**Meet the editor**

Ravinder Mohan, MD, PhD, teaches and practices family medicine in Virginia and also works as a hospitalist physician. He became interested in research on health-related quality of life in Belgium, where he studied outcomes of cardiac surgery. He did similar research in the United States in survivorship of patients newly diagnosed with localized prostate cancer. This is his third book as editor

on clinical outcomes in cancer.

### Contents

#### **Preface XI**



Chapter 8 **Redefining Androgen Receptor Function: Clinical Implications in Understanding Prostate Cancer Progression and Therapeutic Resistance 93**

Miltiadis Paliouras, Carlos Alvarado and Mark Trifiro


### Preface

Chapter 8 **Redefining Androgen Receptor Function: Clinical Implications**

Miltiadis Paliouras, Carlos Alvarado and Mark Trifiro

Yasuyoshi Miyata, Yohei Shida, Tomohiro Matsuo, Tomoaki

Zorana Nikolić, Dušanka Savić Pavićević and Goran Brajušković

Eswar Shankar, Mario Candamo, Gregory T. MacLennan and Sanjay

Chapter 9 **Reconsideration of Hormonal Therapy in the Era of Next‐**

**Generation Hormonal Therapy 125**

Chapter 10 **Genetic Association Studies on Prostate Cancer 145**

Chapter 11 **Key Genes in Prostate Cancer Progression: Role of MDM2, PTEN, and TMPRSS2-ERG Fusions 179** Appu Rathinavelu and Arkene Levy

Chapter 12 **Maspin Expression and its Metastasis Suppressing Function in**

Chapter 13 **The Emerging Role of PARP Inhibitors in the Treatment of**

Jue Wang, Brent B. Freeman and Paul Mathew

Chapter 14 **Rehabilitation of Patients with Prostate Cancer 227** Meral Huri, Burcu Semin Akel and Sedef Şahin

Hakariya and Hideki Sakai

**Prostate Cancer 197**

**Prostate Cancer 213**

Gupta

**Resistance 93**

**VI** Contents

**in Understanding Prostate Cancer Progression and Therapeutic**

Prostate cancer is a common cancer that is usually slow growing and may be mistakenly perceived as harmless. In nonsmoking American men, it is the commonest cause of cancer death. In the past two decades, the norm was to screen for this cancer actively in any man over 50, find it, and treat it with surgery or radiotherapy. Many patients then suffered erec‐ tile dysfunction and urinary incontinence, and large studies questioned whether their sur‐ vival had been prolonged even slightly. In the last five years, the pendulum has swung extremely to the opposite side; national recommendations have declared that screening for this cancer is harmful, and screening for the cancer has almost halted—at times even in Afri‐ can-American men who have the cancer more commonly and die due to it more commonly. Whether this approach will lead to a large increase in prostate cancer mortality remains to be seen.

At such a time, we have to again review the usefulness of newer screening protocols and biomarkers that can help in identifying cancers that we should treat before they spread rap‐ idly and also be ready to treat more advanced cancers with newer, more effective, and safer methods of hormone therapy, chemotherapy, and radiotherapy. In this book, some of the best investigators in the world present their cutting-edge research, clinical practice, and dis‐ cussions in both of these aspects.

> **Prof. Ravinder Mohan** Associate Professor, Eastern Virginia Medical School, Norfolk, Virginia, USA

## **Prostate-Specific Antigen-Based Prostate Cancer Screening**

Naoki Sakai

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63751

#### **Abstract**

Serum prostate-specific antigen (PSA) testing is a simple and effective method for diagnosing prostate cancer. The widespread PSA screening resulted in increased diagnosis of early-staged, localized prostate cancer and marked reduction in advanced, metastatic cancer, which contributed to subsequent reduction in prostate cancer mortality. Most patients with localized prostate cancer, especially low-grade cancer, have an indolent clinical course. In addition, the rate of death from prostate cancer itself is very low. Therefore, early diagnosis of prostate cancer can lead to overdiagnosis and overtreatment. There has been a controversy regarding the effect of PSA screening on prostate cancer mortali‐ ty. Results of the two largest randomized trials concerning PSA screening were totally contrary. European countries-based trial showed a significant prostate cancer mortality reduction, whereas the USA-based trial showed no benefit in reducing prostate cancer mortality. In 2013, based on these arguments, the American Urological Association updated a guideline regarding PSA screen‐ ing, which did not recommend routine PSA screening but a selective screen‐ ing, according to patient's age, coexisting medical condition, and risks, such as family history. The guideline also emphasized shared decision making.

However, older patients have been shown to be more likely to have high-risk prostate cancer at diagnosis. Older patients more often have other medical conditions and are more likely to die from causes other than prostate cancer. Prostate cancer is more often diagnosed with older patients. These facts bring PSA-based screening for prostate cancer more and more complex. In this chapter, problems concerning PSA screening are presented.

**Keywords:** prostate-specific antigen, prostate cancer, screening, mortality, evidence

© 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**

Prostate cancer is the most common cancer, accounting for up to 29% of all incident cases and is the second most common cause of cancer death among men in the United States in 2012 [1]. The lifetime probability of being diagnosed as prostate cancer is approximately 16% [1]. Serum prostate-specific antigen (PSA) measurement is an extremely effective tool for identifying men with prostate cancer. Today, almost all prostate cancer patients are being diagnosed with an elevated PSA. It is well known that PSA screening has increased the incidence of prostate cancer but decreased a rate of advanced, metastatic prostate cancer and resulted in a subsequent drastic reduction in prostate cancer mortality.

In the United States, following the introduction of widespread PSA screening in the late 1980s, there was a 70% increase in prostate cancer incidence. In 1992, there was a peak of incidence, as if it can be called the prostate cancer incidence surge. Several years after the introduction of PSA testing, from around 1994, mortality rate began to fall [1].

In European countries, PSA screening decreased the absolute risk of being diagnosed with advanced prostate cancer [2]. A population-based, prospective, randomized, controlled screening study for prostate cancer, with biennial PSA testing group and control group, showed that after a follow-up of 10 year, the diagnosis of prostate cancer increased by 1.8-fold; however, the risk of being diagnosed with metastatic prostate cancer was reduced by 48.9% (*p* = 0.0084) [2].

The incidence of prostate cancer in Japan rapidly increased between 2000 and 2003, while the mortality rate started to decrease in 2004, immediately after the increase in incidence [3]. The proportion of localized prostate cancer rapidly increased between 1997 and 2003, from 40 to 60% or higher. These facts are explained by early diagnosis using serum PSA measurement [3].

The primary aim of PSA-based prostate cancer screening should be the early detection of asymptomatic patients with potentially fatal cancers resulting in reduction of cancer-specific mortality [4]. However, early diagnosis of the prostate cancer due to widespread PSA screening has also led to increased diagnosis of clinically indolent cancers. Almost all localized cancers, especially low-grade disease, rarely progress to advanced disease and even fewer have a fatal clinical course [5, 6]. Prostate biopsy as well as any treatment has some adverse events and harms. Thus, PSA screening itself is associated with overdiagnosis and overtreatment. PSA screening in asymptomatic individuals involves a trade-off of benefit and harm.

Randomized trial is the most valuable study design for making evidence in medical fields. To address whether there is a mortality benefit in PSA screening, several randomized trials were performed. However, results of the two largest randomized trials were extremely confusing. The USA-based, the prostate component of the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO trial) showed no benefit in reducing mortality rate [7]; however, the European-based, the European Randomized study of Screening for Prostate Cancer (ERSPC trial) showed a significant mortality reduction [8].

The United States Preventive Health Services Task Force (USPSTF) has regarded harms associated PSA screening more important and has recommended against PSA screening in asymptomatic men regardless of age [9]. The USPSTF stated that current empiric evidence is insufficient to assess the risks and benefits of prostate cancer screening. Potential harms from screening and treatment are much more significant and clinicians should recommend against PSA screening for prostate cancer [9].

**1. Introduction**

(*p* = 0.0084) [2].

reduction in prostate cancer mortality.

2 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

PSA testing, from around 1994, mortality rate began to fall [1].

Prostate cancer is the most common cancer, accounting for up to 29% of all incident cases and is the second most common cause of cancer death among men in the United States in 2012 [1]. The lifetime probability of being diagnosed as prostate cancer is approximately 16% [1]. Serum prostate-specific antigen (PSA) measurement is an extremely effective tool for identifying men with prostate cancer. Today, almost all prostate cancer patients are being diagnosed with an elevated PSA. It is well known that PSA screening has increased the incidence of prostate cancer but decreased a rate of advanced, metastatic prostate cancer and resulted in a subsequent drastic

In the United States, following the introduction of widespread PSA screening in the late 1980s, there was a 70% increase in prostate cancer incidence. In 1992, there was a peak of incidence, as if it can be called the prostate cancer incidence surge. Several years after the introduction of

In European countries, PSA screening decreased the absolute risk of being diagnosed with advanced prostate cancer [2]. A population-based, prospective, randomized, controlled screening study for prostate cancer, with biennial PSA testing group and control group, showed that after a follow-up of 10 year, the diagnosis of prostate cancer increased by 1.8-fold; however, the risk of being diagnosed with metastatic prostate cancer was reduced by 48.9%

The incidence of prostate cancer in Japan rapidly increased between 2000 and 2003, while the mortality rate started to decrease in 2004, immediately after the increase in incidence [3]. The proportion of localized prostate cancer rapidly increased between 1997 and 2003, from 40 to 60% or higher. These facts are explained by early diagnosis using serum PSA measurement [3]. The primary aim of PSA-based prostate cancer screening should be the early detection of asymptomatic patients with potentially fatal cancers resulting in reduction of cancer-specific mortality [4]. However, early diagnosis of the prostate cancer due to widespread PSA screening has also led to increased diagnosis of clinically indolent cancers. Almost all localized cancers, especially low-grade disease, rarely progress to advanced disease and even fewer have a fatal clinical course [5, 6]. Prostate biopsy as well as any treatment has some adverse events and harms. Thus, PSA screening itself is associated with overdiagnosis and overtreatment. PSA

screening in asymptomatic individuals involves a trade-off of benefit and harm.

trial) showed a significant mortality reduction [8].

Randomized trial is the most valuable study design for making evidence in medical fields. To address whether there is a mortality benefit in PSA screening, several randomized trials were performed. However, results of the two largest randomized trials were extremely confusing. The USA-based, the prostate component of the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO trial) showed no benefit in reducing mortality rate [7]; however, the European-based, the European Randomized study of Screening for Prostate Cancer (ERSPC

The United States Preventive Health Services Task Force (USPSTF) has regarded harms associated PSA screening more important and has recommended against PSA screening in In 2013, based on these arguments, the American Urological Association (AUA) updated clinical guideline for PSA screening, "Early detection of Prostate cancer", which recommended against an organized PSA screening but for a selective PSA screening according to age, comorbidities, and risks such as family history [10]. In addition, the AUA strongly recom‐ mended shared decision making, a thorough discussion between physician and patient regarding the risks and benefits of PSA-based screening [10].

In general, the rate of prostate cancer incidence increases with age [1, 11]. Compared with younger men, older men are more frequently diagnosed with high grade and high risk prostate cancer [12]. In addition, coexisting medical conditions are also more prevalent in older patients. These facts bring PSA screening much more confusing.

As an alternative data source, a population level data, cohort study, concerning PSA screening is presented. Although cohort study is considered a lower level of evidence compared to the randomized study, it has some merit in reflecting "real world" clinical data.

#### **2. Conflicting two large randomized trials concerning PSA screening**

In 2009, totally conflicting two large randomized trials addressing the effect of PSA screening on the prostate cancer-specific mortality rate were published at the same time in the same journal, and consecutively [7, 8]. The USA-based, the prostate component of the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (the PLCO trial), showed no benefit in reducing mortality rate, after 7–10 years of follow-up (rate ratio (RR) 1.13; 95% confidence interval (CI) 0.75–1.70) [7]. In contrast, European countries-based trial, the European Randomized study of Screening for Prostate Cancer (the ERSPC trial), showed a benefit of PSA screening in reducing prostate cancer mortality [8]. The ERSPC trial reported that during a median follow-up of 9 years, the rate ratio for death from prostate cancer in the screening group, as compared with the control group, was 0.80 (95% CI, 0.65–0.98; adjusted *p* = 0.04) [8].

The prostate component of the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO trial) is a multicenter, randomized trial designed to evaluate the effect of PSA screening for prostate cancer-specific mortality. A total of 76,685 men, aged 55–74 years, were randomly assigned to the intervention arm (offered annual PSA testing for 6 years and annual digital rectal examination for 4 years) and to the control arm (usual, opportunistic PSA examination) at 10 centers in the United States between 1993 and 2001. A PSA cutoff 4.0 ng/ml was used for prostate biopsy. The PLCO trial extended follow-up period to 13 years and concluded that there was also no evidence of a mortality benefit for organized annual screening compared with opportunistic screening (RR, 1.09; 95% CI, 0.87–1.36) [13].

On the other hand, the European Randomized study of Screening for Prostate Cancer (ERSPC trial) is also a randomized multicenter trial with core age group (55–69 years) evaluating the benefit of screening for prostate cancer with serum PSA in eight European countries, including the Netherlands, Belgium, Sweden, Finland, Italy, Spain, Switzerland, and France. Briefly, a total of 162,388 aged 55–69 men were randomly assigned as an intervention arm (invited to PSA screening once every 4 years in seven countries and once every 2 years in Sweden) or a control arm with no PSA testing offered. A PSA cutoff 3.0 ng/ml was used for prostate biopsy. The ERSPC group also extended follow-up to 11–13 years and reported that the rate ratio (RR) of prostate cancer incidence between the intervention and control arms was 1.91, 1.66, and 1.57 after 9, 11, and 13 years, respectively, and the RR of prostate cancer mortality was 0.85, 0.78, and 0.79 at 9, 11, and 13 years, respectively (95% confidence interval 13 years 0.69–0.91, *p* = 0.001), corresponding to a relative risk reduction of 21% at 13 years [14, 15]. The updated ERSPC study concluded a substantial prostate cancer mortality reduction due to PSA testing. In the ERSPC trial, a significant decrease in metastatic disease at diagnosis was demonstrated. The rate of advanced group (M1 and/or PSA > 100 ng/ml) was 3.4 and 9.6%, in the intervention and control arm, respectively.

The reasons why the results of these two trials were totally different might be due to differences in study design: screening protocol and PSA cutoff value. For example, the ERSPC trial compared screening group to no screening group, while the PLCO trial compared annual PSA screening to opportunistic PSA testing in the control arm [16].

Randomized trial has some unavoidable limitations: compliance in the intervention arm, nonparticipation in the screening arm, and PSA contamination, opportunistic PSA testing in the control arm. Because investigated individuals have the will, PSA contamination cannot be excluded completely. The contamination rates were 20–25% in the ERSPC trial and 40% in the first year and increased to 52% in the sixth years in the PLCO trial [7, 8]. Increased exposure to opportunistic PSA testing in the control arm reduces the reliability of the randomized study. The Rotterdam section of the ERSPC trial demonstrated that after correction for both nonat‐ tendance in the screening arm and PSA contamination in the control arm, PSA-based screening conducted in the Dutch center reduced the risk of dying from prostate cancer up to 51% (RR of 0.49; 95% CI, 0.27–0.87) in men who undergo organized screening at a median follow-up of 13 years [17].

In addition, randomized trial has another limitation. The primary end point of the trial is prostate cancer-specific mortality. Therefore, randomized trials would require a considerably long time to withdraw some conclusions. These trials randomly registered asymptomatic, healthy men and had followed until assigned men developed prostatic cancer and, further, died from prostate cancer or other causes. When a result is provided, the diagnostic method that is prostate biopsy method as well as treatment methods usually have progressed.

In addition, the rate of the death from prostate cancer is very low and there is a difficulty in assessing the effect of PSA screening on the prostate cancer-specific mortality. For example, the PLCO trial reported that after 7–10 years of follow-up, the incidence of death per 10,000 person-years was only 2.0 (50 deaths) in the screening group and 1.7 (44 deaths) in the control group (rate ratio, 1.13; 95% CI, 0.75–1.70) [7].

#### **3. Localized, low-grade prostate cancer most often has an indolent character**

On the other hand, the European Randomized study of Screening for Prostate Cancer (ERSPC trial) is also a randomized multicenter trial with core age group (55–69 years) evaluating the benefit of screening for prostate cancer with serum PSA in eight European countries, including the Netherlands, Belgium, Sweden, Finland, Italy, Spain, Switzerland, and France. Briefly, a total of 162,388 aged 55–69 men were randomly assigned as an intervention arm (invited to PSA screening once every 4 years in seven countries and once every 2 years in Sweden) or a control arm with no PSA testing offered. A PSA cutoff 3.0 ng/ml was used for prostate biopsy. The ERSPC group also extended follow-up to 11–13 years and reported that the rate ratio (RR) of prostate cancer incidence between the intervention and control arms was 1.91, 1.66, and 1.57 after 9, 11, and 13 years, respectively, and the RR of prostate cancer mortality was 0.85, 0.78, and 0.79 at 9, 11, and 13 years, respectively (95% confidence interval 13 years 0.69–0.91, *p* = 0.001), corresponding to a relative risk reduction of 21% at 13 years [14, 15]. The updated ERSPC study concluded a substantial prostate cancer mortality reduction due to PSA testing. In the ERSPC trial, a significant decrease in metastatic disease at diagnosis was demonstrated. The rate of advanced group (M1 and/or PSA > 100 ng/ml) was 3.4 and 9.6%, in the intervention and

The reasons why the results of these two trials were totally different might be due to differences in study design: screening protocol and PSA cutoff value. For example, the ERSPC trial compared screening group to no screening group, while the PLCO trial compared annual PSA

Randomized trial has some unavoidable limitations: compliance in the intervention arm, nonparticipation in the screening arm, and PSA contamination, opportunistic PSA testing in the control arm. Because investigated individuals have the will, PSA contamination cannot be excluded completely. The contamination rates were 20–25% in the ERSPC trial and 40% in the first year and increased to 52% in the sixth years in the PLCO trial [7, 8]. Increased exposure to opportunistic PSA testing in the control arm reduces the reliability of the randomized study. The Rotterdam section of the ERSPC trial demonstrated that after correction for both nonat‐ tendance in the screening arm and PSA contamination in the control arm, PSA-based screening conducted in the Dutch center reduced the risk of dying from prostate cancer up to 51% (RR of 0.49; 95% CI, 0.27–0.87) in men who undergo organized screening at a median follow-up of

In addition, randomized trial has another limitation. The primary end point of the trial is prostate cancer-specific mortality. Therefore, randomized trials would require a considerably long time to withdraw some conclusions. These trials randomly registered asymptomatic, healthy men and had followed until assigned men developed prostatic cancer and, further, died from prostate cancer or other causes. When a result is provided, the diagnostic method that is prostate biopsy method as well as treatment methods usually have progressed.

In addition, the rate of the death from prostate cancer is very low and there is a difficulty in assessing the effect of PSA screening on the prostate cancer-specific mortality. For example, the PLCO trial reported that after 7–10 years of follow-up, the incidence of death per 10,000 person-years was only 2.0 (50 deaths) in the screening group and 1.7 (44 deaths) in the control

screening to opportunistic PSA testing in the control arm [16].

4 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

group (rate ratio, 1.13; 95% CI, 0.75–1.70) [7].

control arm, respectively.

13 years [17].

The majority of prostate cancer patients with early, localized, low-grade cancer with proper treatments most often have an indolent clinical course. An analysis of 828 case records of men treated conservatively (with observation and delayed hormone therapy) for clinically localized prostate cancer showed that 10 years after diagnosis, disease-specific survival was 87% for men with grade 1 or 2 tumors and 34% for those with grade 3 tumors [5]. Most early-stage (T0- T2 NX M0 classification), initially untreated prostate cancer, had an indolent course during the first 10–15 years [6].

#### **4. Patients with some coexisting conditions are more likely to die from causes other than prostate cancer**

Patients with some comorbidities were more likely to die from causes other than prostate cancer [18]. The Surveillance, Epidemiology, and End Results (SEER) group conducted a 10 year competing risk analysis of men 66 years age and older with localized prostate cancer and received no surgery and radiation within 180 days of diagnosis and found that relatively few men died as a result of prostate cancer within 10 years of diagnosis [19]. Patients were more likely to die from comorbid causes other than prostate cancer during the first 10 years after diagnosis. Depending on patient age, Gleason score, and number of coexisting conditions, 10 year overall mortality rates increased from 28.8% (95% CI, 25.3–32.6%) to 94.3% (95% CI, 87.4– 100%), in contrast, prostate cancer-specific mortality rates varied from 2.0% (95% CI, 0.0–5.3%) to 27.5% (95% CI, 21.5–36.5%). The group reported that most patients with localized prostate cancer older than 65 years would not die from prostate cancer within 10 years of diagnosis. Most prostate cancer patients with either no or one comorbidity would survive at least 10 years; in contrast, those with two or more comorbid conditions would have a substantial risk of dying from a coexisting disease within this time period [19]. Another randomized study demon‐ strated a similar result. After 10-year follow-up of 76,693 men with prostate cancer, a significant decrease in the risk of prostate cancer-specific mortality was observed in patients with no or minimal comorbidity (adjusted hazard ratio (HR), 0.56; 95% CI, 0.33–0.95; *p* = 0.03); however, no reduction of that was observed in patients with at least one significant comorbidity (adjusted hazard ratio, 1.43; 95% CI, 0.96–2.11; *p* = 0.08). The group concluded that selective use of PSA screening for men in good health appeared to reduce the risk of prostate cancerspecific mortality [20].

#### **5. The AUA updated a guideline concerning early detection of prostate cancer**

In 2013, the American Urological Association (AUA) updated clinical guideline regarding PSA screening, "Early detection of Prostate cancer", which recommended a selective PSA screening, for example, according to age, coexisting medical conditions, or risks such as family history [10]. In addition, the AUA guideline strongly recommended "shared-decision making", which is a thorough discussion between clinicians and patients concerning the risks and benefits of PSA screening and treatment.

Above-mentioned arguments were the main reasons why the AUA recommended against routine PSA screening but for a selected screening and emphasized "shared-decision making". For example, the AUA guideline did not recommend routine PSA screening in 70 years of age or older men.

However, we must pay attention to the fact that the AUA guideline does not address diagnosis of symptomatic patients, where symptoms imply those that could be related to locally advanced or metastatic disease, but detection of disease at an early, pre-symptomatic stage when a man would have no reason to seek medical care.

#### **6. Prostate cancer incidence increases with age**

The rate of prostate cancer incidence increases with age. The incidence rate of prostate cancer increases, especially older than 70 years. In the United States, probability of developing prostate cancer for 60–69 years of age, 70 and older, and birth to death were 6.84, 12.54, and 16.48%, respectively [1]. As for in Japan, the Japan Cancer Surveillance Research Group reported that the prostate cancer incidence rate of all ages, crude rate, was 63.1 per 100,000 population in 2004; however, age-adjusted incidence rate increased from 32.2 in the 55- to 59 year-old group to 401.2 in the 80- to 84-year-old group [11].

#### **7. Older men are more likely to be presented with high-risk prostate cancer**

Older men are more likely to be presented with high-risk prostate cancer and to have lower cancer-specific survival [12]. A study from the Cancer of the Prostate Strategic Urologic Research Endeavor (CaPSURE) demonstrated that 26% of men above 75 years of age presented with high-risk prostate cancer and older men were less likely to receive curative treatment, partially explaining the higher prostate cancer-specific mortality rate [12]. Furthermore, post PSA screening follow-up study showed that compared with cancers detected during the screening period for men up to 69 year of age, cancers diagnosed after screening for men beyond 69 year of age were shifted toward more advanced, high-risk cancers with a low rate of low-risk cancers [21]. In addition, an extended follow-up study revealed that even patients with initially low risk tumors showed local progression and distant metastasis and eventually resulted in lethal outcome. A comparative study of early-staged prostate cancer patients between the first 15 years and the next 15–20 years demonstrated a substantial decrease in progression-free survival, from 45.0 to 36.0%; survival without metastases, from 76.9 to 51.2%; and prostate cancer-specific survival, from 78.7 to 54.4% [6]. In addition, the prostate cancerspecific mortality rate increased from 15 per 1000 person-years (95% CI, 10–21) during the first 15 years to 44 per 1000 person-years (95% CI, 22–88) following the first 15 years (*p* = 0.01) [6]. After 32 years of follow-up of this series, 90 (41.4%) cases showed local progression and 41 (18.4%) exhibited progression to distant metastasis [22]. These facts imply that localized prostate cancer most often has an indolent course; however, local progression and distant metastasis could develop over the long term, even among patients considered as low-risk disease at diagnosis.

for example, according to age, coexisting medical conditions, or risks such as family history [10]. In addition, the AUA guideline strongly recommended "shared-decision making", which is a thorough discussion between clinicians and patients concerning the risks and benefits of

Above-mentioned arguments were the main reasons why the AUA recommended against routine PSA screening but for a selected screening and emphasized "shared-decision making". For example, the AUA guideline did not recommend routine PSA screening in 70 years of age

However, we must pay attention to the fact that the AUA guideline does not address diagnosis of symptomatic patients, where symptoms imply those that could be related to locally advanced or metastatic disease, but detection of disease at an early, pre-symptomatic stage

The rate of prostate cancer incidence increases with age. The incidence rate of prostate cancer increases, especially older than 70 years. In the United States, probability of developing prostate cancer for 60–69 years of age, 70 and older, and birth to death were 6.84, 12.54, and 16.48%, respectively [1]. As for in Japan, the Japan Cancer Surveillance Research Group reported that the prostate cancer incidence rate of all ages, crude rate, was 63.1 per 100,000 population in 2004; however, age-adjusted incidence rate increased from 32.2 in the 55- to 59-

**7. Older men are more likely to be presented with high-risk prostate cancer**

Older men are more likely to be presented with high-risk prostate cancer and to have lower cancer-specific survival [12]. A study from the Cancer of the Prostate Strategic Urologic Research Endeavor (CaPSURE) demonstrated that 26% of men above 75 years of age presented with high-risk prostate cancer and older men were less likely to receive curative treatment, partially explaining the higher prostate cancer-specific mortality rate [12]. Furthermore, post PSA screening follow-up study showed that compared with cancers detected during the screening period for men up to 69 year of age, cancers diagnosed after screening for men beyond 69 year of age were shifted toward more advanced, high-risk cancers with a low rate of low-risk cancers [21]. In addition, an extended follow-up study revealed that even patients with initially low risk tumors showed local progression and distant metastasis and eventually resulted in lethal outcome. A comparative study of early-staged prostate cancer patients between the first 15 years and the next 15–20 years demonstrated a substantial decrease in progression-free survival, from 45.0 to 36.0%; survival without metastases, from 76.9 to 51.2%; and prostate cancer-specific survival, from 78.7 to 54.4% [6]. In addition, the prostate cancerspecific mortality rate increased from 15 per 1000 person-years (95% CI, 10–21) during the first

PSA screening and treatment.

when a man would have no reason to seek medical care.

6 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

**6. Prostate cancer incidence increases with age**

year-old group to 401.2 in the 80- to 84-year-old group [11].

or older men.

#### **8. Population-based cohort study reflects the real -world clinical outcome**

Population-based cohort study is the second best study design for evidenced-based decision making in medicine. Randomized study searches for an idealized world data, while cohort study uses a real-world clinical data. Different from the randomized trials, the cohort study targets the patients with prostate cancer from the beginning.

Authors conducted a population-based cohort study of pathologically diagnosed prostate cancer patients in Yokosuka City, Japan, between 2001 and 2010 and compared their clinical outcomes until 2013. Prostate cancer patients were divided into two groups: 524 detected by PSA-based screening in Yokosuka City (screening, S group) versus 1044 those detected by opportunistic PSA examinations (non-screening, NS group) [23]. Median age at diagnosis in the S group was significantly lower than that in the NS group: 71 and 73 years of age, respec‐ tively (*p* < 0.001). The rate of Gleason score (GS) 8–10 in the S group was significantly lower than that in the NS group: 9.7 and 16.7%, respectively (*p* < 0.001). The rate of patients with metastasis or PSA 100 ng/ml or more in the S group was also significantly lower than that in the NS group: 7.8 and 23.0%, respectively (*p* < 0.001). The group reported 8 (1.5%) prostate cancer deaths in the S group, whereas 70 (6.7 %) deaths in the NS group during the follow-up period. There were 42 (8.0%) deaths from other causes in the S group and 119 (11.4%) such deaths in the NS group. In the study, the group found a significantly higher 10-year cancerspecific survival rate in the S group than in the NS group: 97 and 86%, respectively (*p* < 0.001). The 10-year overall survival rate in the S group was also significantly higher than the NS group, 77 and 64%, respectively (*p* < 0.001). Multivariate Cox regression analysis showed that GS 8– 10 was significantly associated with cancer-specific survival rate (HR, 4.808; 95% CI, 1.044– 22.14; *p* = 0.044). The significantly lower prevalence of advanced prostate cancer, especially lower prevalence of GS 8–10 in the S group, was considered to be associated with higher cancerspecific survival. Older patients are more likely to have high-risk prostate cancer at diagnosis [12, 21]. The difference in the median age between the two groups was only 2 years; however, this difference might be responsible for the significantly lower prevalence of GS 8–10 prostate cancer in the S group.

