as the radiotracer was injected.

The first step of the surgery is to look for the sentinel lymph node. The sentinel lymph nodes are identified by tracing the lymphatic chains with the gamma probe, identifying nodes with radioactive counts greater than five times the background count (Figure 1). The pelvic sidewalls, presacral, and para-aortic lymph chains should be scanned to identify "hot spots" by the gamma probe and/or by identifying blue-stained lymphatic channels and lymph nodes (Figures 2 and 3). Lymph nodes that appear "hot", blue, or both are identified as sentinel lymph nodes, and are removed (Figures 4 and 5). The sentinel lymph nodes are sent for intraoperative pathological review. Lymph nodes that appear to be grossly abnormal should be also removed, whether "hot", blue, or not, since the lymphatic channels may be obstructed by tumor, and the lymphatic drainage and tracer may be bypassing such nodes.

**Figure 1.** Detection of the sentinel lymph node with the gamma probe.

**Figure 2.** Blue chain of a sentinel lymph node.

**Figure 3.** Blue sentinel lymph node.

**Figure 4.** Resection of a blue and "hot" sentinel lymph node.

**Figure 2.** Blue chain of a sentinel lymph node.

302 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

**Figure 3.** Blue sentinel lymph node.

The cervix is a midline structure presenting a bilateral lymphatic drainage. The sentinel lymph node must be detected bilaterally in order to reduce the false negative rate. If no sentinel node is detected on one side, a complete lymphadenectomy must be performed on that side.

Sentinel lymph node detection in early stage cervical cancer presents several advantages over common pelvic lymphadenectomy [21]. First, it permits an intraoperative analysis of the node. Second, this technique can detect aberrant lymphatic drainage. And third, it permits ultra‐ staging of the sentinel lymph node and detection of micrometastasis and isolated tumor cells (ITCs).

As mentioned earlier, the association of radiotherapy and radical hysterectomy causes a higher risk of radiotherapy-related complications. To avoid the increase of morbidity caused by the association of treatments, a possibility is to perform the complete surgery in two phases instead of one. First, the pelvic lymphadenectomy can be carried out, waiting one or two weeks to obtain the definitive pathological report. If the lymph nodes are reported as negative a second surgery, a radical hysterectomy or trachelectomy, is performed. With the sentinel lymph node technique, information on the lymph node status is available in the operating room during the surgical procedure, permitting changes in the therapeutic management of the patient if necessary. If the sentinel lymph node is informed as metastatic, it is possible to complete the para-aortic lymphadenectomy as one procedure, not perform the hysterectomy, and avoid increased morbidity. Sentinel lymph node detection permits triaging patients toward surgery or chemo-radiation therapy, as well as selecting candidates for fertility-preserving surgery.

Aberrant lymphatic drainage or unusual locations of metastatic lymph nodes are due to those sentinel lymph nodes that are detected in lymphatic chains, which are not typically removed with the standard pelvic lymphadenectomy, as can be the presacral nodes or the common iliac nodes. Consequently, if a standard pelvic lymphadenectomy were to be performed without the sentinel lymph node detection technique, these metastatic nodes would not be detected. Bats et al. detected metastatic sentinel lymph nodes in an unexpected territory in up to 15% of the patients in which the sentinel lymph node technique was performed, and they concluded that the sentinel lymph node detection technique contributed to improved nodal staging [22].

Ultrastaging is the pathological process of studying the sentinel lymph nodes, consisting of a multiple serial sectioning with immunohistochemical assessment. Pathological ultrastaging permits the detection of low volume disease, which includes micrometastasis and ITC, as defined for breast cancer by the American Joint Committee of Cancer (AJCC). Macrometastasis was defined as tumor deposit greater than 2mm in diameter, micrometastasis was defined as tumor deposit between 0.2 to 2mm in diameter, and isolated tumor cells were defined as tumor deposits no larger than 0.2mm [23]. The importance of the detection of low volume disease in cervical cancer is its relationship with poor prognosis. In a study published in 2012 by Cibula et al. [24] that included 645 patients, it was observed that the presence of micrometastasis was associated with a significant reduction in the overall survival similar to those patients that presented macrometastasis, while no increased risk was found in those patients that presented ITC. Micrometastases are being detected in the sentinel lymph node in 4–15% of the patients, depending on the study [25].