Prostate cancer patients are in general more likely to die from coexisting diseases other than prostate cancer [18–20]. This study also demonstrated that overall 5.0% of patients died from prostate cancer, whereas 10.3% died from other causes. However, we must pay attention to the fact that in patients with advanced disease group, metastasis positive, or PSA 100 ng/ml or more, especially in the NS group, the incidence of death from prostate cancer was higher than those from other causes, 22.5 and 13.8%, respectively.

The ERSPC trial demonstrated similar overall survival rates between the intervention and the control arms [8]. In contrast, this real-world analysis revealed a significantly higher 10-year overall survival rate in the S group. The higher cancer-specific survival rate in the S group and the 2 years difference in median age between the two groups might be partly responsible for the higher overall survival rate. In addition, a plausible reason for this difference is considered as follows: the S group might consist of health-conscious men, who are willing to seek PSA screening at their own will, in other words, who are eager to be in good health. PSA screening itself is not a national duty. In fact, the group noticed a significantly lower Charlson comor‐ bidity score in the S group than in the NS group, 0.5 and 1.3, respectively (*p* < 0.001). These factors might be associated with a higher overall survival rate in the S group [23]. PSA-based population screening in Yokosuka City could contribute to reduce the rate of advanced prostate cancer and might help to reduce prostate cancer mortality rate.

Cancer statistical analysis reported that among men, prostate cancer was the most common cancer, accounting for up to 29% of all incident cases and was the second most common (9%) cause of cancer death in the United States in 2012 [1]. While lung and bronchus cancer was the second most common (14%) estimated new cases and the most common (29%) cause of cancer death among men in the United States in 2012 [1]. These figures suggest that at least prostate cancer, totally, is less aggressive than lung and bronchus cancer. In this stat‐ istical analysis, disease duration was not clearly shown; however, prostate cancer patients would seem to survive much longer than lung and bronchus patients. Prostate cancer is a highly heterogeneous cancer, ranging from low-grade, slow-growing, and indolent tumors to high grade, rapidly growing, and fatal carcinomas associated with significant morbidity [24].

An ideal tumor marker should be such that it could mainly detect asymptomatic patients with potentially fatal cancers; however, in fact, this would be rather impossible for PSA screening. Because PSA screening is an extremely sensitive examination, it inevitably de‐ tects indolent tumors as well as fatal carcinomas. Rather, if PSA screening could not detect a variety of prostate cancers, PSA would have no value as a screening marker for prostate cancer.

Prostate cancer is an extremely heterogeneous cancer, from low-grade, slow-growing, and indolent tumors to high-grade, rapidly growing, and fatal carcinomas. Prostate cancer incidence increases with age. Older men are more likely to be presented with high-grade prostate cancer. Older men are more likely to have comorbid medical conditions, such as diabetes, cardiovascular diseases, and cerebrovascular diseases. Health-conscious men would seek PSA screening. According to these facts, prostate cancer management should be tailored based on an individual patient's health status, coexisting medical conditions, life expectancy, and tumor characteristics [25]. One man argued as follows: the time has come from a "one size fits all" approach to a tailored approach based on an individual patient's health condition [26]. Physicians should be aware of these facts and should use PSA screening to offer the most appropriate approach to prostate cancer management for each patient.

#### **Author details**

Naoki Sakai

The ERSPC trial demonstrated similar overall survival rates between the intervention and the control arms [8]. In contrast, this real-world analysis revealed a significantly higher 10-year overall survival rate in the S group. The higher cancer-specific survival rate in the S group and the 2 years difference in median age between the two groups might be partly responsible for the higher overall survival rate. In addition, a plausible reason for this difference is considered as follows: the S group might consist of health-conscious men, who are willing to seek PSA screening at their own will, in other words, who are eager to be in good health. PSA screening itself is not a national duty. In fact, the group noticed a significantly lower Charlson comor‐ bidity score in the S group than in the NS group, 0.5 and 1.3, respectively (*p* < 0.001). These factors might be associated with a higher overall survival rate in the S group [23]. PSA-based population screening in Yokosuka City could contribute to reduce the rate of advanced

Cancer statistical analysis reported that among men, prostate cancer was the most common cancer, accounting for up to 29% of all incident cases and was the second most common (9%) cause of cancer death in the United States in 2012 [1]. While lung and bronchus cancer was the second most common (14%) estimated new cases and the most common (29%) cause of cancer death among men in the United States in 2012 [1]. These figures suggest that at least prostate cancer, totally, is less aggressive than lung and bronchus cancer. In this stat‐ istical analysis, disease duration was not clearly shown; however, prostate cancer patients would seem to survive much longer than lung and bronchus patients. Prostate cancer is a highly heterogeneous cancer, ranging from low-grade, slow-growing, and indolent tumors to high grade, rapidly growing, and fatal carcinomas associated with significant morbidity

An ideal tumor marker should be such that it could mainly detect asymptomatic patients with potentially fatal cancers; however, in fact, this would be rather impossible for PSA screening. Because PSA screening is an extremely sensitive examination, it inevitably de‐ tects indolent tumors as well as fatal carcinomas. Rather, if PSA screening could not detect a variety of prostate cancers, PSA would have no value as a screening marker for prostate

Prostate cancer is an extremely heterogeneous cancer, from low-grade, slow-growing, and indolent tumors to high-grade, rapidly growing, and fatal carcinomas. Prostate cancer incidence increases with age. Older men are more likely to be presented with high-grade prostate cancer. Older men are more likely to have comorbid medical conditions, such as diabetes, cardiovascular diseases, and cerebrovascular diseases. Health-conscious men would seek PSA screening. According to these facts, prostate cancer management should be tailored based on an individual patient's health status, coexisting medical conditions, life expectancy, and tumor characteristics [25]. One man argued as follows: the time has come from a "one size fits all" approach to a tailored approach based on an individual patient's health condition [26]. Physicians should be aware of these facts and should use PSA screening to offer the most

appropriate approach to prostate cancer management for each patient.

prostate cancer and might help to reduce prostate cancer mortality rate.

8 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

[24].

cancer.

Address all correspondence to: sakai@wakakusa.saiseikai.or.jp

Department of Urology, Saiseikai Wakakusa Hospital, Yokohama, Kanagawa, Japan

#### **References**


### **Advances in Prostate Cancer Diagnosis: Triggers for Prostate Biopsy**

John W. Davis and Chinedu Mmeje

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64402

#### **Abstract**

[12] Bechis SK, Carroll PR and Cooperberg MR. Impact of age at diagnosis on prostate

[13] Andriole GL, Crawford ED, Grubb IIIRL, et al. Prostate cancer screening in the randomized prostate, lung, colorectal, and ovarian cancer screening trial: mortality

[14] Schroder FH, Hugosson J, Roobol MJ, et al. Prostate-cancer mortality at 11 years of

[15] Schroder FH, Hugosson J, Roobol MJ, et al. The European randomized study of screening for prostate cancer-prostate cancer mortality at 13 years of follow-up. Lancet.

[16] Kim EH and Andriole GL. Prostate-specific antigen-based screening: controversy and

[17] Bokhorst LP, Bangma CH, van Leederd GJLH, et al. Prostate-specific antigen–based prostate cancer screening: reduction of prostate cancer mortality after correction for nonattendance and contamination in the Rotterdam section of the European random‐

[18] Lu-Yao GL, Albertson PC, Moore DF, et al. Outcomes of localized prostate cancer

[19] Albertsen PC, Moore DF, Shih W, et al. Impact of comorbidity on survival among men

[20] Crawford ED, Grubb III R, Black A, et al. Comorbidity and mortality results from a randomized prostate cancer screening trial. J Clin Oncol. 2011; 29: 355–361.

[21] Bergdahl AG, Holmberg E, Moss S, et al. Incidence of prostate cancer after termination of screening in a population-based randomised trial. Eur Urol. 2013; 64: 703–709.

[22] Popiolek M, Rider JR, Andrén O, et al. Natural history of early, localized prostate cancer: a final report from three decades of follow-up. Eur Urol. 2013; 63: 423–435.

[23] Sakai N, Taguri M, Kobayashi K, et al. Clinical outcomes of prostate cancer patients in Yokosuka City, Japan: a comparative study between cases detected by prostate-specific antigen-based screening in Yokosuka and those detected by other means. Int J Urol.

[24] Roobol MJ and Carlsson SV. Risk stratification in prostate cancer screening. Nat Urol

[25] Hoffman KE. Management of older men with clinically localized prostate cancer: the significance of advanced age and comorbidity. Semin Radiat Oncol. 2012; 22: 284–294.

[26] McNaughton MF and Barry MJ. One man at a time-resolving the PSA controversy. N

ized study of screening for prostate cancer. Eur Urol. 2014; 65: 329–336.

following conservative management. JAMA. 2009; 302: 1202–1209.

with localized prostate cancer. J Clin Oncol. 2011; 29: 1335–1341.

results after 13 years of follow-up. J Natl Cancer Inst. 2012; 104: 125–132.

cancer treatment and survival. J. Clin Oncol. 2011; 29: 235–241.

follow-up. N Engl J Med. 2012; 366: 981–990.

10 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

guidelines. BMC Med. 2015; 13: 61–64.

2014; 384: 2027–2035.

2015; 22: 747–753.

Rev. 2013; 10: 38–48.

Engl J Med. 2011; 365: 1951–1953.

In the early years of screening for prostate cancer with serum PSA, absolute cutoffs were typically utilized such as greater than 4.0 ng/mL or even 2.5 ng/mL. A biopsy of the prostate would commonly be recommended in a man with greater than 10-year life expectancy who had a confirmed elevation above such a threshold or in the presence of an abnormal digital rectal examination. The unmet need, however, is to be more selective in recommending a prostate biopsy, due to the risk of complications and the high rate of false-positive PSAs. More recently, various clinical nomograms can be used to refine selection. In addition, clinicians can now utilize various advanced serum biomarkers that have enhanced specificity—especially for the patient with a rising PSA with prior negative biopsy. In this chapter, we will focus on the biomarkers PCA3, Prostate Health Index, and 4 K score to illustrate key concepts in biomarker develop‐ ment and clinical utility.

**Keywords:** prostate cancer, prostate biopsy, PCA3, urine, Prostate Health Index, se‐ rum, 4 K score, serum

#### **1. Introduction—a narrative on contemporary management of prostate cancer risk with ordinary clinical tools**

The male disease process "prostate cancer" is a heterogeneous entity at multiple subtop‐ ics including epidemiology, screening, diagnosis, treatment options, side effects, and cancer control results. It is common to introduce a peer-reviewed article or text chapter fo‐ cused on any aspect of prostate cancer with the observation that the disease is common but a much smaller subset result in a disease-specific mortality—almost to the point of

© 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.

useless repetition. The heterogeneity of prostate cancer extends well beyond population statistics, and in this chapter, we will focus on one aspect of heterogeneity—diagnosis. The key paradigm in contemporary practice is a male screened for prostate cancer with serum prostate specific antigen (PSA) and digital rectal examination (DRE). The alternate/ related paradigm is the male with lower urinary tract or other pelvic symptoms who is evaluated for prostate cancer with serum PSA/DRE as a differential diagnosis. Perform‐ ing a biopsy and subsequent treatment has certainly altered key statistics in prostate cancer compared to pure clinical detection. The incidence has increased as well as treatment numbers. Recently, Welch et al. demonstrated with SEER/Medicare data that the inci‐ dence of metastatic disease at diagnosis significantly declined with the introduction of PSA, compared with the relatively flat effect of mammography screening to breast cancer staging [1].

The problem faced from this paradigm is that the results of a prostate biopsy are frustrating for a growing list of reasons:


The concepts can be illustrated in the following case vignettes:


Physicians managing patients at risk for prostate cancer can certainly use clinical features to frame many questions. In the asymptomatic patient who might have clinically localized disease, a life expectancy of less than 10 years would be treated differently than a life expect‐ ancy greater than 10 years. Family history of prostate cancer (father, brothers, uncles) and African American race are well-known adverse risk features for prostate cancer. The PSA has its statistical problems with specificity (related to false positives), but does have sensitivity (related to true positives) related to its value—higher PSAs have more risk of cancer, but hard to determine a clear line to draw where you call the test clearly normal versus abnormal. Translating these narrated elements into summary numerical estimates (noncited as widely available):

useless repetition. The heterogeneity of prostate cancer extends well beyond population statistics, and in this chapter, we will focus on one aspect of heterogeneity—diagnosis. The key paradigm in contemporary practice is a male screened for prostate cancer with serum prostate specific antigen (PSA) and digital rectal examination (DRE). The alternate/ related paradigm is the male with lower urinary tract or other pelvic symptoms who is evaluated for prostate cancer with serum PSA/DRE as a differential diagnosis. Perform‐ ing a biopsy and subsequent treatment has certainly altered key statistics in prostate cancer compared to pure clinical detection. The incidence has increased as well as treatment numbers. Recently, Welch et al. demonstrated with SEER/Medicare data that the inci‐ dence of metastatic disease at diagnosis significantly declined with the introduction of PSA, compared with the relatively flat effect of mammography screening to breast cancer

The problem faced from this paradigm is that the results of a prostate biopsy are frustrating

**2.** Too many negative or indeterminate biopsies that are found to be falsely negative with

**4.** Too many positive biopsies for low grade disease that with future radical prostatectomy

**5.** Cost, discomfort, and an occasional septic infection as the side effects of this effort to make

**•** A patient with Gleason Score 3+3 on biopsy has a radical prostatectomy, and the final

**•** A patient with three negative biopsies has a continuous rising PSA—should he have a 4th

**•** A patient with a negative biopsy and stable PSA—should he continue screening? What age

**•** An elderly patient with Gleason 7 prostate cancer is unsure of overall longevity—does he

Physicians managing patients at risk for prostate cancer can certainly use clinical features to frame many questions. In the asymptomatic patient who might have clinically localized disease, a life expectancy of less than 10 years would be treated differently than a life expect‐ ancy greater than 10 years. Family history of prostate cancer (father, brothers, uncles) and African American race are well-known adverse risk features for prostate cancer. The PSA has its statistical problems with specificity (related to false positives), but does have sensitivity (related to true positives) related to its value—higher PSAs have more risk of cancer, but hard

**3.** Too many positive biopsies for low grade disease—leads to overtreatment.

or repeat biopsies are found to have missed higher grade disease.

need curative therapy or just watchful waiting based on symptoms?

The concepts can be illustrated in the following case vignettes:

staging [1].

to stop?

for a growing list of reasons:

future evaluations.

**1.** Too many negative or indeterminate biopsies.

12 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

the *correct* diagnosis for a patient.

biopsy or accept the cause as benign?

pathology showed Gleason 4+4.


Moving forward, the unmet needs in prostate cancer evaluation are to increase the detection of clinically significant disease when it is present, and to effectively rule out significant disease when it is not present, such that subsequent monitoring can be reduced or eliminated. *To emphasize*—*both of these needs are critical and equal: the need to diagnose cancer that is present and potentially lethal, and the need to eliminate diagnostic attempts when cancer is absent or nonlethal.*

#### **2. Prostate biopsy triggers—mild improvement from mathematics and clinical trials**

In a state-of-the-art lecture at the 2015 American Urological Association Annual Meeting, Stacy Loeb (New York, USA) made the key analogy that the first 10 or more years of use of PSA was analogous to a pregnancy test—in search of when to call the test positive or negative. The initial cut-point was 4.0, and many labs still flag a result in red at >4.0, and subsequent proposals were for >2.5. Clinical experience clearly showed the fallacy of this version of laboratory medicine—a man's risk of prostate cancer does not go from zero at PSA 3.9 to 100% at PSA of 4.1. The point was well illustrated by a follow-up report from the Prostate Cancer Prevention Trial that showed the biopsy results of men with different PSA cut-points who were all biopsied and on a placebo medication—sample size of 2950 participants and 449 cancers [2].**Table 1** shows a sample of their report. Note that the pregnancy illustration was carried forward in this trial, as men with a PSA > 4.0 were biopsied "for cause" and the data represent the men followed for the 7-year duration of the trial who were considered clinically "normal range". Prior to this publication, there was very limited data available on the results of a prostate biopsy in men with a normal DRE and PSA < 4.0.

With this data, you can certainly counsel a man on the concept of a relative range of prostate cancer risk rather than oversimplify it to a positive/negative result. This same dataset was refined further into an online calculator tool where you can input multiple clinical variables and receive an estimate on overall cancer detection and high grade (Gleason 7 or higher) cancer. The web link is: http://deb.uthscsa.edu/URORiskCalc/Pages/uroriskcalc.jsp.

Here are two example cases:



**Table 1.** Data from the Prostate Cancer Prevention Trial [2]. This cohort of men were biopsied as part of the trial design and were on a placebo. Men with a PSA > 4.0 would have been biopsied earlier "for cause".

The results also remind you that regardless of the biopsy result, there may be a 2–4% chance of an infection requiring hospitalization. Thus, the absolute risks of cancer and side effects can be discussed and a personalized choice made. We should not forget why we are considering these efforts. The third update to the Bill-Axelson trial of radical prostatectomy versus watchful waiting reminds us that the treatment arm had a 12.7% absolute difference in overall mortality, with a relative risk of 0.71 in favor of surgery and number needed to treat to avoid a death of 1:8 [3]. Additional benefits were observed in palliation, metastatic progression, and androgen deprivation utilization.

#### **3. Biomarkers in prostate cancer—highlights of evaluation and early improvement of specificity**

As established thus far, PSA is the gold standard for prostate cancer detection. Many experts have voiced the opinion that this might change in the next generation [4]. First, we should review key biomarker nomenclature recognized by the FDA—whether a biomarker is serum, urine, or tissue base. **Table 2** shows four key distinctions and a biomarker such as PSA has elements of all four.

Data on biomarkers is a separate and vast topic and we will not review it. But the key headings would be whether a study is in preclinical exploratory trials, assay development, assay validation, retrospective use/repositories, prospective screening, or randomized controlled. Biomarkers can be described based on their validity with a number of statistical expression and eventually need to be described based upon clinical utility, i.e., strength of ability to alter key clinical decisions and add value to health care.


**Table 2.** FDA biomarker classification based on context of use.

and receive an estimate on overall cancer detection and high grade (Gleason 7 or higher) cancer.

**1.** Race Caucasian, age 59, PSA 2.1, no family history, abnormal DRE, no prior biopsy. Result:

**2.** Race African American, age 59, PSA 9.8, positive family history for prostate cancer, DRE abnormal, no prior biopsy. Result: 35% high-grade cancer, 20% low-grade cancer, 45%

**Table 1.** Data from the Prostate Cancer Prevention Trial [2]. This cohort of men were biopsied as part of the trial design

The results also remind you that regardless of the biopsy result, there may be a 2–4% chance of an infection requiring hospitalization. Thus, the absolute risks of cancer and side effects can be discussed and a personalized choice made. We should not forget why we are considering these efforts. The third update to the Bill-Axelson trial of radical prostatectomy versus watchful waiting reminds us that the treatment arm had a 12.7% absolute difference in overall mortality, with a relative risk of 0.71 in favor of surgery and number needed to treat to avoid a death of 1:8 [3]. Additional benefits were observed in palliation, metastatic progression, and androgen

**3. Biomarkers in prostate cancer—highlights of evaluation and early**

As established thus far, PSA is the gold standard for prostate cancer detection. Many experts have voiced the opinion that this might change in the next generation [4]. First, we should review key biomarker nomenclature recognized by the FDA—whether a biomarker is serum, urine, or tissue base. **Table 2** shows four key distinctions and a biomarker such as PSA has

Data on biomarkers is a separate and vast topic and we will not review it. But the key headings would be whether a study is in preclinical exploratory trials, assay development, assay validation, retrospective use/repositories, prospective screening, or randomized controlled.

The web link is: http://deb.uthscsa.edu/URORiskCalc/Pages/uroriskcalc.jsp.

3% high-grade cancer, 14%, low-grade cancer, and 83% negative biopsy.

**PSA level Percentage positive biopsy Percentage of positive biopsies with high grade**

and were on a placebo. Men with a PSA > 4.0 would have been biopsied earlier "for cause".

Here are two example cases:

negative biopsy.

deprivation utilization.

elements of all four.

**improvement of specificity**

≤0.5 6.6 12.5 0.6–1.0 10.1 10.0 1.1–2.0 17.0 11.8 2.1–3.0 23.9 19.1 3.1–4.0 26.9 25.0

14 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

As stated, there are many statistical advantages to PSA screening compared to using prostatic acid phosphatase (PAP) [5], or clinical exam [6]. However, there is significant room to improve accuracy and especially specificity. Early efforts to make progress were numerous such as adding the percent-free PSA to PSA ratio [7], age adjustment [8], PSA velocity of ≥0.75 ng/mL/ year [9], and PSA density of 0.15 [10]. These methods all made incremental practice, but perhaps the most significant is in making decisions about a repeat biopsy versus a primary biopsy. Another useful contribution to PSA screening interval questions came from Lilja et al., who showed that a single PSA before age 50 could be predictive of lifetime prostate cancer incidence [11].

#### **4. A focus on novel diagnostic biomarkers: prostate Health Index and 4 K score**

Similar themes in advancing future prostate cancer screening have come from focusing on "isomers" or molecular variants of the PSA molecule. Another variant of PSA is called Pro-PSA and can be more prevalent in the free-fraction within cancer versus noncancer [12]. In a validation study of Pro-PSA versus percent-free PSA, the area under the curve (AUC) was 0.68 for %ProPSA and 0.567 for %free PSA. At sensitivity of 75%, the %ProPSA would eliminate 59% of negative biopsies versus 33% with %fPSA [13]. Catalona et al. [14] also did a serum bank study on biopsied patients with elevated PSA and found the %proPSA can eliminate 19– 33% of biopsies while holding 90% sensitivity.

The next iteration came from the discovery of an isoform p2PSA, and the concept of combining the information with free PSA and total PSA into an equation: ([−2]pPSA/fPSA)/√(tPSA). This is now the Prostate Health Index and licensed by Beckman Coulter laboratories. Jansen et al. [15] reported a multisite study showing that prostate cancer patients had higher PHI levels and %p2PSA. A U.S. validation study of 829 patients holding sensitivity at 95% had specificity of 16% for PHI and 8.5% for %fPSA [16]. Relevant to our case vignettes in the introductory narrative, the higher PHI values were also correlating with *a higher risk of Gleason score ≥7.*

A PHI score is currently reported in four "brackets" of results: 0–24.9, 25.0–34.9, 35.0–54.9, and >55. The PHI, as stated will include a total PSA, the free PSA, and the Pro-PSA. The PHI result is then translated into a percentage risk of prostate cancer. The source reference [16] includes the high grade numbers, but an actual test result for some reason does not. **Table 3** shows a commercial report range. The study listed ranges for Gleason ≥7 were PHI 0–24.9 = 26.1%, PHI 25.0–34.9 of 28.2%, PHI 35.0–54.9 of 30.1%, and PHI >55.0 of 42.1%. Thus, the trends are strongest comparing the lowest versus highest PHI brackets.


**Table 3.** PHI result reporting as of 2015.

A European cohort was published by Lazzeri et al. [17] and showed an AUC of 0.67 for PHI as well as %proPSA—both superior to total PSA and %free PSA. At a PHI cutoff of 27.6, biopsies could have been avoided in 15.5%. The Gleason ≥7 trend was also observed in their statistical analyses.

Another research direction developing in parallel has been human glandular kallikrein 2 hK2. It is also part of the serine protease family along with PSA. Nam et al. [18], for example, looked at hK2 and hK2 to %free PSA ratios could find trends elevated in PCa. Vickers et al. then expanded the concept as a panel of Kallikrein markers: total PSA, free PSA, intact PSA, and hK2. This coined the phrase 4 K score [19]. The process developed a nomogram that includes the four markers, age, DRE, and determines a probability of cancer. The model adds to a clinical base model and increases prediction for high grade prostate cancer. The AUC for the full model was 0.832 and high-grade cancer was 0.870, and a decision curve analysis is presented to propose a threshold value of 20% as a biopsy trigger that would spare a significant number of biopsies and only miss 3% high-grade cancers. Parekh et al. [20] then published a U.S. prospective trial that used the PCPT risk calculator above as a control. Again, the full model had an AUC of 0.821 and higher AUC for Gleason ≥7, as well as decision curve benefit. As an illustration, a 4 K score cutoff of 9% led to 43% of biopsies avoided and 2.4% risk of delay in Gleason ≥7 diagnosis.

A PHI score is currently reported in four "brackets" of results: 0–24.9, 25.0–34.9, 35.0–54.9, and >55. The PHI, as stated will include a total PSA, the free PSA, and the Pro-PSA. The PHI result is then translated into a percentage risk of prostate cancer. The source reference [16] includes the high grade numbers, but an actual test result for some reason does not. **Table 3** shows a commercial report range. The study listed ranges for Gleason ≥7 were PHI 0–24.9 = 26.1%, PHI 25.0–34.9 of 28.2%, PHI 35.0–54.9 of 30.1%, and PHI >55.0 of 42.1%. Thus, the trends are

> **%Free PSA <60 60**–**70 >70** <7 85% 95% 96% 7–15 25% 50% 60% 16–25 11% 27% 35% >25 2% 6% 10%

strongest comparing the lowest versus highest PHI brackets.

At risk ≥ 2.0

%free PSA 15 % Free PSA prostate cancer probability by age

PHI 49.9 **PHI Cancer probability**

0–24.9 11.0% 25.0–34.9 18.1% 35.0–54.9 32.7% >55.0 52.1%

A European cohort was published by Lazzeri et al. [17] and showed an AUC of 0.67 for PHI as well as %proPSA—both superior to total PSA and %free PSA. At a PHI cutoff of 27.6, biopsies could have been avoided in 15.5%. The Gleason ≥7 trend was also observed in their statistical

Another research direction developing in parallel has been human glandular kallikrein 2 hK2. It is also part of the serine protease family along with PSA. Nam et al. [18], for example, looked at hK2 and hK2 to %free PSA ratios could find trends elevated in PCa. Vickers et al. then expanded the concept as a panel of Kallikrein markers: total PSA, free PSA, intact PSA, and hK2. This coined the phrase 4 K score [19]. The process developed a nomogram that includes the four markers, age, DRE, and determines a probability of cancer. The model adds to a clinical base model and increases prediction for high grade prostate cancer. The AUC for the full model was 0.832 and high-grade cancer was 0.870, and a decision curve analysis is

**Biomarker Sample result Reference interval** Total PSA 7.8 Normal < 2.0

16 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

Free PSA 1.16 See below Pro2PSA 20.78 See PHI

**Table 3.** PHI result reporting as of 2015.

analyses.

For the clinician, the question then becomes which test to use? PHI had a slight advantage in being earlier in regulatory approval. Nordstrom et al. presented a comparison study of PHI versus 4 K [21]. The PHI and 4 K both had improved prediction for overall and high grade prostate cancer. The AUC for 4 K was 0.69 and for PHI was 0.704, and for high-grade prostate cancer was 0.718 and 0.711, respectively. Comparable metrics were observed with a 4 K cutoff of 10% and PHI of 39.


**Table 4.** Comparison of serum biomarkers PHI, 4K, and urine biomarker PCA3.

Another biomarker with a little more clinical experience and validation data is the PCA3 score —different in being a urinary marker rather than serum as for PHI and 4 K. The assay uses a ratio of messenger RNA for the PCA3 molecule with a PSA ratio built in. The AUCs for PCA3 are generally in the 0.0.68–75 range [22]. In the validation study for repeat biopsy patients, Marks et al. showed an AUC of 0.68 for PCA3 versus 0.52 for PSA [23]. The cutoffs recom‐ mended are in the 25–35 range. In the Marks study, a PCA3 cutoff of 35 showed 58% sensitivity and 72% specificity. A particular advantage of PCA3 has been that it is not affected by prostate volume and performed well across multiple PSA levels [24].

Back to comparative studies, Ferro et al. compared PCA3 to PHI in a prospective observational study [25]. The diagnostic accuracy was similar at 90% sensitivity: PHI specificity of 40% and PCA3 of 40% with 31.6 and 22 cutoffs, respectively. In a decision curve analysis, PHI had slightly higher benefit at probability of 25%. The Scattoni study [26] looked at these markers in initial and repeat biopsy populations and found a slight benefit to PHI but not significant. A comparison of AUC for initial biopsy showed PSA of 0.54, %fPSA of 0.67, PCA3 of 0.57, and PHI of 0.69. In repeat biopsy, it showed PSA of 0.60, %fPSA of 0.52, PCA3 of 0.63, and PHI of 0.72.