Ultrastaging is a time-consuming and costly technique, not feasible for the analysis of all the lymph nodes obtained after a pelvic lymphadenectomy, but it is possible if only two to four nodes are studied with this technique. Detection of SLN and subsequent ultrastaging may detect a group of patients that would be overlooked with the standard pathological study of the pelvic lymphadenectomy nodes, although they present prognosis similar to those patients with macrometastasis. These findings highlight the importance of the SLN detection in early stage cervical cancer.

The presence of non-diagnosed micrometastasis or aberrant metastatic lymph nodes could explain the 15% of patients with an early stage cervical cancer with apparently no poor prognosis factors at diagnosis, that recur in the follow-up.

The sentinel lymph node detection has the potential to increase sensitivity in the detection of lymph node metastasis by detecting aberrant lymphatic drainage and micrometastasis [26]. Data of more than 2000 patients have been subjected to the sentinel node technique In a review published by Eiriksson et al. [9], sentinel lymph node detection in tumors of less than 2 cm presents a sensitivity of 98.2% and a negative predictive value of 99.6%, with a false negative rate of less than 5% when each hemipelvis is interpreted independently.

#### **6. Conclusion**

Sentinel lymph node detection permits minimizing surgical morbidity while maximizing the pathologic information of nodal status in patients with cervical cancer. Sentinel lymph node detection could become the standard of care in early stage cervical cancer in a close the future.

#### **Author details**

Elisa Moreno-Palacios\* , Elsa Delgado, Javier De Santiago and Ignacio Zapardiel

\*Address all correspondence to: elimorepal@gmail.com

Gynecologic Oncology Unit, La Paz University Hospital, Madrid, Spain

#### **References**

permits the detection of low volume disease, which includes micrometastasis and ITC, as defined for breast cancer by the American Joint Committee of Cancer (AJCC). Macrometastasis was defined as tumor deposit greater than 2mm in diameter, micrometastasis was defined as tumor deposit between 0.2 to 2mm in diameter, and isolated tumor cells were defined as tumor deposits no larger than 0.2mm [23]. The importance of the detection of low volume disease in cervical cancer is its relationship with poor prognosis. In a study published in 2012 by Cibula et al. [24] that included 645 patients, it was observed that the presence of micrometastasis was associated with a significant reduction in the overall survival similar to those patients that presented macrometastasis, while no increased risk was found in those patients that presented ITC. Micrometastases are being detected in the sentinel lymph node in 4–15% of the patients,

Ultrastaging is a time-consuming and costly technique, not feasible for the analysis of all the lymph nodes obtained after a pelvic lymphadenectomy, but it is possible if only two to four nodes are studied with this technique. Detection of SLN and subsequent ultrastaging may detect a group of patients that would be overlooked with the standard pathological study of the pelvic lymphadenectomy nodes, although they present prognosis similar to those patients with macrometastasis. These findings highlight the importance of the SLN detection in early

The presence of non-diagnosed micrometastasis or aberrant metastatic lymph nodes could explain the 15% of patients with an early stage cervical cancer with apparently no poor

The sentinel lymph node detection has the potential to increase sensitivity in the detection of lymph node metastasis by detecting aberrant lymphatic drainage and micrometastasis [26]. Data of more than 2000 patients have been subjected to the sentinel node technique In a review published by Eiriksson et al. [9], sentinel lymph node detection in tumors of less than 2 cm presents a sensitivity of 98.2% and a negative predictive value of 99.6%, with a false negative

Sentinel lymph node detection permits minimizing surgical morbidity while maximizing the pathologic information of nodal status in patients with cervical cancer. Sentinel lymph node detection could become the standard of care in early stage cervical cancer in a close the future.

, Elsa Delgado, Javier De Santiago and Ignacio Zapardiel

prognosis factors at diagnosis, that recur in the follow-up.

304 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

\*Address all correspondence to: elimorepal@gmail.com

rate of less than 5% when each hemipelvis is interpreted independently.

depending on the study [25].

stage cervical cancer.

**6. Conclusion**

**Author details**

Elisa Moreno-Palacios\*


[25] Daraï E, Rouzier R, Ballester M, et al. Sentinel lymph node biopsy in gynaecological cancers: the importance of micrometastases in cervical cancer. Surg Oncol. 2008 Sep; 17(3):227–235.

[13] Kim HS, Sardi JE, Katsumata N, et al. Efficacy of neoadjuvant chemotherapy in pa‐ tients with FIGO stage IB1 to IIA cervical cancer: an international collaborative meta-

[14] Gonçalves V. Long-term quality of life in gynecological cancer survivors. Curr Opin

[15] Peters WA, Liu PY, Barrett RJ, et al. Concurrent chemotherapy and pelvic radiation therapy compared with pelvic radiation therapy alone as adjuvant therapy after radi‐ cal surgery in high-risk early-stage cancer of the cervix. J Clin Oncol. 2000 Apr;18(8):

[16] Stryker JA, Mortel R. Survival following extended field irradiation in carcinoma of cervix metastatic to para-aortic lymph nodes. Gynecol Oncol. 2000 Dec;79(3):399–405.

[17] Cabanas RM. An approach for the treatment of penile carcinoma. Cancer. 1977 Feb;

[18] Selman TJ, Mann C, Zamora J, et al. Diagnostic accuracy of tests for lymph node sta‐ tus in primary cervical cancer: a systematic review and meta-analysis. CMAJ. 2008

[19] Diaz JP, Gemignani ML, Pandit-Taskar N et al. Sentinel lymph node biopsy in the management of early-stage cervical carcinoma. Gynecol Oncol. 2011 Mar;120(3):347–

[20] Bats AS, Frati A, Froissart M, et al. Feasibility and performance of lymphoscintigra‐ phy in sentinel lymph node biopsy for early cervical cancer: results of the prospec‐

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[22] Bats AS, Mathevet P, Buenerd A, et al. The sentinel node technique detects unexpect‐ ed drainage pathways and allows nodal ultrastaging in early cervical cancer: insights from the multicenter prospective SENTICOL study. Ann Surg Oncol. 2013 Feb;20(2):

the role of sentinel lymph node biopsy in carcinoma of the breast, April 19–22, 2001,

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## **Adenocarcinoma of the Endometrium — The Art of Its Diagnosis**

Manoel Afonso Guimarães Gonçalves and Fernando Anschau

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61578

#### **Abstract**

The diagnostic process begins at the first meeting with the patient, where we must relate the symptoms and signs associated with endometrial disease. Communication skills are fundamental for excellence in medical care. Even with the development and improve‐ ment of new technologies in recent decades, be it endoscopy, ultrasound, computed to‐ mography or magnetic resonance imaging, the communication is also essential. We must have skills to recognize and elucidate a wide variety of signs and symptoms when we take a history and do a physical examination of the patient, where abnormal uterine bleeding is the first main sign that can lead to an early diagnosis of endometrial cancer. The endometrium, as every target organ of steroid hormones, shows involutional changes during ovarian failure. In peri-menopause, however, tissue hyperactivity stages occur with some frequency, showing a marked endometrial sensitivity to hormonal fluc‐ tuations, whether on an absolute or relative level. Irregular blood loss occurs in many women during this period, and although being most times of functional origin, it requires investigation. It is noteworthy that the most frequent cause of abnormal bleeding of or‐ ganic origin in menopause is endometrial. Endometrial pathologies appear with advanc‐ ing age. Therefore an appropriate workup should diagnose or rule out disease at this site. Thus, preventive measures should be adopted, such as screening and early diagnosis, and the best treatment for the patient should be established.