In **Table 4**, we consolidate these statistics into a final comparison with existing data.

The cost comparison certainly favors PHI, although the PCA3 test has more clinical experience and strong metrics in repeat biopsy decisions. It remains to be determined whether the 4 K cost is justified; however, when this test is up and running and through regulation, it may give a more objective reporting of specific high grade prostate cancer risk that clinicians may prefer.

### **5. Moving forward—similar themes in imaging, biopsy techniques, and tissue biomarkers**

In this chapter, we have outlined the PSA problem with emphasis on triggers for a biopsy. A separate but related topic is the technique of biopsy. The gold standard for all of these biomarker studies has been the 10–12 core transrectal ultrasound-guided biopsy. Emerging data, however, demonstrate that a transperineal template biopsy may sample the apex and anterior zones better. Other areas of research are looking at improvements in multiparametric MRI staging, and commercial software platforms that fuse the images such that an ultrasound biopsy now has a more accurate target to sample rather than random sampling by anatomic region. The endpoints are the same—ability to improve Gleason score ≥7 and the ability to trust that a negative test is actually negative. For diagnosed patients, commercial genetic profiling products can then look at specific grades of prostate cancer and offer additional prognostic information such as risk of upgrading/upstaging at surgery, mortality rates untreated, biochemical relapse after surgery, or metastatic relapse after surgery [27]. These topics can be separate chapters, but the themes are consistent—solving heterogeneity in prostate cancer diagnosis such that the downstream monitoring and treatment decisions are optimized.

#### **Author details**

ratio of messenger RNA for the PCA3 molecule with a PSA ratio built in. The AUCs for PCA3 are generally in the 0.0.68–75 range [22]. In the validation study for repeat biopsy patients, Marks et al. showed an AUC of 0.68 for PCA3 versus 0.52 for PSA [23]. The cutoffs recom‐ mended are in the 25–35 range. In the Marks study, a PCA3 cutoff of 35 showed 58% sensitivity and 72% specificity. A particular advantage of PCA3 has been that it is not affected by prostate

Back to comparative studies, Ferro et al. compared PCA3 to PHI in a prospective observational study [25]. The diagnostic accuracy was similar at 90% sensitivity: PHI specificity of 40% and PCA3 of 40% with 31.6 and 22 cutoffs, respectively. In a decision curve analysis, PHI had slightly higher benefit at probability of 25%. The Scattoni study [26] looked at these markers in initial and repeat biopsy populations and found a slight benefit to PHI but not significant. A comparison of AUC for initial biopsy showed PSA of 0.54, %fPSA of 0.67, PCA3 of 0.57, and PHI of 0.69. In repeat biopsy, it showed PSA of 0.60, %fPSA of 0.52, PCA3 of 0.63, and PHI of

In **Table 4**, we consolidate these statistics into a final comparison with existing data.

The cost comparison certainly favors PHI, although the PCA3 test has more clinical experience and strong metrics in repeat biopsy decisions. It remains to be determined whether the 4 K cost is justified; however, when this test is up and running and through regulation, it may give a more objective reporting of specific high grade prostate cancer risk that clinicians may prefer.

**5. Moving forward—similar themes in imaging, biopsy techniques, and**

In this chapter, we have outlined the PSA problem with emphasis on triggers for a biopsy. A separate but related topic is the technique of biopsy. The gold standard for all of these biomarker studies has been the 10–12 core transrectal ultrasound-guided biopsy. Emerging data, however, demonstrate that a transperineal template biopsy may sample the apex and anterior zones better. Other areas of research are looking at improvements in multiparametric MRI staging, and commercial software platforms that fuse the images such that an ultrasound biopsy now has a more accurate target to sample rather than random sampling by anatomic region. The endpoints are the same—ability to improve Gleason score ≥7 and the ability to trust that a negative test is actually negative. For diagnosed patients, commercial genetic profiling products can then look at specific grades of prostate cancer and offer additional prognostic information such as risk of upgrading/upstaging at surgery, mortality rates untreated, biochemical relapse after surgery, or metastatic relapse after surgery [27]. These topics can be separate chapters, but the themes are consistent—solving heterogeneity in prostate cancer diagnosis such that the downstream monitoring and treatment decisions are

volume and performed well across multiple PSA levels [24].

18 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

0.72.

**tissue biomarkers**

optimized.

John W. Davis\* and Chinedu Mmeje

\*Address all correspondence to: johndavis@mdanderson.org

University of Texas MD Anderson Cancer Center, Houston, Texas, USA

#### **References**


[23] Marks LS, Fradet Y, Deras IL, et al. PCA3 molecular urine assay for prostate cancer in men undergoing repeat biopsy. Urology 2007; 69: 532–535.

[11] Lilja H, Cronin AM, Dahlin A, et al. Prediction of significant prostate cancer diagnosed 20 to 30 years later with a single measure of prostate-specific antigen at or before age

[12] Mikolajczyk SD, Millar LS, Wang TJ, et al. A precursor form of prostate-specific antigen is more highly elevated in prostate cancer compared with benign transition zone

[13] Sokioll LJ, Chan DW, Mikolajczyk SD, et al. Proenzyme psa for the early detection of prostate cancer in the 2.5-4.0 ng/ml total psa range: preliminary analysis. Urology 2003;

[14] Catalona WJ , Bartsch G, Rittenhouse HG, et al. Serum pro prostate specific antigen improves cancer detection compared to free and complexed prostate specific antigen

[15] Jansen FH, van Schaik RH, Kurstjens J, et al. Prostate-spcirfic antigen (PSA) isoform p2PSA in combination with total PSA and free PSA improves diagnostic accuracy in

[16] Le BV, Griffin CR, Loeb S, et al. [−2]Proenzyme prostate specific antigen is more accurate than total and free prostate specific antigen in differentiating prostate cancer from benign disease in a prospective prostate cancer screening study. J Urol 2010; 183:

[17] Lazzeri M, Haese A, de la Taille A, et al. Serum isoform [−2] proPSA derivatives significantly improve prediction of prostate cancer at initial biopsy in a total PSA range

[18] Nam RK, Diamandis EP, Toi A, et al. Serum human glandular kallikrien-2 protease levels predict the presence of prostate cancer among men with elevated prostate-

[19] Vickers AJJ, Cronin AM, Aus G, et al. A panel of kallikrein markers can reduce unnecessary biopsy for prostate cancer: data from the European Randomized Study of

[20] Parekh DJ, Punnen S, Sjoberg D, et al. A multi-institutional prospective trial in the USA confirms that the 4 K score accurately identifies men with high grade prostate cancer.

[21] Nordstrom T, Vickers A, Assel M, et al. Comparison between the four-kallikrein panel and the prostate health index for predicting prostate cancer. Eur Urol 2015; 68: 139–146.

[22] Groskopf J, Aubin SM, Deras IL, et al. APTIMA PCA3 molecular urine test: develop‐ ment of a method to aid in the diagnosis of prostate cancer. Clin Chem 2006; 52: 1089–

of 2–10 ng/ml: a multicentric European study. Eur Urol 2013; 63: 986–994.

Prostate Cancer Screening on Goteborg, Sweden. BMC Med 2008; 6: 19.

in men with prostate specific antigen 2 to 4 nl/ml. J Urol 2003; 170: 2181–2185.

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20 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

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1095.


## **The Role of Prostate‐Specific Antigen (PSA) and PSA Kinetics in the Management of Advanced Prostate Cancer**

Jeremy Teoh and Ming‐Kwong Yiu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64180

#### **Abstract**

Prostate‐specific antigen (PSA) plays an important role in the diagnosis and manage‐ ment of prostate cancer. The utility of PSA has been extended to a number of parame‐ ters which may guide clinical decision‐making in subsequent treatment. This book chapter systematically reviewed the current evidence of PSA and PSA kinetics in the management of advanced prostate cancer. Results showed that the prognostic significance of pre‐treatment PSA level is uncertain. PSA nadir predicts survival outcomes but may be confounded by the pre‐treatment PSA level, and the PSA nadir may only be known after there is a PSA rise in subsequent follow‐up. Time to PSA nadir has some prognostic significance but is limited by the potential immortal bias. Evidence on the use of PSA doubling time is limited and the different calculation methodolo‐ gies render difficulties in generalization of such parameter. PSA progression is the best surrogate marker of survival and can be considered as the primary endpoint in future clinical trials. PSA response predicts survival but has not been shown prospectively to be a surrogate of clinical benefit. PSA and its kinetics should play an important role in the management of advanced prostate cancer and should be utilized in a more standardized manner.

**Keywords:** prostate cancer, prostate‐specific antigen, prostate‐specific antigen nadir, time to prostate‐specific antigen nadir, prostatic‐specific antigen doubling time, pros‐ tate‐specific antigen progression, prostate‐specific antigen response

© 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 first study investigating tissue‐specific antibodies in the human prostate can be traced back to 1969 by Ablin et al. [1]. Nadji et al. [2] later characterized prostate‐specific antigen (PSA) as a potential immunohistologic marker for prostatic neoplasms. The landmark article by Stamey et al. [3] showed that serum PSA has a much better performance than prostatic acid phospha‐ tase in the detection of prostate cancer, and appeared to be useful in detecting residual or early recurrence of tumour, and in monitoring response to primary treatment. It has led to exten‐ sive researches in this area, and the discovery of PSA has revolutionized the management of prostate cancer, from early detection to definitive treatment and monitoring of the disease. The utility of PSA has been extended to a number of parameters that may have prognostic signifi‐ cance in prostate cancer and hence has gained wide interest in the past two decades.

#### **2. Objectives**

In this chapter, we systemically reviewed and appraised the current role of PSA and PSA kinetics in the management of advanced prostate cancer. We further discussed the potential benefits and controversies of the different PSA‐related parameters.

#### **3. Methods**

A systematic search was conducted in the PubMed database through November 2015 using the following terms: 'prostate cancer', 'prostate‐specific antigen', 'prostate‐specific antigen nadir', 'time to prostate‐specific antigen nadir', 'prostatic‐specific antigen doubling time', 'prostate‐specific antigen progression' and 'prostate‐specific antigen response'. Only original full research articles published in English with full‐length text available were reviewed. A manual search using the Web‐based search engine Google Scholar was also performed. Reference lists of the retrieved articles were reviewed for other relevant studies.

#### **4. Results**

#### **4.1. Pre‐treatment PSA level**

To a certain extent, the pre‐treatment PSA level may reflect the volume of cancer cells, and hence, it is a parameter of interest in predicting disease prognosis. However, it does not reflect the sensitivity of cancer cells in response to subsequent therapy, in particular hormonal therapy in the context of metastatic disease. The prognostic significance of pre‐treatment PSA level is uncertain. Some studies showed that higher pre‐treatment PSA level was associated with disease progression, cancer‐specific mortality and all‐cause mortality [4–14], while other studies either showed no association or failed to demonstrate statistical significance upon multivariate analyses [15–32]. The wide range of pre‐treatment PSA level in metastatic disease also limited its clinical application.

#### **4.2. PSA nadir**

**1. Introduction**

24 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

**2. Objectives**

**3. Methods**

**4. Results**

**4.1. Pre‐treatment PSA level**

The first study investigating tissue‐specific antibodies in the human prostate can be traced back to 1969 by Ablin et al. [1]. Nadji et al. [2] later characterized prostate‐specific antigen (PSA) as a potential immunohistologic marker for prostatic neoplasms. The landmark article by Stamey et al. [3] showed that serum PSA has a much better performance than prostatic acid phospha‐ tase in the detection of prostate cancer, and appeared to be useful in detecting residual or early recurrence of tumour, and in monitoring response to primary treatment. It has led to exten‐ sive researches in this area, and the discovery of PSA has revolutionized the management of prostate cancer, from early detection to definitive treatment and monitoring of the disease. The utility of PSA has been extended to a number of parameters that may have prognostic signifi‐

cance in prostate cancer and hence has gained wide interest in the past two decades.

benefits and controversies of the different PSA‐related parameters.

In this chapter, we systemically reviewed and appraised the current role of PSA and PSA kinetics in the management of advanced prostate cancer. We further discussed the potential

A systematic search was conducted in the PubMed database through November 2015 using the following terms: 'prostate cancer', 'prostate‐specific antigen', 'prostate‐specific antigen nadir', 'time to prostate‐specific antigen nadir', 'prostatic‐specific antigen doubling time', 'prostate‐specific antigen progression' and 'prostate‐specific antigen response'. Only original full research articles published in English with full‐length text available were reviewed. A manual search using the Web‐based search engine Google Scholar was also performed.

To a certain extent, the pre‐treatment PSA level may reflect the volume of cancer cells, and hence, it is a parameter of interest in predicting disease prognosis. However, it does not reflect the sensitivity of cancer cells in response to subsequent therapy, in particular hormonal therapy in the context of metastatic disease. The prognostic significance of pre‐treatment PSA level is uncertain. Some studies showed that higher pre‐treatment PSA level was associated with disease progression, cancer‐specific mortality and all‐cause mortality [4–14], while other

Reference lists of the retrieved articles were reviewed for other relevant studies.

PSA nadir was defined as the lowest PSA level achieved after the initiation of treatment. An undetectable PSA nadir level reflects that most if not all of the prostate cancer cells are androgen‐sensitive, while any detectable PSA level reflects the presence of androgen‐insensi‐ tive prostate cancer cells. This was supported by a study which showed that patients who had biochemical relapse following 3 months of neoadjuvant androgen deprivation therapy and radical prostatectomy had greater PSA mRNA levels and more intense PSA immunostaining despite castrate levels of testosterone, than patients who did not relapse, yet they had similar levels of androgen receptor gene expression and protein staining [33]. The majority of the literature showed that PSA nadir is consistent in predicting disease prognosis. A higher PSA nadir level has been shown to be associated with biochemical or disease progression [4, 15, 19, 25, 31, 32, 34–37], prostate cancer‐specific mortality [6, 7, 16, 22, 25, 38–40] and all‐cause mortality [6, 15, 16, 24, 26, 29, 30, 41–44]. However, there is no absolute threshold level for PSA nadir being recognized by any regulatory agency, and cut‐off values at 0.2 ng/mL, 1.0 ng/mL and 4.0 ng/mL have been proposed in various studies. In particular, the drop of PSA to < 4.0  ng/mL has commonly been recognized as PSA normalization, and similar to PSA nadir, PSA normalization was associated with better progression‐free survival, cancer‐specific survival and overall survival [35, 39, 43, 44]. However, PSA nadir may be affected by the pre‐treatment PSA level, rendering difficulty in clinical application, and the PSA nadir may only be known after there is a PSA rise in subsequent follow‐up.

#### **4.3. Time to PSA nadir**

Time to PSA nadir was defined as the duration needed for the PSA level to reach its nadir after the initiation of treatment. Upon hormonal therapy, one may expect the PSA level to drop to its nadir within a shorter period of time in case of hormone‐sensitive prostate cancer, but the ability to have sustained continuous suppression over a longer period of time may be as important. The majority of the studies showed that a longer time to PSA nadir was associated with better outcomes including biochemical or disease progression, cancer‐specific survival and overall survival [15, 16, 26, 29, 34, 36]. The other studies either showed the contrary or did not detect any associations between them [6, 22, 23, 31]. Due to the potential immortal time bias, the relationship between time to PSA nadir and survival has to be interpreted with caution [45]. For example, one must have survived 12 months in order to have a time to PSA nadir of 12 months. Hence, this immortal time bias favours a positive correlation between time to PSA nadir and survival outcomes. In order to minimize this potential bias, one study attempted to investigate the prognostic significance of time to PSA nadir using survival beyond time to PSA nadir as an alternative outcome measurement. It has been shown that a longer time to PSA nadir was associated with better survival beyond time to PSA nadir [45]. A longer time to PSA nadir was also shown to be associated with a lower PSA velocity after progression, but whether PSA velocity after progression can be a surrogate for survival is doubtful [46].

#### **4.4. PSA doubling time**

PSA doubling time can generally be interpreted as the time needed for the PSA level to double itself. It assumes an exponential increase in serum PSA and first‐order kinetics and can be calculated by natural logarithm of 2 divided by the slope of the relationship between the logarithm of PSA and time of PSA measurement [47]. However, several other calculation models have been proposed, and there is no standardization in the calculation of PSA doubling time. A shorter PSA doubling time has been shown to predict metastasis after prior radical prostatectomy [27, 28, 47], disease progression [32, 48], prostate cancer‐specific mortality [6, 7, 22, 23, 25, 40, 49–51] and all‐cause mortality [6, 9, 24, 27, 38, 52–54]. The utility of PSA doubling time has been widespread, yet the inconsistencies in the methodologies in calculating PSA doubling time [55] and the complicated logarithm calculations involved limited its use in clinical practice. Small deviations from the different methods of calculations may also lead to wide variations in the calculated PSA doubling time [55]. In 2008, the Prostate Cancer Clinical Trials Working Group (PCWG2) discourages the use of PSA doubling time as the primary endpoint in clinical trials because its significance is uncertain [56]. A subsequent systematic review also concluded that the evidence on PSA doubling time is limited and there is no justification for the use of PSA doubling time to guide decision‐making in subsequent treatment [57].

#### **4.5. PSA progression**

PSA progression is commonly used as an endpoint in clinical trials, and it was generally thought to represent disease progression and hence reflect the survival outcomes. However, in particular for metastatic disease, multiple definitions of PSA progression have been proposed; they rendered difficulties comparing the results between different studies and limited the generalization of the utility of PSA progression. In 1999, Prostate‐specific Antigen Working Group (PCWG1) made consensus recommendations for different outcome measures in clinical trials in prostate cancer [58]. PCWG1 defined PSA progression as a >50% increase from nadir and an increase of at least 5 ng/mL, or back to baseline, whichever was lowest. In 2008, PCWG2 proposed another definition for PSA progression, recognizing that early changes in PSA should not be used for clinical decision‐making [56]. For those with PSA decline from baseline, PSA progression was defined as an increase in PSA by ≥25% and ≥2 ng/mL above the nadir, which should be confirmed by a second value 3 or more weeks later; for those with no PSA decline, PSA progression was defined as an increase in PSA ≥25% and ≥2 ng/mL after 12 weeks. Hussain et al. [44] reviewed the data from two large‐scale clinical trials, namely the Southwest Oncology Group (SWOG) 9346 trial on intermittent ADT and the SWOG 9916 trial on docetaxel. It was shown that both PCWG1 and PCWG2 definitions of PSA progression predicted a 2.4‐fold increase in risk of death and a more than 4‐fold increase in the risk of death if PSA progression occurred in the first 7 months. This important study demonstrated that PSA progression is a significant predictor of survival in patients who have newly diagnosed metastatic hormone‐sensitive prostate cancer as well as in those with castration resistant prostate cancer treated with chemotherapy. The authors suggested that the PCWG2 definition might be more appealing as patients are identified with progression relatively earlier on. Pooling data from 9 cancer and leukaemia Group B trials [11], both PCWG1 and PCWG2 definitions of PSA progression were shown to be significant predictors of overall survival with hazard ratios of 1.44 (95% CI 1.28–1.62, *P* < 0.001) and 1.43 (95% CI 1.27–1.61, *P* < 0.001), respectively. The above evidence formed the basis of using PSA progression as the primary endpoint in various clinical trials.

#### **4.6. PSA response**

nadir was also shown to be associated with a lower PSA velocity after progression, but whether

PSA doubling time can generally be interpreted as the time needed for the PSA level to double itself. It assumes an exponential increase in serum PSA and first‐order kinetics and can be calculated by natural logarithm of 2 divided by the slope of the relationship between the logarithm of PSA and time of PSA measurement [47]. However, several other calculation models have been proposed, and there is no standardization in the calculation of PSA doubling time. A shorter PSA doubling time has been shown to predict metastasis after prior radical prostatectomy [27, 28, 47], disease progression [32, 48], prostate cancer‐specific mortality [6, 7, 22, 23, 25, 40, 49–51] and all‐cause mortality [6, 9, 24, 27, 38, 52–54]. The utility of PSA doubling time has been widespread, yet the inconsistencies in the methodologies in calculating PSA doubling time [55] and the complicated logarithm calculations involved limited its use in clinical practice. Small deviations from the different methods of calculations may also lead to wide variations in the calculated PSA doubling time [55]. In 2008, the Prostate Cancer Clinical Trials Working Group (PCWG2) discourages the use of PSA doubling time as the primary endpoint in clinical trials because its significance is uncertain [56]. A subsequent systematic review also concluded that the evidence on PSA doubling time is limited and there is no justification for the use of PSA doubling time to guide decision‐making in subsequent

PSA progression is commonly used as an endpoint in clinical trials, and it was generally thought to represent disease progression and hence reflect the survival outcomes. However, in particular for metastatic disease, multiple definitions of PSA progression have been proposed; they rendered difficulties comparing the results between different studies and limited the generalization of the utility of PSA progression. In 1999, Prostate‐specific Antigen Working Group (PCWG1) made consensus recommendations for different outcome measures in clinical trials in prostate cancer [58]. PCWG1 defined PSA progression as a >50% increase from nadir and an increase of at least 5 ng/mL, or back to baseline, whichever was lowest. In 2008, PCWG2 proposed another definition for PSA progression, recognizing that early changes in PSA should not be used for clinical decision‐making [56]. For those with PSA decline from baseline, PSA progression was defined as an increase in PSA by ≥25% and ≥2 ng/mL above the nadir, which should be confirmed by a second value 3 or more weeks later; for those with no PSA decline, PSA progression was defined as an increase in PSA ≥25% and ≥2 ng/mL after 12 weeks. Hussain et al. [44] reviewed the data from two large‐scale clinical trials, namely the Southwest Oncology Group (SWOG) 9346 trial on intermittent ADT and the SWOG 9916 trial on docetaxel. It was shown that both PCWG1 and PCWG2 definitions of PSA progression predicted a 2.4‐fold increase in risk of death and a more than 4‐fold increase in the risk of death if PSA progression occurred in the first 7 months. This important study demonstrated that PSA progression is a significant predictor of survival in patients who have newly diagnosed

PSA velocity after progression can be a surrogate for survival is doubtful [46].

26 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

**4.4. PSA doubling time**

treatment [57].

**4.5. PSA progression**

PSA response was determined by the degree of decline from its pre‐treatment level. The PCWG1 [58] defined PSA response as a decline of >50% from baseline, measured twice 3–4 weeks apart. Several studies have shown that a PSA decline of >50% was associated with better cancer‐specific survival [39, 59] and overall survival [17, 18, 60–62]. However, post hoc analyses in both SWOG 9916 [63] and TAX 327 [43] trials on the use of docetaxel showed that a PSA decline of >30% might be a better surrogate marker for survival than a PSA decline of >50% based on the proportion of treatment effect and the proportion of variation. A subsequent combined analysis on the SWOG 9346 and SWOG 9916 trials [44] showed that a PSA decline of >30% was associated with better overall survival. However, PCWG2 [56] advised against reporting PSA response rates in clinical trials. Concerns were raised about the strength of association between PSA decline and survival, and no criterion, be it >50% or >30% decline in PSA, has been shown prospectively to be a surrogate of clinical benefit [64]. Instead, PCWG2 recommended the use of waterfall plot to provide a broader and more sensitive display of data. On the other hand, following the discovery of AR‐V7 splice variant [65], it was proposed that the lack of PSA response after initial hormonal manipulation might represent primary resistance to hormonal therapy. This is particularly important, as other non‐hormonal treatment such as chemotherapy should be considered early on, based on the prediction of poor response to further hormonal manipulation.

#### **5. Conclusions**

PSA and PSA kinetics may provide additional information about the biological behaviour of prostate cancer and may aid the treatment decision in an individualized approach. One should be aware of the pros and cons of the different PSA‐related parameters and should be cautious when interpreting the results from different studies. PSA and its kinetics should play an important role in the management of advanced prostate cancer, and generalization can only be achieved if definitions of the different parameters can be utilized in a more standardized manner. Among the different parameters discussed, PSA progression appeared to be the most consistent and reliable surrogate marker of survival and can serve as the primary endpoint in future clinical trials.

#### **Author details**

Jeremy Teoh1,2\* and Ming‐Kwong Yiu1,2

\*Address all correspondence to: jeremyteoh@surgery.cuhk.edu.hk

1 Division of Urology, Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China

2 Division of Urology, Department of Surgery, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China

#### **References**


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**Author details**

Jeremy Teoh1,2\* and Ming‐Kwong Yiu1,2

28 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

University of Hong Kong, Hong Kong, China

Hong Kong, Hong Kong, China

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1 Division of Urology, Department of Surgery, Prince of Wales Hospital, The Chinese

2 Division of Urology, Department of Surgery, Queen Mary Hospital, The University of

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[49] D'Amico AV, Halabi S, Tempany C, Titelbaum D, Philips GK, Loffredo M, et al. Tumor volume changes on 1.5 tesla endorectal MRI during neoadjuvant androgen suppression therapy for higher‐risk prostate cancer and recurrence in men treated using radiation therapy results of the phase II CALGB 9682 study. Int J Radiat Oncol Biol Phys 2008;

[50] Freedland SJ, Humphreys EB, Mangold LA, Eisenberger M, Dorey FJ, Walsh PC, et al. Risk of prostate cancer‐specific mortality following biochemical recurrence after radical

[51] Svatek RS, Shulman M, Choudhary PK, Benaim E. Critical analysis of prostate‐specific antigen doubling time calculation methodology. Cancer 2006; 106:1047–53.

androgen deprivation therapy. Ann Surg Oncol 2015; 22:1385–91.

therapy. Asian J Androl 2015. (Epub ahead of print).

refractory prostate cancer. J Clin Oncol 2007; 25:3965–70.

0162) and 9916. J Clin Oncol 2009; 27:2450–6.

(Epub ahead of print).

32 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

281:1591–7.

7.

71:9–15.

prostatectomy. JAMA 2005; 294:433–9.


**Chapter 4**

**Transperineal Targeted Biopsy with Real-Time Fusion Image of Multiparametric Magnetic Resonance Image and Transrectal Ultrasound Image for the Diagnosis of Prostate Cancer**

#### Sunao Shoji

[63] Petrylak DP, Ankerst DP, Jiang CS, Tangen CM, Hussain MH, Lara PN, Jr., et al. Evaluation of prostate‐specific antigen declines for surrogacy in patients treated on

[64] Scher HI, Morris MJ, Basch E, Heller G. End points and outcomes in castration‐resistant prostate cancer: from clinical trials to clinical practice. J Clin Oncol 2011; 29:3695–704.

[65] Antonarakis ES, Lu C, Wang H, Luber B, Nakazawa M, Roeser JC, et al. AR‐V7 and resistance to enzalutamide and abiraterone in prostate cancer. N Engl J Med 2014;

SWOG 99‐16. J Natl Cancer Inst 2006; 98:516–21.

34 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

371:1028–38.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64603

#### **Abstract**

**Objectives**: To report clinical results of early experience of manually controlled targeted biopsy with real-time multiparametric magnetic resonance image (mpMRI)-transrec‐ tal ultrasound (TRUS) fusion images for the diagnosis of prostate cancer.

**Methods**: One hundred sixty-eight patients who were suspected of prostate cancer from mpMRI scans were recruited prospectively. We performed targeted biopsies for each cancer-suspicious lesion and 12 systematic biopsies using the BioJet® system. Patho‐ logical findings of targeted and systematic biopsies were analyzed.

**Results**: Median age of the 168 patients was 67 years (range: 52–89). Median preopera‐ tive prostate specific antigen (PSA) value was 6.9 ng/ml (range: 3.54–20). Median preoperative prostate volume was 37 ml (range: 22–68). The number of the cancerdetected cases was 99 (59%). The median biopsy time, included the MRI-TRUS fusion time and needle-punctured time without the anesthesia, was 8 minutes (range: 5–65). Cancer-detected rates of the systematic and targeted biopsy cores were 5.9 and 38%, respectively (*p* < 0.0001). In 25 patients who underwent radical prostatectomy, the geographic locations and pathological grades of clinically significant cancers and index lesions corresponded to the pathological results of the targeted biopsies.

**Conclusion**: The cancer cores detected by targeted biopsies with manually controlled targeted biopsy with real-time mpMRI-TRUS fusion image had significantly higher grades and larger length compared with those detected by the systematic biopsies. The further study of the comparisons with pathological findings of whole-gland speci‐ mens will give a larger role to the present biopsy method.