**Keywords:** Endometrial cancer, diagnosis, new technologies, staging, prognosis, treat‐ ment

#### **1. Introduction**

The diagnostic process begins at the first meeting with the patient, where we must relate the symptoms and signs associated with endometrial disease.

© 2016 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.

Communication skills are fundamental for excellence in medical care. Even with the devel‐ opment and improvement of new technologies in recent decades, be it endoscopy, ultrasound, computed tomography, or magnetic resonance imaging, the communication is also essential.

We must have skills to recognize and elucidate a wide variety of signs and symptoms when we take a history and do a physical examination of the patient, where abnormal uterine bleeding is the first main sign that can lead to an early diagnosis of endometrial cancer.[1]

The endometrium, as every target organ of steroid hormones, shows involutional changes during ovarian failure. In perimenopause, however, tissue hyperactivity stages occur with some frequency, showing a marked endometrial sensitivity to hormonal fluctuations, whether on an absolute or relative level. Irregular blood loss occurs in many women during this period, and although being most times of functional origin, it requires investigation. It is noteworthy that the most frequent cause of abnormal bleeding of organic origin in menopause is endo‐ metrial. Endometrial pathologies appear with advancing age. Therefore an appropriate workup should diagnose or rule out disease at this site. Thus, preventive measures should be adopted, such as screening and early diagnosis, and the best treatment for the patient should be established.[2]

The annual incidence of endometrial carcinoma is 2 in 100,000 women under 40 years and 40 to 50 per 100,000 women between the sixth and eighth decades, and it is expected to gradually increase due to obesity and increased longevity, especially in North America and Western Europe. In Brazil, the highest incidences are in the South and Southeast regions.[3, 4] In the United States, endometrial cancer is the most common gynecologic malignancy, and it accounted for about 39,080 new cases and 7,400 deaths from cancer in 2007.[5] The signs are early and the most common is vaginal bleeding after menopause. When diagnosed early, about 80% are confined to the uterus, in the early stages, with good outcome and low mortality. In Brazil, it is the second most frequent pelvic malignancy, with an incidence of 5.7 per 100,000 women and mortality estimated at 1.6 per 100,000 women.[6] Staging of the International Federation of Gynecology and Obstetrics (FIGO), introduced in 1988 and updated in 2009, is defined by total abdominal hysterectomy, bilateral salpingo-oophorectomy, pelvic lympha‐ denectomy, and periaortic and peritoneal cytology, where prognostic factors include age, grade and histological type of tumor, depth of invasion into the myometrium, cervical involvement, and the presence of lymph node metastases.

#### **2. Diagnosis**

The diagnosis is histological but should be considered based on the symptoms and physical examination. The main symptom is abnormal uterine bleeding. Other findings associated with the disease are: heaviness in the lower abdomen, pelvic pain, presence of pyometra, hemato‐ metra, presence of atypical glandular cells in cervical Pap smear, menorrhagia, and inter‐ menstrual bleeding. Later symptoms are pain in the lower abdomen, foul-smelling secretion, urinary or intestinal disorders, and weight loss.

In postmenopausal women with uterine bleeding, premenopausal women with abnormal uterine bleeding and before hematometra and pyometra especially in older women, it is imperative to evaluate the endometrial cavity. This evaluation can be performed by blind endometrial biopsy or through hysteroscopy or curettage after gynecological examination. Endometrial biopsy is simple to perform and should be considered of value only when positive for malignancy, because it could give false-negative results. Hysteroscopy has better perform‐ ance, which surpasses curettage in the diagnosis, where possible visualization of the uterine cavity leads to fewer false-negative results as curettage. If the diagnostic biopsy is atypical hyperplasia, it is necessary to evaluate the whole endometrial cavity to rule out carcinoma.

Communication skills are fundamental for excellence in medical care. Even with the devel‐ opment and improvement of new technologies in recent decades, be it endoscopy, ultrasound, computed tomography, or magnetic resonance imaging, the communication is also essential.

310 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

We must have skills to recognize and elucidate a wide variety of signs and symptoms when we take a history and do a physical examination of the patient, where abnormal uterine bleeding is the first main sign that can lead to an early diagnosis of endometrial cancer.[1]

The endometrium, as every target organ of steroid hormones, shows involutional changes during ovarian failure. In perimenopause, however, tissue hyperactivity stages occur with some frequency, showing a marked endometrial sensitivity to hormonal fluctuations, whether on an absolute or relative level. Irregular blood loss occurs in many women during this period, and although being most times of functional origin, it requires investigation. It is noteworthy that the most frequent cause of abnormal bleeding of organic origin in menopause is endo‐ metrial. Endometrial pathologies appear with advancing age. Therefore an appropriate workup should diagnose or rule out disease at this site. Thus, preventive measures should be adopted, such as screening and early diagnosis, and the best treatment for the patient should

The annual incidence of endometrial carcinoma is 2 in 100,000 women under 40 years and 40 to 50 per 100,000 women between the sixth and eighth decades, and it is expected to gradually increase due to obesity and increased longevity, especially in North America and Western Europe. In Brazil, the highest incidences are in the South and Southeast regions.[3, 4] In the United States, endometrial cancer is the most common gynecologic malignancy, and it accounted for about 39,080 new cases and 7,400 deaths from cancer in 2007.[5] The signs are early and the most common is vaginal bleeding after menopause. When diagnosed early, about 80% are confined to the uterus, in the early stages, with good outcome and low mortality. In Brazil, it is the second most frequent pelvic malignancy, with an incidence of 5.7 per 100,000 women and mortality estimated at 1.6 per 100,000 women.[6] Staging of the International Federation of Gynecology and Obstetrics (FIGO), introduced in 1988 and updated in 2009, is defined by total abdominal hysterectomy, bilateral salpingo-oophorectomy, pelvic lympha‐ denectomy, and periaortic and peritoneal cytology, where prognostic factors include age, grade and histological type of tumor, depth of invasion into the myometrium, cervical

The diagnosis is histological but should be considered based on the symptoms and physical examination. The main symptom is abnormal uterine bleeding. Other findings associated with the disease are: heaviness in the lower abdomen, pelvic pain, presence of pyometra, hemato‐ metra, presence of atypical glandular cells in cervical Pap smear, menorrhagia, and inter‐ menstrual bleeding. Later symptoms are pain in the lower abdomen, foul-smelling secretion,

involvement, and the presence of lymph node metastases.

urinary or intestinal disorders, and weight loss.

be established.[2]

**2. Diagnosis**

Transvaginal ultrasound in postmenopausal women, taking into account a cutoff of 5 mm endometrial thickness, has a 96% sensitivity for endometrial cancer detection. However, there is no evidence showing that the use of ultrasound in screening asymptomatic women decreases mortality.[7]

A cervical Pap smear should not be considered a screening method or diagnosis of endometrial cancer.[8] There is no indication for screening for endometrial carcinoma by any method in asymptomatic women with or without medium or high risk factors for endometrial carcinoma, such as hormone therapy with estrogen, tamoxifen users, late menopause, nulliparity, infertility or chronic anovulation, obesity, diabetes, hypertension, or metabolic syndrome. It is recommended to inform these women about the risk factors and symptoms of endometrial carcinoma, such as abnormal uterine bleeding in premenopause and any bleeding after menopause, and to advise them to seek immediate medical attention.[4, 6]