**Keywords:** prostate cancer, targeted biopsy, magnetic resonance image, transrectal ul‐ trasound, fusion image

© 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**

Multiparametric magnetic resonance imaging (mpMRI) improves the imaging of prostate cancer lesion [1, 2], and several methods use MRI to guide the biopsy needle to target the cancer lesion. MRI-TRUS fusion image-guided biopsy achieved accurate prostate biopsy based on MRI, combining the superior sensitivity of MRI for targeting suspicious lesions with the practicality and familiarity of TRUS. MRI-TRUS fusion methods are used as visual registration [1, 3, 4] and fusion biopsy devices [5–9]. In visual registration, the TRUS operator identified the geographic location of the lesions in the prostate on the MRI, and then identify and biopsy viewing real-time TRUS [10]. In previous reports, the visual registration biopsy method improved accuracy over systematic biopsy [11–14]. However, the disadvantages of visual registration lie in human error when the targeted lesion was less than 10 mm in diameter [15]. Therefore, the visual registration is regarded as the prostate biopsy method for experts [10– 14]. With the MRI-TRUS fusion devices, the stored MRI and real-time TRUS are superimposed using computer software to enable targeted biopsy of cancer-suspicious lesions [16]. MRI-TRUS fusion biopsy device "BioJet®" was approved by FDA after the evaluation of the accuracy with phantoms. We report the BioJet® experience of the manually controlled targeted biopsy using real-time fusion image from mpMRI and TRUS.

#### **2. Methods**

#### **2.1. Population**

From November 2013 to October 2015, after receiving the approval of institutional review board, the patients with PSA level greater than 4.0 ng/ml and less than 20 ng/ml were per‐ formed mpMRI prospectively. No patients had any previous history of prostate biopsy.

#### **2.2. Multiparametric MRI**

The MRI examination was carried out using a 1.5-Tesla magnet (Signa HDx®; GE Healthcare, Amersham Place, UK) with an 8-channel cardiac coil. T1-weighted fat-saturated axial fast spinecho images (TR, 450 ms; TE, 8.8 ms; slice thickness, 3 mm; resolution, 0.9 × 1.3 mm) were obtained before injection. An intravenous bolus of 0.2 ml/kg of meglumine gadopentetate (Magnevist Syringe®; Bayer HealthCare Pharmaceuticals, Berlin, Germany) was then injected. All MRI examinations were performed using the same protocol, and included non-enhanced T2-weighted images (T2WI) (TR, 5000 ms; TE, 125 ms; slice thickness, 3 mm; resolution, 0.6 × 0.9 mm) acquired in the axial and sagittal planes, diffusion weighted image (DWI) and apparent diffusion coefficient (ADC) maps (b-value = 1500 s/mm2 ), and dynamic-contrastenhanced (DCE) MRI (resolution, 0.9 × 1.3 mm) using a fat-saturated T1-weighted fast-field echo sequence in the axial plane.

#### **2.3. Image analysis**

**1. Introduction**

36 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

**2. Methods**

**2.1. Population**

**2.2. Multiparametric MRI**

echo sequence in the axial plane.

Multiparametric magnetic resonance imaging (mpMRI) improves the imaging of prostate cancer lesion [1, 2], and several methods use MRI to guide the biopsy needle to target the cancer lesion. MRI-TRUS fusion image-guided biopsy achieved accurate prostate biopsy based on MRI, combining the superior sensitivity of MRI for targeting suspicious lesions with the practicality and familiarity of TRUS. MRI-TRUS fusion methods are used as visual registration [1, 3, 4] and fusion biopsy devices [5–9]. In visual registration, the TRUS operator identified the geographic location of the lesions in the prostate on the MRI, and then identify and biopsy viewing real-time TRUS [10]. In previous reports, the visual registration biopsy method improved accuracy over systematic biopsy [11–14]. However, the disadvantages of visual registration lie in human error when the targeted lesion was less than 10 mm in diameter [15]. Therefore, the visual registration is regarded as the prostate biopsy method for experts [10– 14]. With the MRI-TRUS fusion devices, the stored MRI and real-time TRUS are superimposed using computer software to enable targeted biopsy of cancer-suspicious lesions [16]. MRI-TRUS fusion biopsy device "BioJet®" was approved by FDA after the evaluation of the accuracy with phantoms. We report the BioJet® experience of the manually controlled targeted

From November 2013 to October 2015, after receiving the approval of institutional review board, the patients with PSA level greater than 4.0 ng/ml and less than 20 ng/ml were per‐ formed mpMRI prospectively. No patients had any previous history of prostate biopsy.

The MRI examination was carried out using a 1.5-Tesla magnet (Signa HDx®; GE Healthcare, Amersham Place, UK) with an 8-channel cardiac coil. T1-weighted fat-saturated axial fast spinecho images (TR, 450 ms; TE, 8.8 ms; slice thickness, 3 mm; resolution, 0.9 × 1.3 mm) were obtained before injection. An intravenous bolus of 0.2 ml/kg of meglumine gadopentetate (Magnevist Syringe®; Bayer HealthCare Pharmaceuticals, Berlin, Germany) was then injected. All MRI examinations were performed using the same protocol, and included non-enhanced T2-weighted images (T2WI) (TR, 5000 ms; TE, 125 ms; slice thickness, 3 mm; resolution, 0.6 × 0.9 mm) acquired in the axial and sagittal planes, diffusion weighted image (DWI) and

enhanced (DCE) MRI (resolution, 0.9 × 1.3 mm) using a fat-saturated T1-weighted fast-field

), and dynamic-contrast-

biopsy using real-time fusion image from mpMRI and TRUS.

apparent diffusion coefficient (ADC) maps (b-value = 1500 s/mm2

All mpMRI images, including T2WI, dynamic, DWI, and ADC map, were reviewed by two experienced radiologists with no prior clinical information. Suspicious areas, the so-called "regions of interest (ROI)", were provided a likelihood score that clinically significant cancer would be present for each ROI from 2 to 5 on the prostate imaging reporting and data system (PI-RAS) classification [17] based on Likert scale according to the European Society of Uro‐ genital Radiology Prostate MR Guidelines 2012 [18]: 1, most probably benign; 2, probably benign; 3, intermediate; 4, probably malignant; and 5, highly suspicious of malignancy [17] The location of each area was determined based on dividing the prostate into 27 regions, as described by Dickson et al. [16]. MRIs were imported into the biopsy fusion system. Segmen‐ tation into a two-dimensional (2D) mpMRI was performed on the workstation to create a 3D model of the MRI, and then fused to the real-time TRUS.

#### **2.4. Biopsy protocol**

A cleaning enema and antibiotics were given before the biopsy. TRUS with power Doppler was performed using a Prosound α7 (Hitachi Aloka Medical, Tokyo, Japan) equipped with a UST-678 transrectum composite probe, in the lithotomy position under spinal anesthesia. On the workstation, the operator fused the real-time TRUS image and 3D MRI model that included the prostate contour and ROI. After the elastic image fusion, an ultrasound probe was fixed to the arm that senses the 3D movement of the probe and exports the information to the work‐ station (**Figure 1a**). Using this device, the 2D image created from the 3D MRI model moves together with the real-time TRUS image on the workstation. The operator performed the biopsy using MRI-TRUS fusion image navigation (**Figure 1b**). During the procedure, the realtime ultrasound image is continuously available. The biopsy started with targeted biopsies to the center of cancer-suspicious lesions, and then 12 systematic biopsies were performed with transperineal technique in all patients. The biopsy used a standard brachytherapy grid with 5-mm spacing, with *x*-axis coordinates A through G and *y*-axis coordinates from 1 through 7, using D as the middle line urethral plane. An 18-gauge automatic biopsy gun with a specimen size of 22 mm (BARD® MAGNUM®, BARD MEDICAL, Covington, USA) was used to take biopsy cores. Using the interactive needle guide system, the biopsy template coordinates were shown on the monitor when the operator marked the target point of the ROI on the workstation (**Figure 2a, b**). The operator inserted the needle at the template coordinates and could get the prostate specimens by viewing the sagittal image of the prostate (**Figure 2c**). Immediately after each biopsy, the spatial punctured needle orbits were recorded in 2D TRUS image of axial and sagittal plane, and in the 3D model of MRI.

#### **2.5. Pathological analysis**

All biopsies were examined by expert pathologists. A significant cancer was defined as follows: at least one core with a Gleason score of 3 + 4 or 6 with a maximum cancer core length larger than 4 mm [19]. The pathological biopsy results were compared between systematic and targeted biopsies. The biopsy-proven index lesion of each patient was defined primarily as the lesion with the highest Gleason score, and secondarily as the lesion with the greatest cancerinvolved core in terms of length or percentage. Geographic location of prostate cancer in the prostate [16] was compared with pathologic step-sectioned prostatectomy specimens in the patients who were performed with radical prostatectomy.

**Figure 1.** (a) BioJet system (D&K Technologies GmbH, Barum, Germany); (b) the set-up of prostate biopsy with BioJet system.

**Figure 2.** Process of prostate biopsy with BioJet system. (a) Fusion image from MRI and TRUS image. (b) Interactive needle guide system. (c) Real-time fusion images of axial and sagittal image.

#### **2.6. Statistical analysis**

involved core in terms of length or percentage. Geographic location of prostate cancer in the prostate [16] was compared with pathologic step-sectioned prostatectomy specimens in the

**Figure 1.** (a) BioJet system (D&K Technologies GmbH, Barum, Germany); (b) the set-up of prostate biopsy with BioJet

**Figure 2.** Process of prostate biopsy with BioJet system. (a) Fusion image from MRI and TRUS image. (b) Interactive

needle guide system. (c) Real-time fusion images of axial and sagittal image.

patients who were performed with radical prostatectomy.

38 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

system.

All statistical analyses were performed using IBM SPSS® Statistics version 19 (IBM, Ar‐ monk, NY, USA). Among systematic and targeted biopsies, cancer-detected rate of biopsy, positive core length, positive core percentage, primary and secondary Gleason grade, and Gleason score were analyzed using the Mann-Whitney U-test. Changes in patient functional data were analyzed using paired *t*-tests. *P*-values of <0.05 were considered to indicate statis‐ tically significant differences.

#### **3. Results**

One-hundred sixty eight patients were suspected of prostate cancer with 2 to 5 of PI-RAD classification. The median age of the 168 patients was 67 years (range: 52–89). The median preoperative PSA value was 6.9 ng/ml (range: 3.54–20). The median preoperative prostate volume was 37 ml (range: 22–68). In the resected prostate specimen of 25 patients, the geo‐ graphic locations and pathological grades of clinically significant cancers and index lesions corresponded to the results of the targeted biopsies.

The results of the prostate biopsies are shown in **Table 1**. The number of the cancer-detected cases was 99 (59%). The median biopsy time included the MRI-TRUS fusion time and needlepunctured time without the anesthesia, which was 8 minutes (range: 5–65). For the systematic and targeted biopsy cores, the total number of cores were 2016 and 372, respectively; the cancerdetected rates, the median positive core lengths, the median positive core percents, the median primary Gleason grades, the median secondary Gleason grades, and the median Gleason scores in systematic and targeted biopsy cores were significantly different.


**Table 1.** Biopsy results.

In targeted lesions of transition zone (TZ) (*n* = 146) and peripheral zone (PZ) (*n* = 226), the rate of cancer detection was 28% (*n* = 40) and 45% (*n* = 101), respectively. The rates of cancer detection and the corresponding scores on the PI-RAD in TZ and PZ are shown in **Table 2**.


TZ, transition zone; PZ, peripheral zone; PI-RADS, prostate imaging and reporting data system.

**Table 2.** The rates of cancer detection and the corresponding scores on the PI-RAD in transition zone and peripheral zone.

#### **4. Discussion**

Our results showed that cancer detection rates using targeted biopsies were significantly better than using systematic biopsies (*p* < 0.0001). Positive core length (*p* < 0.0001), positive core percent (*p* < 0.0001), primary (*p* < 0.0001) and secondary (*p* = 0.0020) Gleason grade, and Gleason score (*p* = 0.0012) were also significantly different between targeted and systematic biopsies. In addition, all biopsy-proven significant cancers were detected in ROIs, and the index lesions corresponded to the largest-sized ROIs. Based on these results, the targeted biopsy method was superior to systematic biopsy, and clinically significant cancers with a spatial relationship were detected accurately in the present study. Although the resected prostate specimens only comprised 25 cases, accuracy of the locations and pathological grades was reliable in our study.

In the present study, we used the T2WI for segmentation of the ROI, but the decision con‐ cerning the selection of ROI was made using multiparametric MRI factors, such as T2WI, DCE, DWI, and ADC maps because T2WI is sensitive but not specific for prostate cancer detection [1]. In mpMRI, the image values of its component techniques are different. T2WI provides the best depiction of the prostate's zonal anatomy and capsule in mpMRI and thus is used for prostate cancer detection and localization [18]. DCE is the most common imaging method for evaluating vascularity in the tumor [20]. DWI involves the quantification of free water motion [21] and allows ADC maps to be calculated, enabling qualitative and quantitative assessment of prostate cancer aggressiveness. Lower ADC corresponds to greater restriction in free water motion, likely on the basis of increased cellularity compared with normal prostate tissue, and cancer shows a lower ADC value than normal prostate tissue [21]. Furthermore, ADC values correlate with Gleason scores [22–24]. However, some normal prostatic tissues, especially in the TZ, such as benign prostatic hyperplasia, chronic inflammation, and atrophic tissue, have similar findings of prostate cancer [16]. Indeed, the detection of prostate cancer in TZ was found difficulty in a previous study [23]. In our results, the cancer detection rate of the patients with a PI-RAD classification of 4 or 5 in TZ (39%) was inferior to that in PZ (92%).

The present device allows manually controlled targeted biopsy using real-time MRI-TRUS fusion images by the sensor arm of 3D movement. In addition, the fusion function has elastic fusion functions. The axial and sagittal view of US and MRI was useful to fuse the images of MRI and TRUS easily during the procedure. In addition, the present biopsy was performed with transperineal technique. Using the transperineal technique with the device, the biopsies were performed accurately to the ROIs. However, our study has limitations. First, our study did not compare biopsy results with pathological findings from whole-gland specimens. Therefore, although locations and pathological grades of clinically significant cancers and index lesions corresponded to the targeted biopsy results, it is difficult to exclude the possi‐ bility that a clinically important cancer has been missed without pathological analysis of whole-gland specimens.

In conclusion, cancers detected by targeted biopsies using manual controlled targeted biopsy with real-time fusion image of mpMRI and TRUS had a significantly higher grade and larger length compared with systematic biopsies. In present study, the cancer detection rate in TZ was significantly lower than in PZ. However, further study would contribute to set the cutoff point of PI-RADS scores in TZ and PZ to detect the prostate cancer at high frequency. The further study of the comparisons with pathological findings of whole-gland specimens will give a larger role to the present biopsy method.

#### **Author details**

Sunao Shoji

**No. of target**

zone.

**4. Discussion**

**PI-RADS classification**

40 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

**Rates of cancer detection**

3 (*n* = 126) 13% (*n* = 16) 10% (*n* = 13) 4 (*n* = 110) 61% (*n* = 67) 58% (*n* = 64) 5 (*n* = 71) 77% (*n* = 55) 77% (*n* = 55)

3 (*n* = 40) 15% (*n* = 6) 10% (*n* = 4) 4 (*n* = 48) 24% (*n* = 12) 21% (*n* = 10) 5 (*n* = 35) 56% (*n* = 20) 56% (*n* = 20)

3 (*n* = 86) 12% (*n* = 10) 11% (*n* = 9) 4 (*n* = 62) 88% (*n* = 55) 87% (*n* = 54) 5 (*n* = 36) 97% (*n* = 35) 97% (*n* = 35)

**Table 2.** The rates of cancer detection and the corresponding scores on the PI-RAD in transition zone and peripheral

Our results showed that cancer detection rates using targeted biopsies were significantly better than using systematic biopsies (*p* < 0.0001). Positive core length (*p* < 0.0001), positive core percent (*p* < 0.0001), primary (*p* < 0.0001) and secondary (*p* = 0.0020) Gleason grade, and Gleason score (*p* = 0.0012) were also significantly different between targeted and systematic biopsies. In addition, all biopsy-proven significant cancers were detected in ROIs, and the index lesions corresponded to the largest-sized ROIs. Based on these results, the targeted biopsy method was superior to systematic biopsy, and clinically significant cancers with a spatial relationship were detected accurately in the present study. Although the resected prostate specimens only comprised 25 cases, accuracy of the locations and pathological grades was reliable in our study. In the present study, we used the T2WI for segmentation of the ROI, but the decision con‐ cerning the selection of ROI was made using multiparametric MRI factors, such as T2WI, DCE, DWI, and ADC maps because T2WI is sensitive but not specific for prostate cancer detection [1]. In mpMRI, the image values of its component techniques are different. T2WI provides the best depiction of the prostate's zonal anatomy and capsule in mpMRI and thus is used for prostate cancer detection and localization [18]. DCE is the most common imaging method for evaluating vascularity in the tumor [20]. DWI involves the quantification of free water motion [21] and allows ADC maps to be calculated, enabling qualitative and quantitative assessment of prostate cancer aggressiveness. Lower ADC corresponds to greater restriction in free water

**TZ + PZ** 372 2 (*n* = 70) 38% (*n* = 141) 4.3% (*n* = 3) 0% (*n* = 0)

**TZ** 146 2 (*n* = 28) 28% (*n* = 40) 7.1% (*n* = 2) 0% (*n* = 0)

**PZ** 226 2 (*n* = 42) 45% (*n* = 101) 2.4% (*n* = 1) 0% (*n* = 0)

TZ, transition zone; PZ, peripheral zone; PI-RADS, prostate imaging and reporting data system.

**Rates of significant cancer detection**

Address all correspondence to: sunashoj@mail.goo.ne.jp

Department of Urology, Tokai University Hachioji Hospital, Hachioji, Tokyo, Japan

#### **References**

[1] Moore CM, Kasivisvanathan V, Eggener S, et al. Standards of reporting for MRItargeted biopsy studies (START) of the prostate: recommendations from an Interna‐ tional Working Group. European Urology. 2013;64(4):544–552.


men with prior negative biopsy. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research. 2010;16(6):1875–1883.

[14] Labanaris AP, Engelhard K, Zugor V, Nutzel R, Kuhn R. Prostate cancer detection using an extended prostate biopsy schema in combination with additional targeted cores from suspicious images in conventional and functional endorectal magnetic resonance imaging of the prostate. Prostate Cancer and Prostatic Diseases. 2010;13(1):65–70.

[2] Sciarra A, Barentsz J, Bjartell A, et al. Advances in magnetic resonance imaging: how they are changing the management of prostate cancer. European Urology. 2011;59(6):

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[5] Natarajan S, Marks LS, Margolis DJ, et al. Clinical application of a 3D ultrasound-

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[7] Mozer P, Roupret M, Le Cossec C, et al. First round of targeted biopsies with magnetic resonance imaging/ultrasound-fusion images compared to conventional ultrasoundguided trans-rectal biopsies for the diagnosis of localised prostate cancer. BJU Inter‐

[8] Wysock JS, Rosenkrantz AB, Huang WC, et al. A prospective, blinded comparison of magnetic resonance (MR) imaging-ultrasound fusion and visual estimation in the performance of MR-targeted prostate biopsy: the PROFUS Trial. European Urology.

[9] Marks L, Young S, Natarajan S. MRI-ultrasound fusion for guidance of targeted

[10] Moore CM, Robertson NL, Arsanious N, et al. Image-guided prostate biopsy using magnetic resonance imaging-derived targets: a systematic review. European Urology.

[11] Haffner J, Lemaitre L, Puech P, et al. Role of magnetic resonance imaging before initial biopsy: comparison of magnetic resonance imaging-targeted and systematic biopsy for significant prostate cancer detection. BJU International. 2011;108(8 Pt 2):E171–E178.

[12] Park BK, Park JW, Park SY, et al. Prospective evaluation of 3-T MRI performed before initial transrectal ultrasound-guided prostate biopsy in patients with high prostatespecific antigen and no previous biopsy. AJR American Journal of Roentgenology.

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### **Oligometastatic Disease in Prostate Cancer: Advances in Diagnosis and Treatment**

Weranja Ranasinghe and Raj Persad

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63370

#### **Abstract**

Prostate cancer (PC) is the second most common cancer in men and the fifth leading cause of death in men worldwide in 2012 [1]. Oligometastatic disease is defined as the presence of five or fewer metastatic or recurrent lesions that could be treated by local therapy to achieve long‐term survival or cure [2]. Androgen deprivation therapy is currently the accepted treatment of metastatic PC. However, the identification of oligometastatic disease in PC with the improvements in diagnostic imaging has lead to early treatment of these isolated metastases showing some benefit [3]. In this chapter, we aim to discuss the newer modalities used in the identification of oligometastatic disease in PC and the advances in treatment.

**Keywords:** Oligometastases, prostate cancer, diagnosis, treatment

#### **1. Introduction**

Although oligometastases forms a recent vogue in prostate cancer, the concept of 'oligometa‐ stases' was originally described by Hellman and Weichselbaum in 1995 [4]. They theorised that metastases occurred as a 'metastatic progression' from localised disease to widespread systemic disease [5]. As such, in some patients with limited metastases, they described an 'oligometa‐ static state' which occurs as a transitional state between localised and systemic disease [5]. Therefore, rather than classifying all metastatic prostate cancer in to a universal cohort with poor outcomes, this defined a group of patients who could be identified and treated with potentially favourable results.

© 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.

#### **1.1. Definitions**

The nomenclature in 'oligometastases' is often used inter changeably and can be sometimes confusing. The term 'oligometastasis' usually refers to metastases (from tumours early in the chain of progression) limited in number and location because the facility for metastatic growth has not been fully developed and the site for growth is restricted, while 'oligometastatic disease' is defined as solitary or few detectable metastatic lesions (<5 metastases) that are usually confined to a single organ [5]. Although sometimes oligometastases can refer to synchronous or metachronous disease, it should be stressed that the key feature determining the behaviour of oligometastases is its metastatic potential. As such, 'true oligometastases' are defined as oligometastases with limited metastatic potential, while 'induced oligometastases' occur following successful systemic treatment have more extensive malignant capacities and were spared from eradication by pharmacological means, local immunological conditions or from the development of resistant clones [6].

In prostate cancer, induced oligometastases can be further divided into those with a rising PSA following primary therapy who has oligometastases on imaging or those with castrate‐ resistant prostate cancer (CRPC) with a rising PSA level and image‐detected oligometastases [7].

#### **2. The evidence for treatment of oligometastases**

Treatment of liver metastases in colorectal cancer, lung metastases from a variety of cancers and adrenal metastases in lung cancer have demonstrated in improved survival and in some cases even cure; forming the basis of treating oligometastases in cancers [6]. Currently, androgen deprivation therapy is the optimal treatment for widespread metastatic prostate cancer. Studies have demonstrated that those men on androgen deprivation therapy for ≤3 metastases had much superior outcomes compared to those with larger number of metastases [8, 9]. A further study demonstrated that men with prostate cancer who developed ≤5 meta‐ static sites had better survival than those with >5 lesions [10]. With the recent shift, the landscape of prostate cancer diagnostics and treatments has changed significantly offering the opportunity to accurately identify and treats the oligometastases. Treatment of oligometasta‐ ses in prostate cancer can offer better local cancer control and reduce the systematic metastatic potential and its complications by reducing seeding of established metastases control of the overall disease burden and perhaps even cure [7]. In addition, the treatment of oligometastatic disease in prostate cancer delays the need for androgen deprivation and its associated systemic side effects.

#### **3. Biology of oligometastases**

As described in Paget's 'seed and soil' hypothesis, metastases occur due to an interaction between the tumour cell and the targeted organ, which supports the secondary growth of the primary tumour cells [6, 11]. This is a complex and selective process which promotes tumour growth by tumour diversity due to the genetic instability of the tumour cells due to the telomere erosion, mutations in tumour‐suppressor and DNA‐repair genes, and intrinsic tumour metabolism (aerobic glycolysis) that is toxic to surrounding normal cell and suppres‐ sion of the host immunity [6]. A number of genes contribute to this metastatic process such as metastasis 'initiation' genes; metastasis 'progression' genes and metastasis 'virulence' genes by altering cell adhesion, intravasation, survival in the circulation, extravasation, seeding in a distant site, invasion, and development of the appropriate microenvironment in host organs and provides a selective advantage of the primary tumour cells to be preserved and amplified during tumour progression [6]. As such, these primary tumour cells that have limited capa‐ bility in one or more of the necessary biological requirements for metastasis form the basis of oligometastases [6].

**1.1. Definitions**

[7].

side effects.

**3. Biology of oligometastases**

from the development of resistant clones [6].

46 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

**2. The evidence for treatment of oligometastases**

The nomenclature in 'oligometastases' is often used inter changeably and can be sometimes confusing. The term 'oligometastasis' usually refers to metastases (from tumours early in the chain of progression) limited in number and location because the facility for metastatic growth has not been fully developed and the site for growth is restricted, while 'oligometastatic disease' is defined as solitary or few detectable metastatic lesions (<5 metastases) that are usually confined to a single organ [5]. Although sometimes oligometastases can refer to synchronous or metachronous disease, it should be stressed that the key feature determining the behaviour of oligometastases is its metastatic potential. As such, 'true oligometastases' are defined as oligometastases with limited metastatic potential, while 'induced oligometastases' occur following successful systemic treatment have more extensive malignant capacities and were spared from eradication by pharmacological means, local immunological conditions or

In prostate cancer, induced oligometastases can be further divided into those with a rising PSA following primary therapy who has oligometastases on imaging or those with castrate‐ resistant prostate cancer (CRPC) with a rising PSA level and image‐detected oligometastases

Treatment of liver metastases in colorectal cancer, lung metastases from a variety of cancers and adrenal metastases in lung cancer have demonstrated in improved survival and in some cases even cure; forming the basis of treating oligometastases in cancers [6]. Currently, androgen deprivation therapy is the optimal treatment for widespread metastatic prostate cancer. Studies have demonstrated that those men on androgen deprivation therapy for ≤3 metastases had much superior outcomes compared to those with larger number of metastases [8, 9]. A further study demonstrated that men with prostate cancer who developed ≤5 meta‐ static sites had better survival than those with >5 lesions [10]. With the recent shift, the landscape of prostate cancer diagnostics and treatments has changed significantly offering the opportunity to accurately identify and treats the oligometastases. Treatment of oligometasta‐ ses in prostate cancer can offer better local cancer control and reduce the systematic metastatic potential and its complications by reducing seeding of established metastases control of the overall disease burden and perhaps even cure [7]. In addition, the treatment of oligometastatic disease in prostate cancer delays the need for androgen deprivation and its associated systemic

As described in Paget's 'seed and soil' hypothesis, metastases occur due to an interaction between the tumour cell and the targeted organ, which supports the secondary growth of the

### **4. Advances in imaging modalities: identification of oligometastases in prostate cancer**

#### **4.1. The conventional modalities: computed tomography (CT) and skeletal scintigraphy (99MTc‐MDP bone scan)**

CT of the abdomen and pelvis forms the main modality of staging patients with intermediate or high‐risk disease generating valuable information of local advancement, lymph node and bony involvement of prostate cancer [12] (**Figure 1**). Studies have demonstrated its specificity and positive predictive value up to 100%, but its sensitivity remains poor [13]. As such, CT is gradually being superseded by MRI and the combination PET/CT in recurrent prostate cancer and oligometastatic disease.

**Figure 1.** The CT scan of the abdomen and pelvis demonstrates a pelvic lymph node denoted by the white arrow.

**Figure 2.** The isotope bone scan demonstrates uptake at the right acetabulum (blue arrow) with several areas of focal uptake in the axial skeleton, in the ribs and in the left scapula.

99mTc‐methylene diphosphonate (MDP) bone scan is the main imaging modality used to assess the burden of skeletal disease in patients with PC in intermediate or high‐risk PC or those with symptoms of bony metastases [12] (**Figure 2**). However, bone scintigraphy can be non‐specific and can show increase bone uptake in degenerative joint disease, benign fractures and inflammation in addition to metastases [14]. However, further functional and anatomical details can be obtained by integrating the SPECT/CT along with skeletal scintigraphy. While the negative predictive value of the bone scan is estimated between 87 and 100% in the literature, its diagnostic yield is highly dependent on the PSA level and clinical stage [12]. As such bone scans have a poor yield in the early detection of prostate cancer recurrences post‐ definitive treatment.

#### **4.2. The newer imaging modalities**

#### *4.2.1. Multi‐parametric magnetic resonance imaging (MP‐MRI)*

MP‐MRI forms an integral role in diagnosis of prostate cancer and localisation for prostatic biopsy. In addition, it is a very useful tool in determining extra prostatic extension, lymph nodes or bony metastases in prostate cancer.

A number of studies have demonstrated promising results in detecting local recurrences post‐ radical prostatectomy using MP‐MRI. In patients with biochemical recurrence post‐radical prostatectomy, MP‐MRI can help determine loco‐regional relapse and small amounts of healthy residual glandular tissue, scar/fibrosis and granulation tissue, and it may even enable assessment of the aggressiveness of nodule recurrence by means of ADC values and help identify tumour deposits and target treatment [15]. One study demonstrated sensitivities and specificities of 84–88 and 89–100%, respectively in detection of recurrences post‐radical prostatectomy using MP‐MRI [16].

One of the limitations of MRI is the poor detection of pelvic lymph nodes at PSA levels <0.5 ng/mL, threshold usually used for salvage therapy. One of the main reasons for this being that 70% of lymph‐node metastases in prostate cancer is <8 mm [17]. In 2008, a meta‐analysis of 24 studies demonstrated that both CT and MRI scans were both poor at detecting pelvic lymph‐ node metastases and there were no differences between the modalities [18]. In fact, they concluded that reliance on either CT or MRI will misrepresent the patient's true status regarding nodal metastases, and thus misdirect the therapeutic strategies offered to the patient [18]. However, there have been significant advances in better anatomical imaging since the introduction of MP–MRI scans and technology such as lymphotropic nanoparticle–enhanced MRI can improve the lymph–node detection as well as for biopsy targeting and guidance of salvage treatment [19].

The increasing use of whole body MP‐MRI may be the future of staging patients with oligo‐ metastatic prostate cancer, as this can also be used to detect bony metastases with good accuracy [20]. However, this technology is currently mainly limited due to cost and needs further validation.

#### *4.2.2. Positron emission tomography (PET) scan*

**Figure 2.** The isotope bone scan demonstrates uptake at the right acetabulum (blue arrow) with several areas of focal

99mTc‐methylene diphosphonate (MDP) bone scan is the main imaging modality used to assess the burden of skeletal disease in patients with PC in intermediate or high‐risk PC or those with symptoms of bony metastases [12] (**Figure 2**). However, bone scintigraphy can be non‐specific and can show increase bone uptake in degenerative joint disease, benign fractures and inflammation in addition to metastases [14]. However, further functional and anatomical details can be obtained by integrating the SPECT/CT along with skeletal scintigraphy. While the negative predictive value of the bone scan is estimated between 87 and 100% in the literature, its diagnostic yield is highly dependent on the PSA level and clinical stage [12]. As such bone scans have a poor yield in the early detection of prostate cancer recurrences post‐

uptake in the axial skeleton, in the ribs and in the left scapula.

48 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

definitive treatment.

Positron emission tomography (PET) scan is a functional scan which commonly uses 18F‐ labeled sodium fluoride (18F‐NaF) and 18F‐labeled 2‐fluoro‐2‐deoxy‐Dglucose (18F‐FDG) as a radiotracer to detect a metabolic process associated with PC and is fused with a CT to determine the anatomic location of this process. Despite the role of 18F–FDG PET/CT in detecting occult metastatic disease in men with biochemical recurrence, and the high detection rates of osseous metastases with 18F–NaF PET/CT compared to standard imaging [21], they are still not recommended as first-line imaging modalities due to poor sensitivities in at low PSA levels and in high-grade tumours [12, 22–24].

A recent meta‐analysis by Evangelista et al. concluded that Choline PET and PET/CT represent high sensitivity and specificity techniques for the detection of loco‐regional and distant metastases in prostate cancer patients with recurrence of disease demonstrating a pooled sensitivity of 85.6% and a pooled specificity of 92.6% for all sites of disease (prostatic fossa, lymph nodes and bone) [25]. They further demonstrated a pooled sensitivity of 100% (95% CI 90.5–100%) and pooled specificity of 81.8% (95% CI 48.2–97.7%) for lymph‐node metastases [25]. In accordance, majority of the studies investigating recurrent oligometastatic prostate cancer utilised Choline PET as the imaging modality of choice [26].

#### *4.2.3. Prostate specific membrane antigen PET/CT (PSMA PET/CT)*

68Ga‐PSMA‐ligand PET/CT utilises the prostate specific membrane antigen which is signifi‐ cantly upregulated in prostate cancer. Although the data for PSMA PET/CT scan in recurrence of prostate cancer is limited, the early results have been promising. 68Ga‐PSMA–PET improves detection of lymph nodes, bone or visceral metastases compared with standard imaging (**Figures 3** and **4**). One study demonstrated a specificity of 98.9% and sensitivity 65% for detection of pelvic lymph‐node disease in prostate cancer with PSMA PET, much better than standard imaging modalities [27]. Furthermore, PSMA–PET–MRI or PSMA–PET–CT enables a complete staging procedure to be performed by a single examination compared with the standard staging combination of CT and bone scan.

**Figure 3.** The PSMA PET scan demonstrates uptake of the tracer in an internal iliac node denoted by the white arrow.

One study using data from 319 patients showed a sensitivity, specificity, negative predictive value and positive predictive value of PSMA PET/CT of 76.6, 100, 91.4 and 100%, respectively, in the detection of recurrent prostate cancer [28]. The PSMA detection of recurrent prostate cancer improved with higher PSA levels and the use of androgen deprivation therapy [28]. A further study of 248 patients replicated the accuracy of PSMA‐PET with an overall detection rate of 89.5% with a mean PSA value of 1.99 ng/ml [29]. As such, PSMA PET/CT is increasingly being used in studies focussed on oligometastatic disease and may form the cornerstone in detection and management of oligometastatic disease.

**Figure 4.** The PSMA PET scan demonstrates uptake of the tracer in a spine at the T9 level denoted by the white arrow.

#### **5. Advances in treatment: treatment of oligometastatic prostate cancer**

The conventional treatment of metastatic prostate cancer of androgen deprivation therapy is associated with a number of systemic side effects most importantly cardiovascular disease, and a large majority of patients will develop resistance to androgen deprivation. As such, metastases directed treatment of oligometastatic disease provides opportunity to select and treat this group of patients, delay the need for androgen deprivation or perhaps even cure.

#### **5.1. Synchronous oligometastatic prostate cancer**

#### *5.1.1. Radical prostatectomy*

metastases in prostate cancer patients with recurrence of disease demonstrating a pooled sensitivity of 85.6% and a pooled specificity of 92.6% for all sites of disease (prostatic fossa, lymph nodes and bone) [25]. They further demonstrated a pooled sensitivity of 100% (95% CI 90.5–100%) and pooled specificity of 81.8% (95% CI 48.2–97.7%) for lymph‐node metastases [25]. In accordance, majority of the studies investigating recurrent oligometastatic prostate

68Ga‐PSMA‐ligand PET/CT utilises the prostate specific membrane antigen which is signifi‐ cantly upregulated in prostate cancer. Although the data for PSMA PET/CT scan in recurrence of prostate cancer is limited, the early results have been promising. 68Ga‐PSMA–PET improves detection of lymph nodes, bone or visceral metastases compared with standard imaging (**Figures 3** and **4**). One study demonstrated a specificity of 98.9% and sensitivity 65% for detection of pelvic lymph‐node disease in prostate cancer with PSMA PET, much better than standard imaging modalities [27]. Furthermore, PSMA–PET–MRI or PSMA–PET–CT enables a complete staging procedure to be performed by a single examination compared with the

**Figure 3.** The PSMA PET scan demonstrates uptake of the tracer in an internal iliac node denoted by the white arrow.

One study using data from 319 patients showed a sensitivity, specificity, negative predictive value and positive predictive value of PSMA PET/CT of 76.6, 100, 91.4 and 100%, respectively, in the detection of recurrent prostate cancer [28]. The PSMA detection of recurrent prostate cancer improved with higher PSA levels and the use of androgen deprivation therapy [28]. A further study of 248 patients replicated the accuracy of PSMA‐PET with an overall detection

cancer utilised Choline PET as the imaging modality of choice [26].

*4.2.3. Prostate specific membrane antigen PET/CT (PSMA PET/CT)*

50 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

standard staging combination of CT and bone scan.

Based on the responses seen by cytoreductive therapy in other cancers such as ovarian, breast and renal cell carcinoma, a few recent studies have investigated the role of radical prostatec‐ tomy in metastatic prostate cancer. Using the SEER database of 8185 men with stage IV M1 prostate cancer, Culp et al. demonstrated that a reduction in cancer specific mortality in men undergoing radical prostatectomy or brachytherapy [30]. They demonstrated a 44.8% im‐ provement in 5‐year overall survival and 27.1% improvement disease‐specific survival in this cohort undergoing radical prostatectomy compared with those who did not have surgery or radiotherapy [30]. However, there were a few significant limitations in this study including the use of systemic therapy. A further study by Engel et al. replicated these findings using the Munich Cancer registry data demonstrating an improved survival in those who underwent a radical prostatectomy in the presence of lymph‐node metastases [31]. While these results appear to be promising, in the absence of prospective randomised controlled study data, radical prostatectomy for oligometastatic disease should be currently considered experimen‐ tal.

#### **5.2. Recurrent disease: oligometastases after primary curative therapy**

#### *5.2.1. Salvage lymph‐node dissection*

Salvage lymph‐node dissection in the setting of oligometastatic prostate cancer is limited to a number of cohort studies, with the largest being 59 patients [32]. Recently, a systematic review combined the results of these smaller series and reported on the results of 151 patients undergoing salvage pelvic, retroperitoneal or pelvic and retroperitoneal lymph‐node dissec‐ tion for oligometastatic disease [26]. Majority of the studies performed an open salvage lymph‐ node dissection with a median two positive nodes removed with 49 patients receiving post‐ operative prophylactic nodal irradiation and adjuvant ADT in 54% [26].

In the reported largest series with the longest follow‐up of 59 patients undergoing salvage lymph‐node dissection for oligometastatic prostate cancer, Suardi et al. reported a 8‐year biochemical recurrence free survival rate of 23% and an overall 8‐year clinical recurrence free survival of 38% and cancer specific mortality free survival rate of 81% [32]. They found that the PSA level at salvage LND, biochemical recurrence and the presence of retroperitoneal lymph‐node metastases all influenced clinical recurrence post‐operative clinical recurrence [32]. Jilg et al., in their study of 47 patients undergoing salvage LND, reported a clinical progression‐free survival of 25.6% and cancer specific survival of 77.7% at 5 years [33]. Notably, the initial disease recurrence post‐salvage lymph‐node dissection occurred again in lymph nodes in 47–59% in these studies [26].

A large proportion of patients (55%) undergoing salvage lymph‐node dissection developed complications with the majority being Clavien grade ≤2 [26]. The most common complications were lymphorrhoea (13%), fever (17%), ileus (10%), and a lymphocele requiring drainage (8%). Grade 3a complications were observed in 11% of the patients. Only one case of grade 3b complication (lymphocele requiring surgical drainage) was reported [26].

The current role of salvage lymph‐node dissection in oligometastatic disease remains experi‐ mental and more robust long‐term data are needed prior to being utilised as an established treatment modality in this setting.

#### *5.2.2. Stereotactic radiotherapy (SBRT)*

SBRT is external beam radiotherapy which is used to deliver a high dose of radiation very precisely to an extra cranial target within the body, as a single dose or a small number of fractions, thus reducing the amount of normal tissue irradiated and potentially offering complete ablation of all tissue in the treated area [34]. Therefore, it is a less invasive alternative to surgery in treating lymph‐node recurrence and bony metastases in prostate cancer.

Similar to salvage lymph‐node dissection, the evidence is based on small cohort studies. In one of the larger studies of 50 men with recurrence, post‐definitive therapy for Schick et al. demonstrated that a short duration androgen deprivation with and high‐dose irradiation to the metastatic lesions median follow‐up of 31 months (range 9–89) the 3‐year biochemical relapse‐free survival, clinical failure‐free survival, and overall survival rates were 54.5, 58.6 and 92%, respectively [35]. In a contrasting large cohort of 50 patients receiving SBRT with a median follow up of 2 years, Decaestecker et al. reported a 35% progression free survival at 2 years [36]. The differences in progression‐free survival rates are attributed to the use of adjuvant ADT and prophylactic nodal irradiation used in the study by Schick, offering better progression‐free survival [31]. A further interesting observation between the studies was the pattern of first progression where, 75% presented with oligometastases in the series of Decaestecker et al. compared with only 10% in the series of Schick et al. The recurring patents then went on to receive second or third course of SBRT in the former study [36]. A short PSA doubling time before SBRT predicted worse PFS in the study by Decaestecker et al. [36]. A recent retrospective series of 19 men who had biochemical recurrence post‐local therapy for prostate cancer with oligometastases (≤3 metachronous metastases) demonstrated a 21 months' median distant progression‐free survival with 3‐ and 5‐year DPFS of 31 and 15%, respectively [37]. Also importantly, this study demonstrated a delay of androgen deprivation by 28 months [37].

A further study by Tabata et al. demonstrated overall survival rates of up to 90.5% in patients receiving radiotherapy for oligometastatic disease of the bones with long‐term pain control in oligometastatic disease and no spinal cord compression or pathological fractures occurring at the radiated sites [38]. CyberKnife‐based stereotactic ablative radiotherapy is newer modality being utilised in oligometastatic disease with early studies also demonstrating good local control and relatively good PSA response [39].

The toxicity rates of SBRT six studies were reviewed in their analysis by Ost et al. [26]. Sixteen per cent of patients had late complications with the majority being grade 2 toxicity, mainly gastrointestinal in 8.5%, with one case of grade 3 toxicity (macroscopic haematuria) [26].

#### **6. Conclusion**

appear to be promising, in the absence of prospective randomised controlled study data, radical prostatectomy for oligometastatic disease should be currently considered experimen‐

Salvage lymph‐node dissection in the setting of oligometastatic prostate cancer is limited to a number of cohort studies, with the largest being 59 patients [32]. Recently, a systematic review combined the results of these smaller series and reported on the results of 151 patients undergoing salvage pelvic, retroperitoneal or pelvic and retroperitoneal lymph‐node dissec‐ tion for oligometastatic disease [26]. Majority of the studies performed an open salvage lymph‐ node dissection with a median two positive nodes removed with 49 patients receiving post‐

In the reported largest series with the longest follow‐up of 59 patients undergoing salvage lymph‐node dissection for oligometastatic prostate cancer, Suardi et al. reported a 8‐year biochemical recurrence free survival rate of 23% and an overall 8‐year clinical recurrence free survival of 38% and cancer specific mortality free survival rate of 81% [32]. They found that the PSA level at salvage LND, biochemical recurrence and the presence of retroperitoneal lymph‐node metastases all influenced clinical recurrence post‐operative clinical recurrence [32]. Jilg et al., in their study of 47 patients undergoing salvage LND, reported a clinical progression‐free survival of 25.6% and cancer specific survival of 77.7% at 5 years [33]. Notably, the initial disease recurrence post‐salvage lymph‐node dissection occurred again in lymph

A large proportion of patients (55%) undergoing salvage lymph‐node dissection developed complications with the majority being Clavien grade ≤2 [26]. The most common complications were lymphorrhoea (13%), fever (17%), ileus (10%), and a lymphocele requiring drainage (8%). Grade 3a complications were observed in 11% of the patients. Only one case of grade 3b

The current role of salvage lymph‐node dissection in oligometastatic disease remains experi‐ mental and more robust long‐term data are needed prior to being utilised as an established

SBRT is external beam radiotherapy which is used to deliver a high dose of radiation very precisely to an extra cranial target within the body, as a single dose or a small number of fractions, thus reducing the amount of normal tissue irradiated and potentially offering complete ablation of all tissue in the treated area [34]. Therefore, it is a less invasive alternative to surgery in treating lymph‐node recurrence and bony metastases in prostate cancer.

Similar to salvage lymph‐node dissection, the evidence is based on small cohort studies. In one of the larger studies of 50 men with recurrence, post‐definitive therapy for Schick et al.

**5.2. Recurrent disease: oligometastases after primary curative therapy**

operative prophylactic nodal irradiation and adjuvant ADT in 54% [26].

complication (lymphocele requiring surgical drainage) was reported [26].

tal.

*5.2.1. Salvage lymph‐node dissection*

52 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

nodes in 47–59% in these studies [26].

treatment modality in this setting.

*5.2.2. Stereotactic radiotherapy (SBRT)*

The concept of oligometastases in prostate cancer offers a newer approach to patients with the presence of five or fewer metastatic or recurrent lesions that could be treated by local therapy to achieve long‐term survival or cure. Furthermore, it offers the advantage of delaying the need for androgen deprivation therapy and its associated side effects. Treatment of oligome‐ tastatic prostate cancer relies on early diagnosis in order to offer the best outcomes for these patients. Therefore, improvements in prostate cancer diagnostics such as choline PET, whole‐ body multi‐parametric MRI, PSMA PET can provide early identification of this group of patients, while surgical and targeted radio‐ablative techniques can deliver advanced thera‐ peutics to the targeted regions. While the future management strategies appear promising for oligometastatic prostate cancer, it currently remains experimental.

#### **Acknowledgements**

We would like to thank Dr Chew‐Lin Yip for the images used in this manuscript.

#### **Author details**

Weranja Ranasinghe1\* and Raj Persad<sup>2</sup>

\*Address all correspondence to: weranja@gmail.com

1 Boxhill Hospital, Melbourne, Australia

2 University Hospitals Bristol NHS Foundation Trust, Bristol, UK

#### **References**


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**Acknowledgements**

Weranja Ranasinghe1\* and Raj Persad<sup>2</sup>

1 Boxhill Hospital, Melbourne, Australia

\*Address all correspondence to: weranja@gmail.com

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### **Targeted Therapy for Metastatic Prostate Cancer with Radionuclides**

Hojjat Ahmadzadehfar

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64016

#### **Abstract**

Progression to androgen-independent status is the main cause of death in patients with metastatic prostate cancer. Prostate-specific membrane antigen (PSMA) is anchored in the cell membrane of prostate epithelial cells. PSMA is highly expressed on prostate epithelial cells and strongly upregulated in prostate cancer. Therefore, it is an appro‐ priate target for diagnosis and therapy of prostate cancer and its metastases. There is growing knowledge about promising response and low toxicity profile of radioligand therapy of metastatic castration-resistant prostate cancer using Lutetium-177-labeled PSMA ligands. For patients with only bone metastases, there are different radionu‐ clides which have been used for decades. In this chapter, different methods of targeted radionuclide therapy of metastatic prostate cancer are described.

**Keywords:** PSMA, prostate cancer, radioligand therapy, metastatic disease, PSA, bone metastasis, radionuclide therapy

#### **1. Introduction**

Almost all patients with metastatic prostate cancer (PC) will initially respond to well-estab‐ lished and innovative anti-androgen treatments including the two recently approved hormone therapy agents, enzalutamide and abiraterone [1, 2], which significantly improve overall survival. However, progression to androgen-independent status is the main cause of death in these patients [3]. Most deaths related to PC are due to metastatic disease, which results from any combination of blood, lymphatic, or local spread. Targeted radionuclide therapy is an attractive and quickly developing therapy option for many different cancers, such as lym‐ phoma, melanoma, and neuroendocrine tumors [4–7]. Radionuclide therapies should be

© 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.

targeted, because this procedure always involves the administration of unsealed sources of radioactivity.

Most therapeutic tracers utilize β-particle emissions due to the ability of these particles to penetrate tissues. The deposition of energy in tissue by β-emitters results in cellular damage. Among the β-emitters, there are several choices regarding the energy of the β-emission. Lower energy β-particles can travel a few cell diameters, or at most in the submillimeter range. Higher energy β-particles, such as those emitted by Yttrium-90 (90Y) or Lutetium-177 (177Lu), have excellent tissue penetration with a range beyond the source of several millimeters [8, 9]. The only routinely used α-emitter for the treatment of metastatic disease is Radium-223 (223Ra), which has been approved for the treatment of bone metastases in patients with prostate cancer and symptomatic disease with no known visceral metastases [10]. The physical half-life of therapeutic radionuclide is an important consideration and an underlying principle for therapy planning [11].

#### **2. PSMA as a target**

Prostate-specific membrane antigen (PSMA), also known as folate hydrolase I or glutamate carboxypeptidase II, is a type II transmembrane protein anchored in the cell membrane of prostate epithelial cells [12]. Several biological characteristics make PSMA an outstanding target for drug development. PSMA is highly expressed on prostate epithelial cells and strongly upregulated in PC. PSMA expression levels are directly correlated to androgen independence, metastasis, and PC progression [13]; thus, PSMA is an attractive target for the diagnosis and therapy of metastasized PC. Its target specificity is maintained after radiolab‐ eling with 68Ga [12, 14].

A commonly used radionuclide is 68Ga-PSMA-11, which has been successfully used for the imaging of PC with high sensitivity and specificity, even in patients with very low prostatespecific antigen (PSA) levels (<2 ng/ml) [15]. Direct comparison studies support the superiority of 68Ga-PSMA-11 in lymph node assessment over CT 3D volumetric-based lymph node assessments [16] and in overall disease assessment compared to 18F-methylcholine, especially in patients with low PSA levels [17]. These positive results will lead to or have already led to a paradigm shift in the use of imaging in primary staging of PC. In a recent study by Hijazi et al., the diagnostic accuracy of 68Ga-PSMA-11 in the preoperative assessment of nodal meta‐ stases was very high for macrometastases and even micrometastases in lymph nodes. Corre‐ lating imaging and tissue specimens of 213 removed nodes provided 94% sensitivity, 99% specificity, 89% positive predictive value, and 99.5% negative predictive value [18].

#### **3. PSMA radioimmunotherapy**

After rather unsuccessful therapy with the 90Y-CYT-356 monoclonal antibody (mAb) recog‐ nizing the intracellular domain of PSMA [19], Phase I and II clinical trials utilizing the PSMA mAb J591, radiolabeled with 177Lu or 90Y, have shown promising results [20–23].

J591 is an anti-PSMA mAb that binds with 1 nM affinity to the extracellular domain of PSMA [24, 25]. Milowsky et al. [26] treated 29 patients in the 90Y-J591 Phase I trial; patients received therapeutic doses of 185, 370, 555, 647.5, and 740 MBq/m2 90Y-J591. Dose-limiting toxicity was seen at 740 MBq/m2 , with two patients experiencing thrombocytopenia with nonlife-threaten‐ ing bleeding episodes requiring platelet transfusions. The 647.5 MBq/m2 dose was determined to be the maximum tolerated dose (MDT).

targeted, because this procedure always involves the administration of unsealed sources of

Most therapeutic tracers utilize β-particle emissions due to the ability of these particles to penetrate tissues. The deposition of energy in tissue by β-emitters results in cellular damage. Among the β-emitters, there are several choices regarding the energy of the β-emission. Lower energy β-particles can travel a few cell diameters, or at most in the submillimeter range. Higher energy β-particles, such as those emitted by Yttrium-90 (90Y) or Lutetium-177 (177Lu), have excellent tissue penetration with a range beyond the source of several millimeters [8, 9]. The only routinely used α-emitter for the treatment of metastatic disease is Radium-223 (223Ra), which has been approved for the treatment of bone metastases in patients with prostate cancer and symptomatic disease with no known visceral metastases [10]. The physical half-life of therapeutic radionuclide is an important consideration and an underlying principle for

Prostate-specific membrane antigen (PSMA), also known as folate hydrolase I or glutamate carboxypeptidase II, is a type II transmembrane protein anchored in the cell membrane of prostate epithelial cells [12]. Several biological characteristics make PSMA an outstanding target for drug development. PSMA is highly expressed on prostate epithelial cells and strongly upregulated in PC. PSMA expression levels are directly correlated to androgen independence, metastasis, and PC progression [13]; thus, PSMA is an attractive target for the diagnosis and therapy of metastasized PC. Its target specificity is maintained after radiolab‐

A commonly used radionuclide is 68Ga-PSMA-11, which has been successfully used for the imaging of PC with high sensitivity and specificity, even in patients with very low prostatespecific antigen (PSA) levels (<2 ng/ml) [15]. Direct comparison studies support the superiority of 68Ga-PSMA-11 in lymph node assessment over CT 3D volumetric-based lymph node assessments [16] and in overall disease assessment compared to 18F-methylcholine, especially in patients with low PSA levels [17]. These positive results will lead to or have already led to a paradigm shift in the use of imaging in primary staging of PC. In a recent study by Hijazi et al., the diagnostic accuracy of 68Ga-PSMA-11 in the preoperative assessment of nodal meta‐ stases was very high for macrometastases and even micrometastases in lymph nodes. Corre‐ lating imaging and tissue specimens of 213 removed nodes provided 94% sensitivity, 99%

specificity, 89% positive predictive value, and 99.5% negative predictive value [18].

mAb J591, radiolabeled with 177Lu or 90Y, have shown promising results [20–23].

After rather unsuccessful therapy with the 90Y-CYT-356 monoclonal antibody (mAb) recog‐ nizing the intracellular domain of PSMA [19], Phase I and II clinical trials utilizing the PSMA

radioactivity.

60 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

therapy planning [11].

**2. PSMA as a target**

eling with 68Ga [12, 14].

**3. PSMA radioimmunotherapy**

Bander et al. [21] treated 35 patients with progressing androgen-independent PC with 177Lu-J591, and 16 of these patients received up to three doses. Myelosuppression was dose-limiting at 2775 MBq/m2 , and the 2590 MBq/m2 dose was determined to be the single-dose MTD. Repeat dosing at 1665–2220 MBq/m2 was associated with dose-limiting myelosuppression [21]. The authors reported good targeting of all known sites of bone and soft tissue metastases in all patients. They found no clear relationship between a history of prior chemotherapy treatment and the degree of toxicity. Biological activity was seen with four patients experiencing ≥50% declines in PSA levels lasting from 3 to 8 months. An additional 16 patients (46%) experienced PSA stabilization for a median of 60 days [21]. Tagawa et al. presented the results of a Phase II study of radionuclide therapy with the 177Lu-PSMA mAb J591 [20], which was based on two published Phase I studies investigating this agent [21, 26]. In this study [20], 47 hormonerefractory patients (55.3% also had received chemotherapy) were treated with 177Lu-J591. They compared two different doses (2405 vs. 2590 MBq/m2 ). About 11% of patients experienced a ≥50% decline in PSA, 36.2% experienced a ≥30% decline in PSA, and 59.6% experienced any PSA decline following a single therapy. All experienced reversible hematological toxicity, with Grade 4 thrombocytopenia occurring in 46.8% without significant hemorrhage. Grade 4 neutropenia happened in a total of 25.5% of patients, with one episode of febrile neutropenia. The 2590 MBq/m2 dose resulted in not only 30% more of PSA decline (46.9 vs. 13.3%, *P* = 0.048) and longer survival (21.8 vs. 11.9 months, *P* = 0.03), but also more Grade 4 hematological toxicity and platelet transfusions. mAb are large molecules, and therefore show poor perme‐ ability in solid tumors and slow clearance from the circulation. This combination leads to suboptimal targeting and an increased absorbed dose in the bone marrow, narrowing the therapeutic window [27]. Thus, radionuclide treatment with 90Y-J591 and 177Lu-J591 is limited by myelosuppression and nonhematological toxicity, with a maximum tolerated activity per cycle of 650 and 2450 MBq/m2 , respectively.

#### **4. PSMA radioligand therapy with a small-molecule inhibitor**

The synthesis and design of a series of small-molecule inhibitors of PSMA have been described by Maresca et al. [28]. On the basis of the work of this group, Hillier et al. [29] performed a preclinical evaluation of two radiopharmaceuticals, 123I-MIP-1072 and 123I-MIP-1095, which were designed to target PSMA in PC cells and tissue. In a recent published study from the Heidelberg group, Zechmann et al. showed the utility of 131I-MIP-1095 PSMA [27]. Therapy with 131I-MIP-1095 PSMA was performed in 25 patients. The patients received a single therapeutic dose of 131I-MIP-1095 (mean activity 4.8 GBq, range 2.0–7.2 GBq). Erythrocyte counts fell below the normal range at the nadir in 21 patients, with 17 patients having lower values prior to therapy. In 14 patients, white blood cell counts fell below the normal range after therapy (one with Grade 3 toxicity). However, five of these 14 patients had levels below normal, prior to therapy (four Grade 1, one Grade 2); 11 patients had a reduction in platelet count below normal after therapy (two Grade 3), and one had a value below normal (Grade 2), prior to therapy. The changes in hematological parameters were not related to the activity adminis‐ tered. The onset of the myelosuppression occurred within 6 weeks after treatment with a quite variable time to recovery, in some cases requiring up to 3–6 months for recovery. White blood cells typically recovered within several weeks, whereas platelets required several months to recover [12, 27]. In contrast to mAb, the low-molecular-weight compounds, with higher permeability into solid tumors, offered a significant advantage in achieving higher uptake per gram of tumor tissue and a higher percentage of specific binding. Moreover, small molecules displayed more rapid tissue distribution and faster blood clearance compared with intact immunoglobulins. These properties often lead to a higher target to nontarget tissue ratio, which is important for successful application of therapeutic absorbed doses [27].