Annual screening tests by endometrial biopsy should be indicated only in women ≥35 years old, with Lynch syndrome (hereditary nonpolyposis colorectal cancer, HNPCC-II) and/or a family history of carrying the mutation in the absence of confirmation of the mutation genetics, or family history with suspicion of autosomal dominant genetic predisposition.[3, 4]

Any postmenopausal bleeding should be investigated because it is the main symptom of endometrial carcinoma, and the assessment should start with ultrasound and/or endometrial biopsy, depending on the choice and ease in carrying out the procedure. The accuracy of ultrasound as to the measurement of normal endometrial thickness of ≤4–5 mm in postmeno‐ pausal women to exclude endometrial disease is very high. If the thickness is ≤4 mm, the negative predictive value (NPV) is 99.79%, and if ≤5 mm, it is 99.47%. It is rare that a woman with endometrial thickness of <4 cm has carcinoma of the endometrium, but in the presence of endometrial thickening, there are difficulties in differentiating between benign and malig‐ nant disease. Prospective studies have shown that the risk of cancer in women with bleeding and endometrial thickness of ≤4 mm is about 1 in 1,000 women.[11, 12, 13]

Endometrial aspiration biopsy (Pipelle being the most common) has been widely used because it is done on an outpatient basis and causes little discomfort to the patient. However, there are important limitations, such as small endometrial area evaluated and very variable diagnostic sensitivity. Studies have shown a rate of false-negative results of 2.5–32.4% in Pipelle biopsies for endometrial carcinoma, especially in tumors occupying <50% of the endometrial cavity, such as polyps.[13, 14]

Women with abnormal uterine bleeding should be investigated with ultrasound (US) and/or biopsy. In menopausal women, ultrasonographic endometrial thickness of ≤4–5 mm, and/or result in a negative aspiration biopsy for endometrial cancer or hyperplasia can be followed up, but should be targeted for further tests in the persistence of any bleeding.

The endoscopic hysteroscopy examination was introduced in 1864 by the English physician Pantaleoni, and with improvements mainly in optics, proved to be since the 1980s an excellent procedure for the diagnosis of diseases of the cervical canal and the uterine cavity.

Hysteroscopy is a procedure that every day reaches a greater importance in medical exami‐ nation and therapeutic arsenals of gynecologists. This is because the necessary equipment has evolved systematically and quickly toward making the procedure more and more delicate and less traumatic. Among the many changes, we can highlight the wide use of hysteroscopic instrumental with 2.0- and 2.9-mm optics.[15]

Hysteroscopy allows the complete examination of the uterine cavity: its distention, morphol‐ ogy, and size; anterior and posterior wall, cones, and tubal ostia; and color, appearance, surface, vascularization, and thickness of the endometrial mucosa. Endometrial sampling is targeted, where the biopsy can be performed via hysteroscopy or immediately after the procedure.

In symptomatic patients, hysteroscopy combined with histological sampling is considered first-line in the diagnostic process; it is also for asymptomatic patients with abnormal endo‐ metrial cytology or ultrasound, or even with normal endometrial cells in cervicovaginal cytology.

A randomized study showed that women with abnormal uterine bleeding can start the investigation with ultrasound and endometrial Pipelle biopsy and only use hysteroscopy and/ or curettage in a second option. A systematic review showed high diagnostic accuracy of hysteroscopy for cancer, with a sensitivity of 86.4% (95% CI, 84.0–88.6%) and specificity of 99.2% (95% CI, 99.1–99.3%). The sensitivity for diagnosis of benign endometrial pathology was 78.0% (95% CI, 76.3–79.6%), while specificity was 95.8% (95% CI, 95.6–96.1%), which would correspond to moderate accuracy. It is a safe procedure, with few complications and good diagnostic performance for endometrial carcinoma in women with abnormal uterine bleeding. [16] Another systematic review compared endometrial biopsy or hysteroscopy and dilatation and curettage (D&C) combined with endometrial cytology and demonstrated high diagnostic sensitivity of hysteroscopy with cytology, but cytology was more associated with sub-staging of the disease, compared with the biopsy or D&C. If the diagnostic biopsy revealed a precursor lesion, that is, atypical hyperplasia, it is necessary to evaluate the whole endometrial cavity to rule out carcinoma. The endometrial cavity should be examined in elderly women in the presence of hematometra and pyometra and in premenopausal women with abnormal uterine bleeding. Later symptoms are pain in the lower abdomen, foul-smelling secretion, urinary or intestinal disorders, and weight loss.[17]