131I has a long half-life of 8.02 days and has a β-particle range in soft tissue of just 0.8 mm. Due to its γ-emitting properties and long half-life, 131I is less attractive from a radiation safety point of view. 90Y has a half-life of 64 h, but only undergoes high-energy β-emission, resulting in a long mean β-particle range of 3.6 mm and a maximum range of 10 mm in soft tissue. Due to its long β-particle range, collateral damage to surrounding tissues is quite high [30].

Recently, a novel theranostic drug, 177Lu-PSMA 617, which is a DOTA-derivative of the Glu– urea–Lys motif, has been developed for the treatment of patients with metastatic PC [29, 31]. 177Lu has a half-life of 6.7 days and undergoes low-energy β-particle emission with a mean range of 1 mm and a maximum range of 2–4 mm in soft tissue. So, the practical issues sur‐ rounding radiation safety with 131I and the limited collateral damage to surrounding tissues compared to 90Y make 177Lu-labeled radionuclide treatment the most attractive option from a physics point of view. Ahmadzadehfar et al. [32] reported on the first 10 consecutive patients who were treated with 177Lu-PSMA-617 in their department (University Hospital Bonn and Muenster, Germany). They showed that 8 weeks after the therapy in these 10 patients, seven patients showed a PSA decline, of whom six experienced a more than 30% and five a more than 50% decline. Three patients showed progressive disease according to the PSA increase. No patients experienced any side effects immediately after injection of 177Lu-PSMA 617 [32]. Relevant hematotoxicity (Grade 3 or 4) occurred 7 weeks after the administration in just one patient. These encouraging results showed again the efficacy of radionuclide therapy in patients who have no other approved therapeutic option. A later study by the group from Bonn showed the efficacy and safety of 177Lu-PSMA 617 therapy in patients who had under‐ gone two cycles of therapy [8]. In this study, 46 cycles of 177Lu-PSMA 617 were performed in 24 consecutive hormone and/or chemorefractory patients. Twenty-two patients received two cycles of therapy. Twenty-two patients had a history of or were on therapy with enzalutamide and/or abiraterone. Twelve patients had received 223Ra (1–6 cycles; median 5 cycles). All patients had multiple bone metastases, and the majority of them also had lymph node metastases. The mean and median PSA levels prior to therapy were 628.3 and 522 ng/ml, respectively (range: 17.1–2360 ng/ml). It was found that 8 weeks after the first cycle of 177Lu-PSMA therapy, 19/24 patients (79.1%) experienced a PSA decline, out of whom 13 experienced a decline of more than 30% and 10 more than 50% (41.6%). Five patients showed progressive disease according to an increase in PSA. Two months after the second cycle in 22 patients who underwent two cycles of 177Lu-PSMA therapy, 15/22 patients (68.2%) experienced a PSA decline in comparison to the baseline PSA value, of whom 15 experienced a decline of more than 30%, and 13 (60%) of more than 50%. Seven patients showed progressive disease accord‐ ing to an increase in PSA or disease progression. Again, in this study, the patients received radioligand therapy as the last therapeutic option [8]. Interestingly, although a majority of prostate cancer patients at such an advanced stage of disease with massive bone marrow infiltration suffer from anemia, relevant hematotoxicity (Grade 3) occurred during the observation period (within 2 months after the last cycle) in just two patients. Apart from some Grade 1 or 2 hematotoxicity, the majority of patients did not show any hematotoxicity during the observation period. Some patients who needed blood transfusions prior to the first cycle needed fewer transfusions after radioligand therapy with 177Lu-PSMA 617 because of the regression of bone marrow involvement [33]. The Nordrhein–Westfalen study group recently published the results of single-dose administration of 177Lu-PSMA 617 in 74 patients. They showed a PSA decline in 47 patients (64%); of these, 23 (31%) had a PSA decline >50%; 35 (47%) had stable disease with a PSA decline from <50% to an increase of <25%; and 17 (23%) showed progressive disease with a PSA increase >25%. Response and tolerability of a single dose of 177Lu-PSMA-617 in patients with metastatic castration-resistant prostate cancer: a multicenter retrospective analysis.

therapy (one with Grade 3 toxicity). However, five of these 14 patients had levels below normal, prior to therapy (four Grade 1, one Grade 2); 11 patients had a reduction in platelet count below normal after therapy (two Grade 3), and one had a value below normal (Grade 2), prior to therapy. The changes in hematological parameters were not related to the activity adminis‐ tered. The onset of the myelosuppression occurred within 6 weeks after treatment with a quite variable time to recovery, in some cases requiring up to 3–6 months for recovery. White blood cells typically recovered within several weeks, whereas platelets required several months to recover [12, 27]. In contrast to mAb, the low-molecular-weight compounds, with higher permeability into solid tumors, offered a significant advantage in achieving higher uptake per gram of tumor tissue and a higher percentage of specific binding. Moreover, small molecules displayed more rapid tissue distribution and faster blood clearance compared with intact immunoglobulins. These properties often lead to a higher target to nontarget tissue ratio,

62 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

which is important for successful application of therapeutic absorbed doses [27].

its long β-particle range, collateral damage to surrounding tissues is quite high [30].

131I has a long half-life of 8.02 days and has a β-particle range in soft tissue of just 0.8 mm. Due to its γ-emitting properties and long half-life, 131I is less attractive from a radiation safety point of view. 90Y has a half-life of 64 h, but only undergoes high-energy β-emission, resulting in a long mean β-particle range of 3.6 mm and a maximum range of 10 mm in soft tissue. Due to

Recently, a novel theranostic drug, 177Lu-PSMA 617, which is a DOTA-derivative of the Glu– urea–Lys motif, has been developed for the treatment of patients with metastatic PC [29, 31]. 177Lu has a half-life of 6.7 days and undergoes low-energy β-particle emission with a mean range of 1 mm and a maximum range of 2–4 mm in soft tissue. So, the practical issues sur‐ rounding radiation safety with 131I and the limited collateral damage to surrounding tissues compared to 90Y make 177Lu-labeled radionuclide treatment the most attractive option from a physics point of view. Ahmadzadehfar et al. [32] reported on the first 10 consecutive patients who were treated with 177Lu-PSMA-617 in their department (University Hospital Bonn and Muenster, Germany). They showed that 8 weeks after the therapy in these 10 patients, seven patients showed a PSA decline, of whom six experienced a more than 30% and five a more than 50% decline. Three patients showed progressive disease according to the PSA increase. No patients experienced any side effects immediately after injection of 177Lu-PSMA 617 [32]. Relevant hematotoxicity (Grade 3 or 4) occurred 7 weeks after the administration in just one patient. These encouraging results showed again the efficacy of radionuclide therapy in patients who have no other approved therapeutic option. A later study by the group from Bonn showed the efficacy and safety of 177Lu-PSMA 617 therapy in patients who had under‐ gone two cycles of therapy [8]. In this study, 46 cycles of 177Lu-PSMA 617 were performed in 24 consecutive hormone and/or chemorefractory patients. Twenty-two patients received two cycles of therapy. Twenty-two patients had a history of or were on therapy with enzalutamide and/or abiraterone. Twelve patients had received 223Ra (1–6 cycles; median 5 cycles). All patients had multiple bone metastases, and the majority of them also had lymph node metastases. The mean and median PSA levels prior to therapy were 628.3 and 522 ng/ml, respectively (range: 17.1–2360 ng/ml). It was found that 8 weeks after the first cycle of 177Lu-PSMA therapy, 19/24 patients (79.1%) experienced a PSA decline, out of whom 13 experienced

**Figure 1.** (**A**) 68Ga-PSMA PET scan of a 66-year-old hormone- and chemorefractory patient with multiple bone and lymph node metastases (*pink arrows*), with a history of chemotherapy, abiraterone, and 223Ra therapies. (**B**) The followup PET scan prior to the second cycle shows a partial response with regression of the metastases and PSA decline. (**C**) The PET scan, 2 months after the third cycle of Lu-PSMA therapy, which shows a very good response with further decline of PSA.

Rahbar K, Schmidt M, Heinzel A, Eppard E, Bode A, Yordanova A, Claesener M, Ahmadza‐ dehfar H. J Nucl Med. 2016 Apr 7. pii: jnumed.116.173757. [Epub ahead of print]

Further research into the efficacy of this therapy is needed. Rahbar et al. showed for the first time the overall survival benefit of RLT in comparison to a historical collective. They showed that the estimated median survival was 29.4 weeks, significantly longer than the survival in the historical control group at 19.7 weeks [hazard ratio: 0.44 (95% confidence interval: 0.20– 0.95); *P* = 0.031] [34] (**Figure 1**).

#### **5. Treatment of bone metastases with radionuclides**

Bone metastases, a major cause of morbidity and mortality in patients with castration-resistant prostate cancer, are associated with pain, pathological fractures, spinal cord compression, and decreased survival [35]. The major mechanism of pain from small metastases appears to be the stimulation of nerve endings in the endosteum by a variety of chemical mediators. Larger bone metastases produce stretching of the periosteum, which leads to pain [36]. The incidence of bone metastases in patients with prostate cancer, according to autopsy studies, is 65–85% [37].

Bone pain palliation with radionuclides has a very long history of using different β-emitters such as phosphorus-32 (32P) [38], strontium-89 (89Sr) [38], rhenium-186-hydroxyethylidene diphosphonate (186Re-HEDP) [39], 188Re-HEDP, samarium-153-EDTMP (153Sm-EDTMP) [39], and recently, lutetium-177-EDTMP (177Lu-EDTMP) [40] and 177Lu-BPAMD [41]. The only approved α-emitter is radium-223 (223Ra) [42].


**Table 1.** Different radionuclides for bone palliation.

Bone-seeking radionuclides are classified into two groups: calcium analogues and radionu‐ clides attached to phosphate (**Table 1**). Different radionuclides have different physical char‐ acteristics, which are shown in **Table 2**.


**Table 2.** Summary of the main physical properties of different radionuclides in clinical use for pain palliation.

#### **5.1. 32Phosphorus**

Rahbar K, Schmidt M, Heinzel A, Eppard E, Bode A, Yordanova A, Claesener M, Ahmadza‐

Further research into the efficacy of this therapy is needed. Rahbar et al. showed for the first time the overall survival benefit of RLT in comparison to a historical collective. They showed that the estimated median survival was 29.4 weeks, significantly longer than the survival in the historical control group at 19.7 weeks [hazard ratio: 0.44 (95% confidence interval: 0.20–

Bone metastases, a major cause of morbidity and mortality in patients with castration-resistant prostate cancer, are associated with pain, pathological fractures, spinal cord compression, and decreased survival [35]. The major mechanism of pain from small metastases appears to be the stimulation of nerve endings in the endosteum by a variety of chemical mediators. Larger bone metastases produce stretching of the periosteum, which leads to pain [36]. The incidence of bone metastases in patients with prostate cancer, according to autopsy studies, is 65–85% [37].

Bone pain palliation with radionuclides has a very long history of using different β-emitters such as phosphorus-32 (32P) [38], strontium-89 (89Sr) [38], rhenium-186-hydroxyethylidene diphosphonate (186Re-HEDP) [39], 188Re-HEDP, samarium-153-EDTMP (153Sm-EDTMP) [39], and recently, lutetium-177-EDTMP (177Lu-EDTMP) [40] and 177Lu-BPAMD [41]. The only

> Rhenium-186-HEDP Rhenium-188-HEDP Lutetium-177-EDTMP Lutetium-177-BPAMD

Bone-seeking radionuclides are classified into two groups: calcium analogues and radionu‐ clides attached to phosphate (**Table 1**). Different radionuclides have different physical char‐

dehfar H. J Nucl Med. 2016 Apr 7. pii: jnumed.116.173757. [Epub ahead of print]

**5. Treatment of bone metastases with radionuclides**

64 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

approved α-emitter is radium-223 (223Ra) [42].

**Table 1.** Different radionuclides for bone palliation.

acteristics, which are shown in **Table 2**.

**Calcium analogues Attached to phosphate**

Radium-223 Samarium-153-EDTMP

Strontium-89 Phosphorus-32

0.95); *P* = 0.031] [34] (**Figure 1**).

<sup>32</sup>P decays by 1.7 MeV (*E*max) β-emission and has a physical half-life of 14.3 days, with a maximum tissue penetration of 8.5 mm (**Table 2**) [43]. During treatment with 32P, pain relief was reported by 50–87% of patients treated with 200–800 MBq of <sup>32</sup>P administered daily in 20– 80 MBq fractions after androgen priming. Pain reduction occurred within 5–14 days, with a mean response duration of 2–4 months [38, 44, 45] (**Table 3**). The main disadvantage of 32P therapy is dose-limiting myelosuppression with reversible pancytopenia maximal at 5–6 weeks after administration [46].


**Table 3.** Bone-seeking radionuclides.

#### **5.2. 89Strontium**

<sup>89</sup>SrCl2 is an element that behaves like calcium and localizes in bone, primarily in areas of osteoblastic activity. It decays by 1.4 MeV (*E*max) β-emission, with a long physical half-life of 50.5 days. The maximum penetration range in tissue is about 7 mm. Excretion is predominantly renal, dictated by the skeletal tumor burden and glomerular filtration rate [47, 48].

The biological half-life in normal bone is around 14 days, compared with more than 50 days in osteoblastic metastases. The first studies using 89Sr demonstrated efficacy for pain reduction as high as 80%. Complete response rates vary widely among studies and have been reported in 8–77% of cases. The overall response rate varied from 33 to 82% [49–54]. It was the first radiopharmaceutical to be approved for systemic radionuclide therapy in the palliation of painful bone metastases. The standard recommended dose of 89Sr is 150–200 MBq (**Table 3**). It was shown to be as effective as both local field and hemibody external-beam radiotherapy in relieving existing bone pain, but delayed the development of new pain at preexisting, clinically silent sites [45, 55].

Toxicity is limited with the common development of thrombocytopenia, with the nadir between the 4th and 6th weeks. Recovery is typically slow over the next 6 weeks, dictated by skeletal tumor extent and bone marrow reserve [45].

The largest study was published by Robinson et al. In this study, 622 patients were included (466 with prostate cancer). About 15% of patients showed complete pain relief, and a partial response was documented in 81% [56–58]. Tu et al. [59] reported improved survival using six weekly administrations of 89SrCl2 combined with doxorubicin after induction chemotherapy, compared with six weekly administrations of doxorubicin alone; however, the follow-up Phase II trial of the same study group did not confirm the positive effect of this combination therapy on overall survival [60].

A nonrandomized study using 12 weekly administrations of estramustine phosphate, vin‐ blastine, and 89SrCl2 recorded effective, durable symptom palliation, more than a 50% reduc‐ tion in PSA in 48% of treated patients, and reduced demand for subsequent palliative radiotherapy [61]. Several patient characteristics could predict a favorable response to 89Sr. A normal serum hemoglobin level prior to treatment is associated with a higher pain response rate [62]. Other predictors of a poor pain response were low performance status, higher serum PSA, more extensive osseous metastases, and a poor PSA response [63–66].

#### **5.3. 186Rhenium-HEDP**

186Re is a medium-energy β-emitter with a physical half-life of 89 h.186Re-1,1-hydroxyethylidene diphosphonate (186Re-HEDP) is a surface bone-seeking radiopharmaceutical used for internal radiotherapy. The maximum tolerated activity is 2960 MBq, but for routine use the recom‐ mended activity is 1285 MBq. Peak skeletal uptake occurs 3 h after intravenous administration [67, 68]. An early study by Maxon et al. [69] using 1285 MBq of 186Re-HEDP documented overall pain relief in 80% of patients, with a mean duration of 7 weeks in hormone refractory PC patients (**Table 3**) [70]. Eighty to ninety percent of patients reported improved symptoms after a single 186Re-HEDP administration. The response was typically rapid, occurring within 24–48 h of activity administration. Placebo-controlled, randomized studies have confirmed the efficacy of 186Re-HEDP [70, 71].186Re-HEDP undergoes rapid urinary excretion and rapid blood clearance (plasma half-life of 41 h) [70]. For the standard applied activity of 1285 MBq, 186Re-HEDP provided a median radiation-absorbed dose of 26 Gy to bone metastases and 1.73 Gy to the red bone marrow [70]. The tumor to marrow dose ratios had a high therapeutic index, with a mean value of 34:1 [69]. Toxicity was limited to temporary myelosuppression, with platelet and neutrophil nadirs at 4 weeks after therapy. Recovery occurred within 8 weeks and was usually complete [72].

### **5.4. 188Rhenium-HEDP**

as high as 80%. Complete response rates vary widely among studies and have been reported in 8–77% of cases. The overall response rate varied from 33 to 82% [49–54]. It was the first radiopharmaceutical to be approved for systemic radionuclide therapy in the palliation of painful bone metastases. The standard recommended dose of 89Sr is 150–200 MBq (**Table 3**). It was shown to be as effective as both local field and hemibody external-beam radiotherapy in relieving existing bone pain, but delayed the development of new pain at preexisting, clinically

Toxicity is limited with the common development of thrombocytopenia, with the nadir between the 4th and 6th weeks. Recovery is typically slow over the next 6 weeks, dictated by

The largest study was published by Robinson et al. In this study, 622 patients were included (466 with prostate cancer). About 15% of patients showed complete pain relief, and a partial response was documented in 81% [56–58]. Tu et al. [59] reported improved survival using six weekly administrations of 89SrCl2 combined with doxorubicin after induction chemotherapy, compared with six weekly administrations of doxorubicin alone; however, the follow-up Phase II trial of the same study group did not confirm the positive effect of this combination therapy

A nonrandomized study using 12 weekly administrations of estramustine phosphate, vin‐ blastine, and 89SrCl2 recorded effective, durable symptom palliation, more than a 50% reduc‐ tion in PSA in 48% of treated patients, and reduced demand for subsequent palliative radiotherapy [61]. Several patient characteristics could predict a favorable response to 89Sr. A normal serum hemoglobin level prior to treatment is associated with a higher pain response rate [62]. Other predictors of a poor pain response were low performance status, higher serum

186Re is a medium-energy β-emitter with a physical half-life of 89 h.186Re-1,1-hydroxyethylidene diphosphonate (186Re-HEDP) is a surface bone-seeking radiopharmaceutical used for internal radiotherapy. The maximum tolerated activity is 2960 MBq, but for routine use the recom‐ mended activity is 1285 MBq. Peak skeletal uptake occurs 3 h after intravenous administration [67, 68]. An early study by Maxon et al. [69] using 1285 MBq of 186Re-HEDP documented overall pain relief in 80% of patients, with a mean duration of 7 weeks in hormone refractory PC patients (**Table 3**) [70]. Eighty to ninety percent of patients reported improved symptoms after a single 186Re-HEDP administration. The response was typically rapid, occurring within 24–48 h of activity administration. Placebo-controlled, randomized studies have confirmed the efficacy of 186Re-HEDP [70, 71].186Re-HEDP undergoes rapid urinary excretion and rapid blood clearance (plasma half-life of 41 h) [70]. For the standard applied activity of 1285 MBq, 186Re-HEDP provided a median radiation-absorbed dose of 26 Gy to bone metastases and 1.73 Gy to the red bone marrow [70]. The tumor to marrow dose ratios had a high therapeutic index, with a mean value of 34:1 [69]. Toxicity was limited to temporary myelosuppression, with platelet and neutrophil nadirs at 4 weeks after therapy. Recovery occurred within 8 weeks and

PSA, more extensive osseous metastases, and a poor PSA response [63–66].

silent sites [45, 55].

on overall survival [60].

**5.3. 186Rhenium-HEDP**

was usually complete [72].

skeletal tumor extent and bone marrow reserve [45].

66 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

188Re has a short physical half-life of 16.8 h and a maximum β-particle energy of 2.1 MeV with a 15% γ-component of 155 keV. The maximum β-range in tissue is approximately 10 mm [73]. Blood clearance is rapid after injection, with 41% renal clearance within 8 h of administration. Absorbed doses for bone metastases are in the range of 3.83 ± 2 mGy/MBq, in comparison with 0.61 ± 0.2 mGy/MBq for bone marrow and 0.07 ± 0.02 mGy/MBq for the whole body. The mean effective whole-body half-life is 11.6 ± 2.1 h compared with 15.9 ± 3.5 h in bone metastases [45]. <sup>188</sup> Re is of special interest in clinical applications because of its excellent availability and costeffectiveness, as it is the product of a 188W (188W/188Re) generator [74].

The short physical half-life and high dose rate are predicted to lead to a rapid symptom response. Fractionated therapy has been shown to prolong response duration and progressionfree survival (PFS). Palmedo et al. [73] randomly assigned 64 patients to two different groups for radionuclide therapy with 188Re-HEDP; patients in group A received a single injection, while patients in group B received two injections with an 8-week interval. In both groups, toxicity was low, with moderate thrombopenia and leukopenia. Repeated 188Re-HEDP therapies (group B) were more effective for pain palliation compared to group A, with a response rate and time of response of 92% and 5.66 months, respectively (*P* = 0.006 and *P* = 0.001). In group B, 11/28 patients (39%) had a PSA decline of more than 50% for at least 8 weeks, compared with 2/30 patients (7%) in group A. The median times to progression in group A and group B were 2.3 months (0–12.2 months) and 7.0 months (0–24.1 months), respectively (*P* = 0.0013), and the median overall survival times were 7.0 months (range, 1.3–36.7 months) and 12.7 months (range, 4.1–32.2 months), respectively (*P* = 0.043) [74].

Liepe et al. [75] reported moderate transient bone marrow toxicity with a decrease in the number of platelets from a baseline value of 286 ± 75 × 109 /l to a maximum of 218 ± 83 × 109 /l with the nadir at 3 weeks. This study group found no evidence of either local or systemic intolerance to treatment with 188Re-HEDP, while a flare reaction with an increase in pain within 14 days after therapy was noted in 16% of patients [75].

Biersack et al. [76] also showed the positive effect of repeated therapy on overall survival. They retrospectively analyzed 60 hormone-refractory patients classified into three different groups according to the number of therapies. Group A comprised patients who had received only one therapy (19 patients), group B included patients who had received two therapies (19 patients), and group C included patients who had received three or more therapies (22 patients). All patients had bone pain and presented with more than five lesions documented by a bone scan. Mean survival after the initial therapy improved from 4.5 months in group A to 9.98 months in group B and 15.7 months in group C [76].

#### **5.5. 153Samarium-EDTMP**

153Sm-EDTMP has a lower β-emission energy [0.81 MeV (20%), 0.71 MeV (49%), and 0.64 MeV (30%)], a 28% abundance of γ-emission at 103 keV (28%) and a physical half-life of 46.3 h. 153Sm forms a stable complex with ethylenediamine tetramethylene phosphonate (EDTMP). Clearance is bi-exponential after administration, comprising rapid bone uptake (half-life of 5.5 min) and plasma renal clearance (half-life of 65 min) [77].

A dose escalation study with 10–36 MBq/kg of 153Sm-EDTMP reported a pain relief rate of 65%, with a duration range from 4 to 35 weeks [78]. A further dose escalation study in 52 patients using administered activities from 37 to 111 MBq/kg had a response rate of 74% with a median duration of 10 weeks [79]. Larger studies with more than 100 patients showed a median therapeutic efficacy of 80%. In a randomized, double-blind, placebo trial (*n* = 152), pain relief was found in 65% of patients after 153Sm-EDTMP treatment compared to 45% in the placebo group [80]. A significant decrease in pain between 153Sm-EDTMP and placebo was reported after 1 week, and the analgesic intake was significantly reduced after 3 and 4 weeks. Two large studies using 153Sm-EDTMP with more than 550 patients reported response rates of 73 and 86% [81, 82].

#### **5.6. Comparing the pain response between different radionuclides**

Dickie et al. compared 89Sr with 153Sm in 57 prostate cancer patients. They found no difference in the pain response rate and toxicity [83]. van der Poel et al. compared 186Re with 89Sr and reported no differences in the response rate or toxicity [54]. A nonrandomized comparison of 188Re-HEDP and 153Sm-EDTMP in patients with painful metastases from prostate and breast cancer by Liepe et al. [84] showed a comparable response and toxicity with both agents. Liepe et al. also performed a comparative study of 188Re-HEDP, 186Re-HEDP, 153Sm-EDTMP, and 89SrCl2 in the treatment of painful bone metastases. They reported that all radiopharmaceuticals were effective in pain palliation, without the induction of severe side effects or significant differences in therapeutic efficacy or toxicity [39].

#### **5.7. 223Radium dichloride**

<sup>223</sup>Ra, an α-emitter, has a half-life of 11.4 days, with a total emitted energy of about 28 MeV. It is the only FDA-approved radiopharmaceutical for the treatment of bone metastases of PC with positive impact on overall survival according to a prospective randomized study [10]. It is a bone stromal-targeted radiopharmaceutical that undergoes α-emission. The α-particle is considerably more destructive to tumor cells than the β-particle. 223Ra has a very high linear energy transfer, and only one to five hits per cell can be fatal. Double-strand breaks are induced even in quiescent cells and at low oxygen levels [85].

The penetration range of α-particles (<100 μm) in tissue is much smaller than that of previously described β-emitters in this chapter; so, despite the high energy, because of the short penetra‐ tion range, bone marrow damage is minimal [86]. Nonhematological toxicities are more commonly observed, and are mild to moderate in intensity. The most common side effects are diarrhea, fatigue, nausea, vomiting, and bone pain, some of which are dose-related [87–89]. These side effects are easily manageable with symptomatic and supportive treatments [90].

Parker et al. [89] performed a randomized, double-blind, dose-finding, Phase II study that included 122 PC patients who were randomized to be treated with three injections of 223Ra at 6-week intervals, at doses of 25 kBq/kg (*n* = 41), 50 kBq/kg (*n* = 39), or 80 kBq/kg (*n* = 42). They compared the proportion of patients in each group with confirmed PSA decline of ≥50%. No patient in the 25 kBq/kg dose group showed a significant PSA decline ≥50%. In the 50 kBq/kg dose group, only two patients (6%) showed a significant PSA decline, whereas in five patients (13%) in the 80 kBq/kg dose group, a significant PSA decline was reported (*P* = 0.0297). A ≥50% decrease in the bone alkaline phosphatase level was reported in 6 patients (16%), 24 patients (67%), and 25 patients (66%), in the 25, 50, and 80 kBq/kg dose groups, respectively (*P* < 0.0001). The most common treatment-related adverse events (≥10%) occurring up to week 24 across all dose groups were diarrhea (21%), nausea (16%), and anemia (14%). No difference in the incidence of hematological events was seen among the dose groups. They concluded that 223Ra had a dose-dependent effect on serum markers of PC activity, suggesting that controlling bone disease with 223Ra may affect cancer-related outcomes [89].

Clearance is bi-exponential after administration, comprising rapid bone uptake (half-life of 5.5

A dose escalation study with 10–36 MBq/kg of 153Sm-EDTMP reported a pain relief rate of 65%, with a duration range from 4 to 35 weeks [78]. A further dose escalation study in 52 patients using administered activities from 37 to 111 MBq/kg had a response rate of 74% with a median duration of 10 weeks [79]. Larger studies with more than 100 patients showed a median therapeutic efficacy of 80%. In a randomized, double-blind, placebo trial (*n* = 152), pain relief was found in 65% of patients after 153Sm-EDTMP treatment compared to 45% in the placebo group [80]. A significant decrease in pain between 153Sm-EDTMP and placebo was reported after 1 week, and the analgesic intake was significantly reduced after 3 and 4 weeks. Two large studies using 153Sm-EDTMP with more than 550 patients reported response rates of 73 and 86%

**5.6. Comparing the pain response between different radionuclides**

Dickie et al. compared 89Sr with 153Sm in 57 prostate cancer patients. They found no difference in the pain response rate and toxicity [83]. van der Poel et al. compared 186Re with 89Sr and reported no differences in the response rate or toxicity [54]. A nonrandomized comparison of 188Re-HEDP and 153Sm-EDTMP in patients with painful metastases from prostate and breast cancer by Liepe et al. [84] showed a comparable response and toxicity with both agents. Liepe et al. also performed a comparative study of 188Re-HEDP, 186Re-HEDP, 153Sm-EDTMP, and

 in the treatment of painful bone metastases. They reported that all radiopharmaceuticals were effective in pain palliation, without the induction of severe side effects or significant

<sup>223</sup>Ra, an α-emitter, has a half-life of 11.4 days, with a total emitted energy of about 28 MeV. It is the only FDA-approved radiopharmaceutical for the treatment of bone metastases of PC with positive impact on overall survival according to a prospective randomized study [10]. It is a bone stromal-targeted radiopharmaceutical that undergoes α-emission. The α-particle is considerably more destructive to tumor cells than the β-particle. 223Ra has a very high linear energy transfer, and only one to five hits per cell can be fatal. Double-strand breaks are induced

The penetration range of α-particles (<100 μm) in tissue is much smaller than that of previously described β-emitters in this chapter; so, despite the high energy, because of the short penetra‐ tion range, bone marrow damage is minimal [86]. Nonhematological toxicities are more commonly observed, and are mild to moderate in intensity. The most common side effects are diarrhea, fatigue, nausea, vomiting, and bone pain, some of which are dose-related [87–89]. These side effects are easily manageable with symptomatic and supportive treatments [90]. Parker et al. [89] performed a randomized, double-blind, dose-finding, Phase II study that included 122 PC patients who were randomized to be treated with three injections of 223Ra at 6-week intervals, at doses of 25 kBq/kg (*n* = 41), 50 kBq/kg (*n* = 39), or 80 kBq/kg (*n* = 42). They

min) and plasma renal clearance (half-life of 65 min) [77].

68 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

differences in therapeutic efficacy or toxicity [39].

even in quiescent cells and at low oxygen levels [85].

**5.7. 223Radium dichloride**

[81, 82].

89SrCl2

The ALSYMPCA trial (ALpharadin in SYMptomatic Prostate CAncer) is the first randomized Phase III study demonstrating improved survival with a bone-seeking radioisotope [42]. The number of PC patients recruited was 921. All patients were required to have progressed with symptomatic bone metastases, with at least two or more metastases on bone scintigraphy with no known visceral metastases. Randomization was 2:1 in a double-blind fashion to receive six cycles of intravenous <sup>223</sup>Ra every 4 weeks with best standard of care or six infusions of placebo with best standard of care. This study demonstrated a significant prolongation of survival (14.9 vs. 11.3 months, respectively; *P* < 0.001). Apart from this, the frequency of skeletal-related events was reduced in the <sup>223</sup>Ra group, and the median time to a skeletal-related event increased (15.6 vs. 9.8 months; *P* < 0.001). <sup>223</sup>Ra was well-tolerated with low rates of grade 3/4 neutropenia (1.8 vs. 0.8%) and thrombocytopenia (4 vs. 2%) [42].

Etchebehere et al. retrospectively reviewed 110 patients with metastatic PC treated with <sup>223</sup>Ra. The end points of this study were overall survival, bone event-free survival, progression-free survival (PFS), and bone marrow failure. They evaluated the following parameters prior to the first therapy cycle: hemoglobin (Hb), PSA, alkaline phosphatase (ALP), ECOG status, pain score, prior chemotherapy, and external beam radiation therapy (EBRT). Furthermore during/ after 223Ra, the PSA doubling time (PSADT), the total number of radium cycles (RaTot), and the use of chemotherapy, EBRT, enzalutamide, and abiraterone were evaluated. A significant reduction of alkaline phosphatase and pain score occurred throughout the <sup>223</sup>Ra cycles. The risk of progression was associated with declining ECOG status and decrease in PSADT. RaTot, initial ECOG(Eastern Cooperative Oncology Group) status, ALP, initial pain score, and the use of abiraterone were associated with OS (*P* ≤ 0.008), PFS (*P* ≤ 0.003), and BeFS (*P* ≤ 0.020). RaTot, initial ECOG status, ALP, and initial pain score were significantly associated with bone marrow failure (*P* ≤ 0.001), as well as Hb (*P* < 0.001) and EBRT (*P* = 0.009). In the multivariable analysis, only RaTot and abiraterone remained significantly associated with OS (*P* < 0.001 and *P* = 0.033, respectively), PFS (*P* < 0.001 and *P* = 0.041, respectively), and BeFS (*P* < 0.001 and *P* = 0.019, respectively). Additionally, RaTot (*P* = 0.027) and EBRT (*P* = 0.013) remained signifi‐ cantly associated with bone marrow failure. They concluded that the concomitant use of abiraterone and 223Ra seems to have a beneficial effect, while EBRT may increase the risk of bone marrow failure [91].

Recently, Pacilio et al. [90] performed a dosimetry study and showed that the lesion uptake of 223Ra was significantly correlated with that of 99mTc-MDP. The DRBE (RBE, relative biological effectiveness; DRBE, RBE-weighted absorbed dose) to lesions per unit administered activity was much higher than that of other bone-seeking radiopharmaceuticals, but considering a standard administration of 21 MBq (six injections of 50 kBq/kg to a 70-kg patient), the mean cumulative value of DRBE was about 19 Gy, and was therefore in a similar range as other radiopharma‐ ceuticals.

Nilsson et al. [92] reported the quality-of-life results of the ALSYMPCA study. It was found that improved survival with 223Ra was accompanied by significant quality-of-life benefits, including a higher percentage of patients with meaningful quality-of-life improvements and a slower decline in quality-of-life over time.

#### **6. Conclusion**

A pain response is seen in approximately one-half of patients treated with radionuclides for painful osseous metastases of prostate cancer. The ALSYMPCA study showed an OS benefit with 223Ra treatment. However, it should be mentioned that this study was supported by the company, Bayer. The other radiopharmaceuticals which are mentioned in this chapter were not tested in prospective multicenter trials with a large number of patients. This means that β-emitters could also have an OS benefit, which was shown in only a few studies. A combi‐ nation of hormone therapy with bone-targeted therapy may be more effective than a single therapy approach. Different combinations of therapies are being studied at the moment. PSMA-targeted therapy has so far shown very promising results. According to the published studies, 177Lu-PSMA therapy after 223Ra is feasible and safe.

#### **Author details**

#### Hojjat Ahmadzadehfar

Address all correspondence to: Hojjat.ahmadzadehfar@ukb.uni-bonn.de; nuclearmedicine@gmail.com

Department of Nuclear Medicine, University Hospital Bonn, Bonn, Germany

#### **References**

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Recently, Pacilio et al. [90] performed a dosimetry study and showed that the lesion uptake of 223Ra was significantly correlated with that of 99mTc-MDP. The DRBE (RBE, relative biological effectiveness; DRBE, RBE-weighted absorbed dose) to lesions per unit administered activity was much higher than that of other bone-seeking radiopharmaceuticals, but considering a standard administration of 21 MBq (six injections of 50 kBq/kg to a 70-kg patient), the mean cumulative value of DRBE was about 19 Gy, and was therefore in a similar range as other radiopharma‐

Nilsson et al. [92] reported the quality-of-life results of the ALSYMPCA study. It was found that improved survival with 223Ra was accompanied by significant quality-of-life benefits, including a higher percentage of patients with meaningful quality-of-life improvements and

A pain response is seen in approximately one-half of patients treated with radionuclides for painful osseous metastases of prostate cancer. The ALSYMPCA study showed an OS benefit with 223Ra treatment. However, it should be mentioned that this study was supported by the company, Bayer. The other radiopharmaceuticals which are mentioned in this chapter were not tested in prospective multicenter trials with a large number of patients. This means that β-emitters could also have an OS benefit, which was shown in only a few studies. A combi‐ nation of hormone therapy with bone-targeted therapy may be more effective than a single therapy approach. Different combinations of therapies are being studied at the moment. PSMA-targeted therapy has so far shown very promising results. According to the published

ceuticals.

**6. Conclusion**

**Author details**

**References**

Hojjat Ahmadzadehfar

nuclearmedicine@gmail.com

a slower decline in quality-of-life over time.

70 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

studies, 177Lu-PSMA therapy after 223Ra is feasible and safe.

Address all correspondence to: Hojjat.ahmadzadehfar@ukb.uni-bonn.de;

Department of Nuclear Medicine, University Hospital Bonn, Bonn, Germany

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### **Samarium-153 Therapy and Radiation Dose for Prostate Cancer**

Yasemin Parlak, Gul Gumuser and Elvan Sayit

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64670

#### **Abstract**

Prostate cancer (PC) is one of the most frequent malignancies in Western countries. At initial diagnosis, bone metastases are present in 15–30% of cases. These metastases cause some complications including bone fracture, hypercalcemia, and bone pain, which significantly affect patients' quality of life. Radionuclide treatment was created as an alternative to external palliative radiotherapy in the treatment of bone pain arising from bone metastasis of PC. The basic principle of the radionuclide treatment of pain is that the uptake of radioactive material is kept in a high amount that is enough to consti‐ tute a proper clinical impact in the tumor, and it is kept at a low dose enough to avoid the occurrence of significant adverse effects in other organs (commonly in the bone marrow). Samarium-153 ethylenediaminetetramethylenephosphonic acid (153Sm-EDTMP) is a radiopharmaceutical compound that has an affinity for skeletal tissue and concentrates in areas of increased bone turnover, localizes in the skeleton, and is excreted via glomerular filtration. Medical staff preparing and administering radio‐ pharmaceuticals in nuclear medicine, whether for diagnostic imaging or for therapeu‐ tic application, may receive significant radiation doses to their hands, particularly the fingers. Sm-153 treatment can be used as an effective and safe treatment alternative in the management of metastatic bone pain. Radiation protection of the public and the environment after Sm-153 EDTMP therapy is important.

**Keywords:** Sm-153 therapy, radiation dose, bone palliation, prostate cancer, radionu‐ clide therapy

#### **1. Introduction**

Prostate cancer is one of the most common malignancies worldwide and the third most common cause of death from cancer in men. In advanced prostate cancer, spread of the disease to the

© 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.

skeleton occurs in the majority of patients, with skeletal metastases being predominantly osteoblastic in nature [1, 2]. Bone metastasis is a common sequela of solid malignant tumors such as prostate, breast, lung, and renal cancers, which can lead to various complications, including fractures, hypercalcemia, and bone pain, as well as reduced performance status and quality of life [3]. A multidisciplinary approach is often required not only to differentiate the specific cause of the pain but also for appropriate patient management. Several radiopharma‐ ceuticals for treating painful bone metastases have been developed [3]. Radiation is of proven benefit for pain palliation, and there is growing interest in the therapeutic potential of boneseeking radiopharmaceuticals [4]. Radionuclide therapy has been proposed as an alternative modality for the management of bone pain. These radiopharmaceuticals localize preferential‐ ly in active bone and, mainly, at metastatic lesions, allowing site-directed radiotherapy [1].

The basic principle of the radionuclide treatment of pain is that the uptake of radioactive material is kept in a high amount that is enough to constitute a proper clinical impact in the tumor, and it is kept at a low dose enough to avoid the occurrence of significant adverse effects in other organs (commonly in the bone marrow where more side effects were seen) [5]. One of the most important advantages of the pain treatment with radionuclide agents is the repeatability of the procedure [3]. The radioactive isotopes of P-32 and Sr-39 are the initial radiopharmaceuticals in radionuclide treatment of painful bone metastasis and most recently, Sm-153[4]. Sm-153 EDTMP is an effective treatment of painful bone metastases from different neoplasms. However, there are few studies describing clinical experience with this therapeutic modality. Medical staff preparing and administering radiopharmaceuticals in nuclear medicine, whether for diagnostic imaging or for therapeutic application, may receive signifi‐ cant radiation doses to their hands, particularly to the fingers. People occupationally exposed to radiation must have the relevant technical knowledge and competence, that is, must at least be aware of radiation protection rules and dose-optimized work practices.

The aim of this chapter was to evaluate the efficacy of Sm-153 EDTMP. The objective is to evaluate extremity doses and dose distributions across the hands of the medical staff working in Nuclear Medicine departments.

#### **2. Main text**

#### **2.1. Prostate cancer**

Prostate cancer is the most commonly diagnosed male malignancy. Prostate cancer is the most prevalent nonskin cancer among men in the United States and is the second leading cause of cancer deaths in men [6].

Prostate-specific membrane antigen (PSMA) is a cell surface protein with a significantly increased expression in prostate cancer cells when compared to other PSMA-expressing tissues such as the small intestine, renal tubular cells, or salivary glands. It therefore provides a promising target for prostate cancer–specific imaging and therapy. Recently, procedures have been developed to label PSMA with 68Ga, 99mTc, and radioiodine for positron emission tomography (PET) or single photon emission computed tomography (SPECT) imaging and therapy [7].

Choline-PET/CT is the most promising whole-body imaging modality in detecting distant metastases of prostate cancer, because of its ability to depict small pathological lymph nodes and bone metastases with a high sensitivity, specificity, and accuracy. This feature is of primary importance on management of patients with prostate cancer and for evaluating their progno‐ sis, thanks to the possibility to assess in a single session both anatomic and metabolic infor‐ mation about the disease [8]. There are several papers about the role of Ch-PET in primary prostate cancer detection and its role in staging prostatic disease before treatment. However, since the Ch uptake can occur in some benignant conditions, such as prostatitis or prostatic hyperplasia, the role of this technique in this field is still not well clear.

The 11C-Ch is characterized by a short half-life (approximately 20.4 min), and for this reason, its use is allowed only in centers provided with a cyclotron. In consideration of the logistical limitations of the use of 11C-Ch, Ch was subsequently labeled with 18F, which, thanks to the increased half-life (109.8 min), allows storage and transport. However, 18F-Ch radiotracer is characterized by an increased urinary excretion compared to 11C-Ch [9].

Bone metastases are the most common and severe complication in patients diagnosed with primary tumors. Skeletal metastases are clinically significant because of associated symptoms, complications such as pathological fracture significance for staging, treatment, and prognosis. It develops in up to 70% of patients diagnosed with prostate cancer and breast cancer. Skeletalrelated events can reduce the health-related quality of life secondary to debilitating pain, paralysis, loss of mobility, and hospitalization. Systemic palliative-targeted therapy with suitable radiopharmaceuticals has emerged as a particularly appealing and efficient treatment modality for patients with multiple skeletal metastases [9].

There are essentially three main types of particulate radiations that are of interest for palliative treatment of bone metastasis using radiopharmaceuticals: beta (β− ) particles, alpha (α) particles, and Auger electrons. Traditionally, tumor-targeted radiotherapy has used β− emitting radionuclides. However, high-energy β− particles, with a range of several millimeters in tissues, can irradiate cells nearby the targeted tumor. Conversely, α particles (typical penetration range of <100 μm) and Auger electrons (penetration range of several nanometers to micrometers) have shorter penetration ranges and higher linear energy transfer (LET) [9].

In recent years, several reports have been published describing the use of multiple radionu‐ clides in the context of palliative treatment of bone metastases.

#### **2.2. Radionuclide therapy**

skeleton occurs in the majority of patients, with skeletal metastases being predominantly osteoblastic in nature [1, 2]. Bone metastasis is a common sequela of solid malignant tumors such as prostate, breast, lung, and renal cancers, which can lead to various complications, including fractures, hypercalcemia, and bone pain, as well as reduced performance status and quality of life [3]. A multidisciplinary approach is often required not only to differentiate the specific cause of the pain but also for appropriate patient management. Several radiopharma‐ ceuticals for treating painful bone metastases have been developed [3]. Radiation is of proven benefit for pain palliation, and there is growing interest in the therapeutic potential of boneseeking radiopharmaceuticals [4]. Radionuclide therapy has been proposed as an alternative modality for the management of bone pain. These radiopharmaceuticals localize preferential‐ ly in active bone and, mainly, at metastatic lesions, allowing site-directed radiotherapy [1].

82 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

The basic principle of the radionuclide treatment of pain is that the uptake of radioactive material is kept in a high amount that is enough to constitute a proper clinical impact in the tumor, and it is kept at a low dose enough to avoid the occurrence of significant adverse effects in other organs (commonly in the bone marrow where more side effects were seen) [5]. One of the most important advantages of the pain treatment with radionuclide agents is the repeatability of the procedure [3]. The radioactive isotopes of P-32 and Sr-39 are the initial radiopharmaceuticals in radionuclide treatment of painful bone metastasis and most recently, Sm-153[4]. Sm-153 EDTMP is an effective treatment of painful bone metastases from different neoplasms. However, there are few studies describing clinical experience with this therapeutic modality. Medical staff preparing and administering radiopharmaceuticals in nuclear medicine, whether for diagnostic imaging or for therapeutic application, may receive signifi‐ cant radiation doses to their hands, particularly to the fingers. People occupationally exposed to radiation must have the relevant technical knowledge and competence, that is, must at least

The aim of this chapter was to evaluate the efficacy of Sm-153 EDTMP. The objective is to evaluate extremity doses and dose distributions across the hands of the medical staff working

Prostate cancer is the most commonly diagnosed male malignancy. Prostate cancer is the most prevalent nonskin cancer among men in the United States and is the second leading

Prostate-specific membrane antigen (PSMA) is a cell surface protein with a significantly increased expression in prostate cancer cells when compared to other PSMA-expressing tissues such as the small intestine, renal tubular cells, or salivary glands. It therefore provides a promising target for prostate cancer–specific imaging and therapy. Recently, procedures have been developed to label PSMA with 68Ga, 99mTc, and radioiodine for positron emission

be aware of radiation protection rules and dose-optimized work practices.

in Nuclear Medicine departments.

cause of cancer deaths in men [6].

**2. Main text**

**2.1. Prostate cancer**

Pain is the most common symptom in the prostate cancer patients. The incidence and severity of pain in the last period of the life is increasing. Patients and their relatives may adversely be affected from the quality of life as a major fear source. However, pain related to prostate cancer can be treated effectively about 85–90% by applying correct approaches. The remaining 5–15% of patients with pain can be achieved by applying appropriate surgical techniques. The severity and frequency of pain in cancer patients depends on many factors such as age of the patients, the stage of the disease, and the site of bone metastases. For effective pain treatment, accom‐ panying medical and psychosocial problems of the patients should be evaluated, and then, appropriate treatment should be done [10].

In recent years, there has been a much greater emphasis on "radionuclide therapies" that are designed to damage only the cancerous cells. At present, effective targeted radiopharmaceut‐ ical therapeutics have been developed. Radionuclide therapy uses ionizing radiation to minimize tumors and kill cancer cells. The basic principles in the treatment of radioactive elements in nuclear medicine, to benefit from the devastating effects generated in the cells. Therefore, many radionuclides have oncological applications of many proven efficacy and safety. Radionuclide therapy uses radioactive isotopes, administered either orally or intrave‐ nously, to deliver highly targeted therapy for a range of disorders, enabling the delivery of a high dose to the target, while minimizing normal tissue toxicity [11].

In targeted radionuclide therapy, the biological effect is obtained by energy absorbed from the radiation emitted by the radionuclide. Whereas the radionuclides used for diagnostic nuclear medicine procedures emit gamma rays, which can penetrate highly into the body, the radio‐ nuclides used for radionuclide therapy must emit radiation with a comparatively short range. Radionuclides emitted beta particles, alpha particle, and Auger electrons for radionuclide therapy use due to short range and high ionization capability. In some cases, mixed emitters are used to perform both imaging and therapy with the same radionuclide (e.g., Samari‐ um-153) [11].

Various radiopharmaceuticals have been advanced for the treatment of painful bone meta‐ stases (**Table 1**). The physical characteristics of these radionuclides are different and have specific benefits. These radionuclides are administered intravenously or orally and localize the painful bone metastases with a high target-to-nontarget tissue ratio and a very low concen‐ tration in the normal bone, especially bone marrow. The therapeutic suitability of the radio‐ nuclides is important. So the penetration range of the radionuclides is concerned with the energy of the electrons. The applications of the radiopharmaceuticals are easily performed without the need for expensive high-technology equipment. Thus, these agents can be applicable not only in major medical centers but also in minor hospitals, and the workers have to be educated to comply with Nuclear Regulatory Commission requirements. These agents target not only osteoblastic lesions but also lesions containing osteolytic and osteoblastic components. Most of the patients who are treated observed reduce of pain, thus reducing their need for analgesics and improving the quality of life and mobility [3, 4].

The studies regarding metastatic bone pain have particularly focused on the metastases of hormone refractory prostate cancer, which was resistant to the treatment of opioid analgesic. While radiotherapy is more appropriate in the palliative treatment of localized, regional metastatic lesions, this management is less applicable in the treatment of diffuse metastatic lesions. The type of metastatic bone pain is different from other somatic pains, such as visceral, neuropathic, arthritic, and neuropathic pain. While the severity of pain is less intensive initially, it will progress a chronicle process including acute pain episodes with increasing severity subsequently. In this process, the pathophysiology of pain cannot be clearly explained, and various theories have been proposed [12].


**Table 1.** Physical properties of therapy isotopes for bone pain palliation.

the stage of the disease, and the site of bone metastases. For effective pain treatment, accom‐ panying medical and psychosocial problems of the patients should be evaluated, and then,

In recent years, there has been a much greater emphasis on "radionuclide therapies" that are designed to damage only the cancerous cells. At present, effective targeted radiopharmaceut‐ ical therapeutics have been developed. Radionuclide therapy uses ionizing radiation to minimize tumors and kill cancer cells. The basic principles in the treatment of radioactive elements in nuclear medicine, to benefit from the devastating effects generated in the cells. Therefore, many radionuclides have oncological applications of many proven efficacy and safety. Radionuclide therapy uses radioactive isotopes, administered either orally or intrave‐ nously, to deliver highly targeted therapy for a range of disorders, enabling the delivery of a

In targeted radionuclide therapy, the biological effect is obtained by energy absorbed from the radiation emitted by the radionuclide. Whereas the radionuclides used for diagnostic nuclear medicine procedures emit gamma rays, which can penetrate highly into the body, the radio‐ nuclides used for radionuclide therapy must emit radiation with a comparatively short range. Radionuclides emitted beta particles, alpha particle, and Auger electrons for radionuclide therapy use due to short range and high ionization capability. In some cases, mixed emitters are used to perform both imaging and therapy with the same radionuclide (e.g., Samari‐

Various radiopharmaceuticals have been advanced for the treatment of painful bone meta‐ stases (**Table 1**). The physical characteristics of these radionuclides are different and have specific benefits. These radionuclides are administered intravenously or orally and localize the painful bone metastases with a high target-to-nontarget tissue ratio and a very low concen‐ tration in the normal bone, especially bone marrow. The therapeutic suitability of the radio‐ nuclides is important. So the penetration range of the radionuclides is concerned with the energy of the electrons. The applications of the radiopharmaceuticals are easily performed without the need for expensive high-technology equipment. Thus, these agents can be applicable not only in major medical centers but also in minor hospitals, and the workers have to be educated to comply with Nuclear Regulatory Commission requirements. These agents target not only osteoblastic lesions but also lesions containing osteolytic and osteoblastic components. Most of the patients who are treated observed reduce of pain, thus reducing their

The studies regarding metastatic bone pain have particularly focused on the metastases of hormone refractory prostate cancer, which was resistant to the treatment of opioid analgesic. While radiotherapy is more appropriate in the palliative treatment of localized, regional metastatic lesions, this management is less applicable in the treatment of diffuse metastatic lesions. The type of metastatic bone pain is different from other somatic pains, such as visceral, neuropathic, arthritic, and neuropathic pain. While the severity of pain is less intensive initially, it will progress a chronicle process including acute pain episodes with increasing severity subsequently. In this process, the pathophysiology of pain cannot be clearly explained,

high dose to the target, while minimizing normal tissue toxicity [11].

need for analgesics and improving the quality of life and mobility [3, 4].

and various theories have been proposed [12].

appropriate treatment should be done [10].

84 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

um-153) [11].

A study reported that the maximal pain response to the treatment occurred at 4–6 weeks after the treatment [13]. In another study, Silberstein [14] declared that the complete or partial response to 153Sm treatment was obtained in 62–74% of patients, and this response was commonly seen 5–10 days after the treatment.

The study investigated the efficacy of 153Sm treatment; complete response rate was found 12.4% and the partial response rate was found 73.4%.

Gul et al. determine the reduction in the analgesic consumption and improvement in the performance and mobility score of the patients (10).

Alleviating of pain can occurred about within 2–7 days depending on the agent after the first injection. The repeating injections should be performed as soon as possible after the occurrence of pain recurrence, if bone marrow reserve is adequate. The clinical usage of radionuclides is not only limited to the opioid resistant pain. As the result of some trials, it was obviously clear that the expected long-term efficacy and tolerance could be achieved without requirement of opioid analgesics. The primary goal of the treatment is to provide better quality of life in daily activities by minimal drug usage [3].

#### *2.2.1. Therapy radionuclides for bone pain palliation*

In the past few years, several radiopharmaceuticals have been improved with bone-seeking properties that provide palliation of pain to multiple bone metastasis. The most of these are beta electrons, depositing highly their energy over up to millimeters in the surrounding tissues. A few of the therapeutic radionuclides emit small amounts of gamma-radiation, allowing for a scintigraphic imaging. The commonly used radiopharmaceutical for pain palliation is samarium-153 HEDP. Hematopoietic suppression is the major side effect of radionuclide therapies, with leukopenia and thrombocytopenia more likely to be clinically significant than anemia. The physical properties of radiopharmaceuticals are discussed in detail in the following sections.

#### *2.2.2. Phosphorus-32*

Phosphorus-32 is pure beta-emitting radionuclide with a physical half-life of 14.3 days. The average beta particle energy is 695 keV. The mean and maximum particle ranges in tissue of phosphorus are 3 and 8 mm, respectively. P-32 is used orthophosphate compound as palliative treatment purposes. Approximately 85% of the total phosphate pool is located in the skeleton bound as inorganic phosphate to the hydroxyapatite matrix. From five to ten percentage of administered activity is excreted via the kidneys within the first 24 h. Total bone doses in the range 0.4–1.7 cGy/MBq have been reported [4, 15].

#### *2.2.3. Strontium-89*

Strontium-89 is a pure beta-emitter with a beta particle energy of 1.46 MeV and a physical halflife of 50.5 days [15]. It localizes in bone primarily in areas of osteoblastic activity. It is an mean particle range in tissue of 2.4 mm. The fix dose is 148 MBq (4 mCi). The radiation dose of metastatic foci is about 1000–5000 cGy. Bone marrow radiation dose is approximately ten percent of metastatic foci. 89Sr can be effective at relieve pain from bone metastases, particu‐ larly for metastatic prostate or breast cancer [4].

#### *2.2.4. Rhenium-186*

Rhenium-186 is a beta- and gamma-emitting radionuclide. The maximum beta particle energy is 1.07 MeV. Re-186 has a 137 keV, 9% abundance gamma-photon with a physical half-life of 89.3 h. It is forms a stable diphosphonate chelate with hydroxyethylidene diphosphonate (HEDP). Rhenium-186 HEDP has rapid urinary excretion. It is excreted about 70% of dose within 6 h in the urine [4].

#### *2.2.5. Sn-117m*

Sn-117m is gamma-emitting radionuclide with 159 keV. Physical half-life of Sn-117m is 13.6 days. It is decays by short-range conversion electrons. Bone marrow toxicity of this radionuclide is low because of its short range. Therefore, Sn-117m can be used to treat bone tumors and rheumatoid arthritis [16].

#### *2.2.6. Samarium-153*

Samarium-153 is a reactor which produced high radionuclidic purity by neutron bombard‐ ment of enriched 152Sm oxide [3]. Samarium-153 is a beta-emitter with a short half-life (46.7 h), which also emits gamma-photons suitable for imaging at 103 keV. Samarium-153 principal radiation emission data are shown in **Table 2**. The isotope is chelated to ethylene diamine tetramethylene phosphonate (EDTMP), which targets the bone matrix as a polyphosphonate (**Figures 1** and **2**). The therapeutic doses administered to patients about 50% settle in the bone excretion are through the kidneys. The proportion of skeletal uptake is the highest for the boneseeking radiopharmaceuticals. The effective range of 153Sm is 2–3 mm in bone [1]. 153Sm-EDTMP is indicated for the relief of pain in patients with osteoblastic metastatic bone lesions at a standard dose of 37 MBq/kg to a maximum of 5550 MBq [1, 2]. Clinical benefit is reported by 60–80% of patients within 2 weeks of administration, frequently within 48 h, with a response duration of 4–40 weeks [4].