Curettage and hysteroscopy have high diagnostic accuracy, but hysteroscopy is the method of choice for small and focal lesions such as polyps and can be performed on an outpatient basis. The diagnostic procedure chosen should be in accordance with its accessibility and the surgeon's experience. Hysteroscopy is considered by many authors as the gold standard for evaluation of the uterine cavity. This examination provides enormous benefits with hits in macroscopic visual information, especially when it identifies organic changes in polyps, submucosal fibroids, complete inactivity states, or atrophic endometrium (figure 1); it also shows a high success in states of high endometrial activity: complex hyperplasia with atypia or endometrial cancer (figure 2).

for endometrial carcinoma, especially in tumors occupying <50% of the endometrial cavity,

Women with abnormal uterine bleeding should be investigated with ultrasound (US) and/or biopsy. In menopausal women, ultrasonographic endometrial thickness of ≤4–5 mm, and/or result in a negative aspiration biopsy for endometrial cancer or hyperplasia can be followed

The endoscopic hysteroscopy examination was introduced in 1864 by the English physician Pantaleoni, and with improvements mainly in optics, proved to be since the 1980s an excellent

Hysteroscopy is a procedure that every day reaches a greater importance in medical exami‐ nation and therapeutic arsenals of gynecologists. This is because the necessary equipment has evolved systematically and quickly toward making the procedure more and more delicate and less traumatic. Among the many changes, we can highlight the wide use of hysteroscopic

Hysteroscopy allows the complete examination of the uterine cavity: its distention, morphol‐ ogy, and size; anterior and posterior wall, cones, and tubal ostia; and color, appearance, surface, vascularization, and thickness of the endometrial mucosa. Endometrial sampling is targeted, where the biopsy can be performed via hysteroscopy or immediately after the

In symptomatic patients, hysteroscopy combined with histological sampling is considered first-line in the diagnostic process; it is also for asymptomatic patients with abnormal endo‐ metrial cytology or ultrasound, or even with normal endometrial cells in cervicovaginal

A randomized study showed that women with abnormal uterine bleeding can start the investigation with ultrasound and endometrial Pipelle biopsy and only use hysteroscopy and/ or curettage in a second option. A systematic review showed high diagnostic accuracy of hysteroscopy for cancer, with a sensitivity of 86.4% (95% CI, 84.0–88.6%) and specificity of 99.2% (95% CI, 99.1–99.3%). The sensitivity for diagnosis of benign endometrial pathology was 78.0% (95% CI, 76.3–79.6%), while specificity was 95.8% (95% CI, 95.6–96.1%), which would correspond to moderate accuracy. It is a safe procedure, with few complications and good diagnostic performance for endometrial carcinoma in women with abnormal uterine bleeding. [16] Another systematic review compared endometrial biopsy or hysteroscopy and dilatation and curettage (D&C) combined with endometrial cytology and demonstrated high diagnostic sensitivity of hysteroscopy with cytology, but cytology was more associated with sub-staging of the disease, compared with the biopsy or D&C. If the diagnostic biopsy revealed a precursor lesion, that is, atypical hyperplasia, it is necessary to evaluate the whole endometrial cavity to rule out carcinoma. The endometrial cavity should be examined in elderly women in the presence of hematometra and pyometra and in premenopausal women with abnormal uterine bleeding. Later symptoms are pain in the lower abdomen, foul-smelling secretion, urinary or

up, but should be targeted for further tests in the persistence of any bleeding.

procedure for the diagnosis of diseases of the cervical canal and the uterine cavity.

such as polyps.[13, 14]

procedure.

cytology.

instrumental with 2.0- and 2.9-mm optics.[15]

312 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

intestinal disorders, and weight loss.[17]

**Figure 1.** (a) Atrophic endometrium, (b) Submucosal fibroids, (c) Polyp

With regard to histological types, the most common is endometrioid adenocarcinoma (75– 80%), where it is the most common variant of squamous differentiation. It is related to hyperestrogenism and the precursor lesion is atypical hyperplasia. Serous papilliferous (10%) and clear-cell (4%) adenocarcinomas are similar to those of the ovary and tube and may show peritoneal spread. These tumors are associated with p53 gene mutation will occur in older women, are often diagnosed at more advanced stages and have a worse prognosis. The other histological types are rarer: mucinous, squamous, and undifferentiated.[18, 19]

(a)

(b)

(a) Complex hyperplasia with atypia, (b) Endometrial cancer **Figure 2.** (a) Complex hyperplasia with atypia, (b) Endometrial cancer

#### **Histological types**

	- **a.** Ciliated adenocarcinoma
	- **b.** Secretory adenocarcinoma
	- **1.** Adenoacanthoma
	- **2.** Adenosquamous

#### **Adenocarcinomas should be grouped according to histopathological differentiation grade:**

G1 (well differentiated); 5% or less with nonsquamous growth pattern

G2 (moderately differentiated); 6–50% with nonsquamous growth pattern

G3 (undifferentiated); more than with nonsquamous growth pattern

*When nuclear atypia is inappropriate for architectural grade, increase G1 to G2 and G2 to G3.*

*Adenocarcinoma with squamous component is graded by the glandular component.*

#### **3. Staging**

(a)

(b)

(a) Complex hyperplasia with atypia, (b) Endometrial cancer

**Figure 2.** (a) Complex hyperplasia with atypia, (b) Endometrial cancer

314 Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives

**a.** Ciliated adenocarcinoma

**b.** Secretory adenocarcinoma

**Histological types**

**i.** Endometrioid (70–80%)

Staging begins with a general physical examination, palpation of supraclavicular and inguinal lymph nodes, vaginal examination, and digital rectal examination, eventually done under analgesia. Sampling for cervical cancer cytology, blood tests, and chest X-ray are routine. In suspected cases of bladder or rectal invasion, cystoscopy and rectosigmoidoscopy with biopsy are indicated. If the parametrium and vagina do not show neoplastic changes, surgical staging is indicated, according to the International Federation of Gynecology and Obstetrics (FIGO) (established in 1988, revised in 2009; Table 1).[8, 9] For women undergoing radiation therapy as initial treatment, FIGO clinical staging (1971) can be used, correlating with the current. The diagnostic biopsy defines the type and histological grade (Table 2); total hysterectomy with adnexectomy defines myometrial, cervical, and adnexal invasion; and peritoneal lavage defines the presence of neoplastic cells. Endometrial ablation can be performed intraopera‐ tively, along with a biopsy of the omentum. It is only possible to prove lymph node metastasis by conducting a pelvic and para-aortic retroperitoneal lymphadenectomy in patients with poor prognostic factors. A lymph node biopsy is indicated in the presence of enlarged lymph nodes. Retroperitoneal lymphadenectomy can increase perioperative morbidity, depending on the clinical conditions of the patient and the training of the surgical team.[10, 11]


FIGO Staging – 1988, revised in 2009 [9, 10]

\* G1, G2 or G3.

\*\* Only endocervical gland involvement should be considered as stage I and no longer as stage II.