\*Maximum energies are listed for beta emissions, and the average beta particle energy is 233 keV

**Table 2.** Samarium-153 principal radiation emission data.

phosphorus are 3 and 8 mm, respectively. P-32 is used orthophosphate compound as palliative treatment purposes. Approximately 85% of the total phosphate pool is located in the skeleton bound as inorganic phosphate to the hydroxyapatite matrix. From five to ten percentage of administered activity is excreted via the kidneys within the first 24 h. Total bone doses in the

Strontium-89 is a pure beta-emitter with a beta particle energy of 1.46 MeV and a physical halflife of 50.5 days [15]. It localizes in bone primarily in areas of osteoblastic activity. It is an mean particle range in tissue of 2.4 mm. The fix dose is 148 MBq (4 mCi). The radiation dose of metastatic foci is about 1000–5000 cGy. Bone marrow radiation dose is approximately ten percent of metastatic foci. 89Sr can be effective at relieve pain from bone metastases, particu‐

Rhenium-186 is a beta- and gamma-emitting radionuclide. The maximum beta particle energy is 1.07 MeV. Re-186 has a 137 keV, 9% abundance gamma-photon with a physical half-life of 89.3 h. It is forms a stable diphosphonate chelate with hydroxyethylidene diphosphonate (HEDP). Rhenium-186 HEDP has rapid urinary excretion. It is excreted about 70% of dose

Sn-117m is gamma-emitting radionuclide with 159 keV. Physical half-life of Sn-117m is 13.6 days. It is decays by short-range conversion electrons. Bone marrow toxicity of this radionuclide is low because of its short range. Therefore, Sn-117m can be used to treat bone

Samarium-153 is a reactor which produced high radionuclidic purity by neutron bombard‐ ment of enriched 152Sm oxide [3]. Samarium-153 is a beta-emitter with a short half-life (46.7 h), which also emits gamma-photons suitable for imaging at 103 keV. Samarium-153 principal radiation emission data are shown in **Table 2**. The isotope is chelated to ethylene diamine tetramethylene phosphonate (EDTMP), which targets the bone matrix as a polyphosphonate (**Figures 1** and **2**). The therapeutic doses administered to patients about 50% settle in the bone excretion are through the kidneys. The proportion of skeletal uptake is the highest for the boneseeking radiopharmaceuticals. The effective range of 153Sm is 2–3 mm in bone [1]. 153Sm-EDTMP is indicated for the relief of pain in patients with osteoblastic metastatic bone lesions at a standard dose of 37 MBq/kg to a maximum of 5550 MBq [1, 2]. Clinical benefit is reported by 60–80% of patients within 2 weeks of administration, frequently within 48 h, with a response

range 0.4–1.7 cGy/MBq have been reported [4, 15].

86 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

larly for metastatic prostate or breast cancer [4].

*2.2.3. Strontium-89*

*2.2.4. Rhenium-186*

*2.2.5. Sn-117m*

*2.2.6. Samarium-153*

duration of 4–40 weeks [4].

within 6 h in the urine [4].

tumors and rheumatoid arthritis [16].

**Figure 1.** A bone scintigraphy that was obtained before the 153Sm-EDTMP treatment.

**Figure 2.** A bone scintigraphy that was obtained after the 153Sm-EDTMP treatment.

This treatment can be repeated several times to the patients. Repeat dosing with 153Sm is both safe and effective. The studied reports showed that the patients with symptomatic bone metastases receiving multiple doses of 153Sm have no significant differences in pain reduction or in myelosuppression after a second or third treatment [17].

Pregnancy, lactation, acute spinal cord compression, single metastatic lesion, renal failure, the long bone that holds more than 50% of the affected bone metastases, risk of fracture and in the presence of disseminated intravascular coagulation are the contraindications of pain palliation with 153Sm therapy.

#### *2.2.7. Radiation dose*

**Figure 2.** A bone scintigraphy that was obtained after the 153Sm-EDTMP treatment.

88 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

or in myelosuppression after a second or third treatment [17].

with 153Sm therapy.

This treatment can be repeated several times to the patients. Repeat dosing with 153Sm is both safe and effective. The studied reports showed that the patients with symptomatic bone metastases receiving multiple doses of 153Sm have no significant differences in pain reduction

Pregnancy, lactation, acute spinal cord compression, single metastatic lesion, renal failure, the long bone that holds more than 50% of the affected bone metastases, risk of fracture and in the presence of disseminated intravascular coagulation are the contraindications of pain palliation The diagnostic and therapeutic procedures have been continuously increasing in most of the nuclear medicine facilities. The risk of radiation exposure of staff is of importance due to increasing procedures. The radiation sources of nuclear medicine Departments are preparing and administering of the radiopharmaceuticals. The workers may receive significant radiation dose to their whole body, especially to the hands.

Therapeutic nuclear medicine requires special consideration due to the high doses of radiation. Therapeutic radionuclides have usually beta electrons. Owing to 153Sm-EDTMP's intermedi‐ ate beta-energy and low tissue penetration, the bone marrow, for the most part, is spared throughout the skeleton. For protection, two radiation safety consideration needs to be paid attention. One of them, external radiation dose of 153Sm, the interaction of high-energy beta particles with high atomic number materials (e.g., lead) will lead to the production of highenergy X-rays (Bremsstrahlung). Parlak et al. reported that external radiation dose of 153Sm is high for the first 8 h (**Figure 3**). They suggest that hospitalizing the patients treated with Sm-153 therapy in an isolated room for 8 h would be helpful for radiation protection of the public. On the other hand, radiation safety consideration is the contamination by excretion of the 153Sm. The variability of isolation times indicates a strong dependency of effective halflife on biological excretion and shows no relationship with administered activity. Certainly, this variability reveals the need to determine these parameters for each patient [1].

**Figure 3.** Excretion of 153Sm-EDTMP through the urinary tract in the first 24 h.

Developments in nuclear medicine show that applications involving beta-emitters will probably increase further. Data on the beta-radiation dose equivalents for the staff performing such treatments are limited. For this reason, additional measures should be provided for the training and continuing education of the staff in order to avoid any further increase in extremity doses.

#### **Author details**

Yasemin Parlak\* , Gul Gumuser and Elvan Sayit

\*Address all correspondence to: yasemin.gultekin@hotmail.com

Department of Nuclear Medicine, Medical School, Celal Bayar University, Manisa, Turkey

#### **References**


treatment of painful bone metastatic disease. Clinical Nuclear Medicine 2006;31:605– 610

[12] Clines GA, Guise TA. Molecular mechanisms and treatment of bone metastasis. Expert Rev Mol Med 2008;10:e7

**Author details**

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**References**

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, Gul Gumuser and Elvan Sayit \*Address all correspondence to: yasemin.gultekin@hotmail.com

90 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

Department of Nuclear Medicine, Medical School, Celal Bayar University, Manisa, Turkey

[1] Parlak Y, Gumuser G, Sayit E, Samarium-153 therapy for prostate cancer: the evaluation of urine activity, staff exposure and dose rate from patients. Radiat Prot Dosimetry

[4] Lewington VJ. Cancer therapy using bone-seeking isotopes. Phys Med Biol

[5] Sahin M, Basoglu T, Bernay I. Radyonuklid Palyatif Agri Tedavisi. On Dokuz Mayis

[6] Hanchette CL ,Schwartz GG. Geographic patterns of prostate cancer mortality. Evidence for a protective effect of ultraviolet radiation. Cancer. 1992;70(12):2861–2869.

[7] Afshar-Oromieh A, Zechmann CM, Malcher A, Eder M, Eisenhut M, Linhart HG, Holland-Letz T, Hadaschik BA, Giesel FL, Debus J, Haberkorn U. Comparison of PET imaging with a 68Ga-labelled PSMA ligand and 18F-choline-based PET/CT for the diagnosis of recurrent prostate cancer. Eur J Nucl Med Mol Imaging 2014;41:11–20

[8] Valeria Panebianco, Flavio Barchetti, Daniela Musio, Francesca De Felice, Camilla Proietti, Elena Lucia Indino, Valentina Megna, Orazio Schillaci, Carlo Catalano, and Vincenzo Tombolini. Advanced imaging for the early diagnosis of local recurrence prostate cancer after radical prostatectomy. BioMed Res Int, 2014;2014;12, Article ID

[9] Guerra Liberal FDC, Tavares AAS, João Manuel RS. Tavares N. Palliative treatment of metastatic bone pain with radiopharmaceuticals: a perspective beyond Strontium-89

[10] Gul SS, Sonmez O, Deveci EK, Ilce HT, Ergonenc JS. Quantitative assessment of radionuclide pain palliation: case report. Türk Onkoloji Dergisi 2013;28(2):75–80

[11] Coronado M, Redondo A, Coya J, Espinosa E, Couto RM, Zamora P, Marin MD, Castelo B, Lillo ME, Frutos L, Barón MG, and Curto LM. Clinical role of Sm-153 EDTMP in the

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[2] Damber JE, Aus G. Prostate cancer. Lancet 2008;371:1710–1721


### **Redefining Androgen Receptor Function: Clinical Implications in Understanding Prostate Cancer Progression and Therapeutic Resistance**

Miltiadis Paliouras, Carlos Alvarado and Mark Trifiro

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64392

#### **Abstract**

The current description of the function of the human androgen receptor (AR), as a transcription factor directing androgen responsive gene expression, is limited in scope and thus is unable to account for the varied cellular and physiological transformation observed in the development and progression of prostate cancer (CaP). The chapter will focus on four important aspects of AR and CaP investigations: (1) a description of AR somatic mutations and the perils of AR-directed therapeutics; (2) our characterization of AR protein interactors that have imbued new functional properties for AR linked to prostatic disease; (3) review of the advances made and shortcomings of AR mouse models in describing CaP onset and progression; and (4) speculate as to the mechanisms by which new mutations can originate and initiate disease onset.

**Keywords:** androgen receptor, prostate cancer, somatic mutations, interactome, mouse models, gain-of-function properties, therapeutic resistance, mutational land‐ scape

#### **1. Introduction**

Advanced DNA sequencing technology and the information garnered from it has ushered a new era especially poignant to the genetics of cancer. In present and next-generation sequenc‐ ing methodologies in conjunction with the establishment of consortiums (COSMIC: and TCGA: http://cancer.sanger.ac.uk/cosmic and TCGA: tcga-data.nci.nih.gov/tcga), whose major efforts are to characterize the cancer genome of a large number of cancers in a systematic fashion,

© 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.

moderncancergeneticshascometotheforefront.These"mutationallandscapes"haveredefined cancer genetics and will dramatically direct cancer research for decades to come [1–10].

Modern cancer genetics has now unequivocally demonstrated extensive somatic DNA alterations many times more than previously envisioned [11–14]. Although dependent on specific tumor types, somatic mutations are in the order of tens of thousands; the present-day technology most likely underestimates the true number of mutations as mutations occurring in less than 10–15% of cells cannot be detected. Advances in single-cell DNA analysis now suggest that indeed many more mutations do exist at in smaller number of cells [15, 16]. More importantly, there is an advanced degree of intertumoral heterogeneity where the same tumor types in different patients share only a few DNA alterations [17, 18]. As well intratumoral heterogeneity is extensive, where in the same individual's tumor, there are many different DNA alterations in specific subpopulation of cells. Also, the DNA sequence defined for a specific tumor is a composite sequence, where an amalgamation of small "bits" of DNA sequence, whose origins are from many different cells, is aligned to generate the "tumor" DNA genome; where in reality, no individual tumor cell most likely has that defined sequence.

Cataloging sequence alterations are the mainstay on present-day consortiums, important in defining tumor heterogeneity and also to help understand what potential effects these alterations may have on neoplastic initiation and evolution. Many mutations evoke specific gain of function properties implying driver capabilities [19]. The true understanding of these mutations is an extremely daunting task; defining these new gain-of-function properties is presently done in the context of the somatically mutated protein in question without any of the mutations of other proteins present; to truly account for real gain of function properties would require the presence of all mutations. The possible permutations and combinations of tens-of-thousands mutations on many proteins and the outcome on cellular physiology are incomprehensible even more so when cell-to-cell functionality is implied.

Nonetheless, the establishment of mutational landscape databases with defining characteris‐ tics, in conjunction with the required systems biology and network analysis, has led to many insights in tumor dynamics. What has been lacking in cancer fundamentals are investigations addressing the origins of these vastly accrued DNA alterations.

Cancer hallmarks defined by Hanahan and Weinberg have more or less been universally agreed upon and now include "enabling hallmarks," those hallmarks that are not descriptive in nature but imply distinct contributions to neoplastic development [20]. One of these enabling hallmarks is referred to as genomic instability. A more apt description would be the connotation of mutator phenotype, originally described by Lawrence Loeb [21–23]. Briefly, the mutator phenotype is a trait shared by all cancer cells that endow cancer cells with the ability to create or enhance new and constant DNA alterations. This hallmark gives neoplastic cells, a constant source of new mutations allowing the genetic background to become widely disparate. Such cellular genetic diversity in turn allows for extreme selection processes to dictate tumoral evolution; selection processes are multiple: microenvironment on tumor cells, tumor cells on the microenvironment, and tumor cells on other tumor cells.

The origins of tumor DNA alterations are indeed critical. Therefore, it is hard to imagine that a tumor and tumor evolution can exist without any DNA alterations. Mutational load directly impacts tumor aggressiveness and metastatic potential. Understanding the origins of somatic DNA alterations is now fundamental to the understanding of tumor initiation and evolution, and the extent of DNA alterations is most likely more critical than the actual single definition and characterization of specific DNA alterations given the tremendous heterogeneity that exists.

#### **2. Somatic mutations and prostate cancer**

moderncancergeneticshascometotheforefront.These"mutationallandscapes"haveredefined cancer genetics and will dramatically direct cancer research for decades to come [1–10].

94 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

Modern cancer genetics has now unequivocally demonstrated extensive somatic DNA alterations many times more than previously envisioned [11–14]. Although dependent on specific tumor types, somatic mutations are in the order of tens of thousands; the present-day technology most likely underestimates the true number of mutations as mutations occurring in less than 10–15% of cells cannot be detected. Advances in single-cell DNA analysis now suggest that indeed many more mutations do exist at in smaller number of cells [15, 16]. More importantly, there is an advanced degree of intertumoral heterogeneity where the same tumor types in different patients share only a few DNA alterations [17, 18]. As well intratumoral heterogeneity is extensive, where in the same individual's tumor, there are many different DNA alterations in specific subpopulation of cells. Also, the DNA sequence defined for a specific tumor is a composite sequence, where an amalgamation of small "bits" of DNA sequence, whose origins are from many different cells, is aligned to generate the "tumor" DNA genome; where in reality, no individual tumor cell most likely has that defined sequence.

Cataloging sequence alterations are the mainstay on present-day consortiums, important in defining tumor heterogeneity and also to help understand what potential effects these alterations may have on neoplastic initiation and evolution. Many mutations evoke specific gain of function properties implying driver capabilities [19]. The true understanding of these mutations is an extremely daunting task; defining these new gain-of-function properties is presently done in the context of the somatically mutated protein in question without any of the mutations of other proteins present; to truly account for real gain of function properties would require the presence of all mutations. The possible permutations and combinations of tens-of-thousands mutations on many proteins and the outcome on cellular physiology are

Nonetheless, the establishment of mutational landscape databases with defining characteris‐ tics, in conjunction with the required systems biology and network analysis, has led to many insights in tumor dynamics. What has been lacking in cancer fundamentals are investigations

Cancer hallmarks defined by Hanahan and Weinberg have more or less been universally agreed upon and now include "enabling hallmarks," those hallmarks that are not descriptive in nature but imply distinct contributions to neoplastic development [20]. One of these enabling hallmarks is referred to as genomic instability. A more apt description would be the connotation of mutator phenotype, originally described by Lawrence Loeb [21–23]. Briefly, the mutator phenotype is a trait shared by all cancer cells that endow cancer cells with the ability to create or enhance new and constant DNA alterations. This hallmark gives neoplastic cells, a constant source of new mutations allowing the genetic background to become widely disparate. Such cellular genetic diversity in turn allows for extreme selection processes to dictate tumoral evolution; selection processes are multiple: microenvironment on tumor cells,

incomprehensible even more so when cell-to-cell functionality is implied.

tumor cells on the microenvironment, and tumor cells on other tumor cells.

addressing the origins of these vastly accrued DNA alterations.

Prostate cancer (CaP) in many ways is unique. It is extremely common; as much as 50% of men will have CaP above the age of 55 and increases in incidence afterwards [24]. It is for the most part slow growing and only in a small percentage can develop advanced and life-threatening disease but still represents a significant number of individuals. However, due to the high incidence rates for CaP and the highly variable and unpredictable effects on morbidity and mortality, CaP is extremely vulnerable to over diagnosis (as aided by screening advocates) and thus overtreatment [25, 26]. Treatment regimens have been extremely controversial with the no clear benefits of endocrine manipulation in early disease; most likely, the era of antiandrogens or androgen deprivation therapy (ADT) in early disease will not be adhered to, the treatment of which may have provoked more aggressive disease and linked to selecting out very worrisome gain-of-function androgen receptor (AR) mutations [27–29]. Surgical prosta‐ tectomy remains the only curative procedure if the disease was localized to the prostate at the time of surgery.

CaP is universally multifocal and is uniformly associated with hypertrophy or hyperplasia. Its pathological scoring (Gleason) is based on the fact that multiple lesions coexist and, by itself, is solely used to assess overall staging [30]. Multifocal cancers are typically genetic in nature, associated with DNA repair deficiencies and somatic loss of heterozygosity. The best example of endocrine genetic cancers is MEN2 syndrome that has been now well studied in all age groups and dramatically displays the hypertrophy to hyperplasia to frank carcinoma evolution [31]. There is no obvious related gene candidate in multifocal CaP.

#### **2.1. Androgen receptor**

The X-linked AR protein is a member of the nuclear receptor superfamily [32, 33]. It is a ligandinducible protein containing a polymorphic N-terminal region, a central DNA-binding domain (DBD), and a C-terminal ligand-binding domain (LBD) [34–36]. Although the AR gene is classically not associated with direct DNA maintenance, it is a single allele (loss of hetero‐ zygosity is not a prerequisite) and remains the most prominent candidate directing CaP initiation and evolutions. Hypogonadal individuals with low levels of 17C steroids or with elements of androgen receptor (AR) deficiency, CaP, are extremely rare. Most if not all molecular endocrinological studies of CaP implement the AR g as being a pivotal player in CaP. In all CaP, AR is highly mutated (androgendb.mcgill.ca) [37–42]. The most recent CaP mutational landscape is very comprehensive and is the new reference for mutational analysis of genes in both initial disease and more advanced disease [39]. In this study, AR remains the most consistent altered gene and is the earliest gene to be altered in localized diseases: AR gene amplifications is then followed by AR splice variants and AR missense mutations, but these alterations are hard pressed to explain multifocality. Other somatic mutations found include AR-associated proteins (ETS fusions, FOXA1, ZBTB16, NCOR1, NCOR2); PIK3 pathway PIK3CA, (PIK3CB, PIK3R1, AKT1); DNA repair (APC, BRCA2); and WNT signaling (RNF43); Cell cycle (RB1) [39].

#### **2.2. AR and the CAG polymorphic tract**

The AR gene has an extremely rare attribute. A polymorphic pure uninterrupted CAG tract in exon 1 is present coding for a polyglutamine tract in the N-terminus of the AR. This tract varies in length in individuals (n = 12–31), and tract length also varies racially [43–49]. This tract also has small but very important effect on AR functionality: smaller length polyglutamine tract ARs have more transcriptionally prowess [50]. The fundamental explanation for the presence of the AR polyglutamine tract within the AR protein itself is not known.

AR CAG tracts are unique to primates and are uninterrupted in almost all species (the exception being mice). It is interesting that humans vs. other primates have the longest tract and thus are the most unstable.

AR CAG tracts are unique to mammals and are uninterrupted in almost all species (the exception being mice) [34, 51]. Another trait related to all trinucleotide repeats is their inherent inability to remain stable; thus, AR CAG tract lengths are known to change in length somati‐ cally in various tissues including primary gonadal tissue [52, 53]. It is interesting that humans vs. other primates have the longest tract and thus are the most unstable. The instability exists at two levels: at cell division with DNA replication and more importantly with AR transcrip‐ tion by the transcription excision repair machinery. Instability is usually biased toward expansion rather than contraction (2:1).

In a study of CaP and AR CAG tract instability, AR CAG tract instability existed in normal tissue to a certain degree but was very much enhanced in adjacent CaP tissue [52, 54]. The CAG tract lengths varied from one foci of CaP to another foci of CaP in the same patient. The instability of the AR CAG tract is many orders of magnitude more than stable random DNA sequence and approaches error rates seen in DNA repair deficiency states. It thus remains a solid candidate for the gene that accounts for the multifocality of CaP. In brief, those cells that undergo the largest AR CAG tract contraction are the most active AR. These cells in turn through overactive AR pathways will provoke new DNA alterations and thus are ordained as a mutator phenotype.

#### **2.3. AR somatic mutations**

It has clear involvement in distinct diseases due to due well-characterized inherited lossof-function or somatic acquired gain-of-function mutations. The one same protein with diverse-heterogeneous mutations, each with clear phenotypes, offers unique complementary structure-functional studies. Exploiting the AR mutational properties found in individuals with androgen resistance syndromes (loss-of-function AR) or CaP (gain-of-function AR), in conjunction with receptor kinetic studies, molecular biology, advanced dynamic structural modeling, and proteomic-coupled network analyses studies, has described many fundamental and new processes to account for disease processes [55–59].

mutational landscape is very comprehensive and is the new reference for mutational analysis of genes in both initial disease and more advanced disease [39]. In this study, AR remains the most consistent altered gene and is the earliest gene to be altered in localized diseases: AR gene amplifications is then followed by AR splice variants and AR missense mutations, but these alterations are hard pressed to explain multifocality. Other somatic mutations found include AR-associated proteins (ETS fusions, FOXA1, ZBTB16, NCOR1, NCOR2); PIK3 pathway PIK3CA, (PIK3CB, PIK3R1, AKT1); DNA repair (APC, BRCA2); and WNT signaling (RNF43);

The AR gene has an extremely rare attribute. A polymorphic pure uninterrupted CAG tract in exon 1 is present coding for a polyglutamine tract in the N-terminus of the AR. This tract varies in length in individuals (n = 12–31), and tract length also varies racially [43–49]. This tract also has small but very important effect on AR functionality: smaller length polyglutamine tract ARs have more transcriptionally prowess [50]. The fundamental explanation for the presence

AR CAG tracts are unique to primates and are uninterrupted in almost all species (the exception being mice). It is interesting that humans vs. other primates have the longest tract

AR CAG tracts are unique to mammals and are uninterrupted in almost all species (the exception being mice) [34, 51]. Another trait related to all trinucleotide repeats is their inherent inability to remain stable; thus, AR CAG tract lengths are known to change in length somati‐ cally in various tissues including primary gonadal tissue [52, 53]. It is interesting that humans vs. other primates have the longest tract and thus are the most unstable. The instability exists at two levels: at cell division with DNA replication and more importantly with AR transcrip‐ tion by the transcription excision repair machinery. Instability is usually biased toward

In a study of CaP and AR CAG tract instability, AR CAG tract instability existed in normal tissue to a certain degree but was very much enhanced in adjacent CaP tissue [52, 54]. The CAG tract lengths varied from one foci of CaP to another foci of CaP in the same patient. The instability of the AR CAG tract is many orders of magnitude more than stable random DNA sequence and approaches error rates seen in DNA repair deficiency states. It thus remains a solid candidate for the gene that accounts for the multifocality of CaP. In brief, those cells that undergo the largest AR CAG tract contraction are the most active AR. These cells in turn through overactive AR pathways will provoke new DNA alterations and thus are ordained as

It has clear involvement in distinct diseases due to due well-characterized inherited lossof-function or somatic acquired gain-of-function mutations. The one same protein with diverse-heterogeneous mutations, each with clear phenotypes, offers unique complementary

of the AR polyglutamine tract within the AR protein itself is not known.

Cell cycle (RB1) [39].

**2.2. AR and the CAG polymorphic tract**

96 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

and thus are the most unstable.

expansion rather than contraction (2:1).

a mutator phenotype.

**2.3. AR somatic mutations**

Given the central role that AR has in prostate biology, it is not unexpected that somatic *AR* mutations may be selected for, adding to the CaP repertoire powerful new functions provoking neoplastic advancement [52, 54, 60, 61] (**Figure 1**). Recent studies in support of initial studies have again demonstrated that although most advanced prostatic cancers are uniformly androgen independent, the AR is still a very important contributor to the more progressive fatal disorder [62, 63]. Nearly, all "androgen-independent" or "castrate-resistant" prostatic tumors express high levels of AR, and levels are predictive of progressive disease [64, 65]. Indeed, as many as one-third of tumors exhibit AR gene amplification [66] and AR somatic prostate missense mutations and splice variants are well documented [38, 67, 68]. A number of somatic CaP AR (e.g., T877A) mutants have unique gain-of-function properties; they can bind several classes of steroids promiscuously with subsequent transactivation, be hyperacti‐ vated by normal ligands [69, 70] or be constitutively active without ligand [71]. Even more surprising is that anti-androgen treatments [e.g., flutamide, cyproterone acetate (CPA) or bicalutamide, and even the latest generation of anti-androgens (enzalutamide)] have selected out specific somatic AR gene (*AR*) mutations [72–75]. Missense mutations also have other related gain of functions beyond their relaxed ligand-binding parameters; normally ligandbinding promotes a dramatic conformation change inducing helix 12 movement creating a new co-activator interacting site. In T877A, helix 12 is slightly misplaced and alters the coactivator binding where co-activator binding motifs preferences are changed. As well another gain of function property is manipulated that is AR N-C-terminal interactions are favored.

**Figure 1.** Schematic illustration of cataloged AR Somatic Mutations from the androgen receptor database. Mutations illustrated with the same color were present in the same cancer specimen. Mutations in red were found in the germline (image is courtesy of http://www.androgenbd.mcgill.ca">www.androgenbd.mcgill.ca, with permission from Dr. Mark Trifiro) [37].

Thus, any somatic mutated AR most likely will inherit multiple new functions, which can affect the whole AR complex itself.

In advanced CaP, new AR variants have been found (**Figure 2**). AR-V7 and ARv567es splice variants have an intact NTD and DBD. The AR-V7 splice variant excludes exon 4 through 8, resulting in a deletion of the LBD and the hinge regions, whereas ARv567es excludes exons 5 through 7 creating a LBD deletion; thus, these variants display "constitutive" ligand-inde‐ pendent transcriptional activity. It has been observed for many years that steroid receptor Cterminal truncated variants have constitutive activity; thus, in full-length steroid receptors, the presence of the C-terminal domains acts as a functional repressor whereupon ligand binding alleviates C-terminal repression.


The repressive AR splice variants differ significantly from full-length AR in their transcrip‐ tional programs and subcellular localization [76, 77], implying different potential functions from wild-type AR (AR-WT). In an analysis of 46 castration-resistant prostate cancers (CRPCs), 80% expressed full-length AR, 73% expressed ARv567es and AR-V7; furthermore, 20% of metastatic cases expressed ARv567es solely [78]. Western blot analysis appears to reveal that AR splice variants are also expressed in a number of different prostatic cancer cell lines [79]; however, it is not quite clear whether these variants are actually active or possess any of the attributed "constitutive" activity. Attention has also been given to the molecular mechanism by which these splice variants may arise. One hypothesis asserts that genomic rearrangements is one mechanism [80, 81], which maybe a valid means for established and immortalized cells lines, but more difficult to account for in a progressive disorder. Such a precise process for DNA deletion/rearrangement to independently and exactly occur so many times to result in the expression of these variants is very unlikely. Most recently, a more valid mechanism has been put forward that involved the overexpression of specific RNA splicing factors, U2AF65 and ASF/SF2, influenced the expression of AR-V7 splice variant in CaP cell lines [82]. Alter‐ native RNA splicing has been shown to change during disease progression, and thus, the expression of specific RNA splicing factors during different stages of disease could more adequately account for the both frequency and temporal incidences of these AR variants. Alternative RNA splicing can also be considered another degree of added genetic heteroge‐ neity to evolving neoplasias [83–86].

Thus, any somatic mutated AR most likely will inherit multiple new functions, which can affect

In advanced CaP, new AR variants have been found (**Figure 2**). AR-V7 and ARv567es splice variants have an intact NTD and DBD. The AR-V7 splice variant excludes exon 4 through 8, resulting in a deletion of the LBD and the hinge regions, whereas ARv567es excludes exons 5 through 7 creating a LBD deletion; thus, these variants display "constitutive" ligand-inde‐ pendent transcriptional activity. It has been observed for many years that steroid receptor Cterminal truncated variants have constitutive activity; thus, in full-length steroid receptors, the presence of the C-terminal domains acts as a functional repressor whereupon ligand binding

the whole AR complex itself.

98 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

alleviates C-terminal repression.

**Figure 2.** Schematic illustration of AR truncated and splice variants.

The repressive AR splice variants differ significantly from full-length AR in their transcrip‐ tional programs and subcellular localization [76, 77], implying different potential functions from wild-type AR (AR-WT). In an analysis of 46 castration-resistant prostate cancers (CRPCs), 80% expressed full-length AR, 73% expressed ARv567es and AR-V7; furthermore, 20% of These gain of functions can extend to other facets of AR activity, namely the ability to attract different interactors or interplay with other pathways and possibly target different genes; these diverse gain-of-function attributes are likely to be manifested by a changed constitution of mutant AR complexes, which may well be cell and ligand specific and lend to the molecular pathological processes. Thus, cumulative analysis still supports the AR as a pivotal role player in prostate cell tumor biology, as it plays a fundamental and decisive role in prostate cell biology including very important prostate cell metabolism; what is left to be assessed is what aspect of wt or mutant AR functionality promotes directly or indirectly the mutator phenotype.

#### **3. AR protein complexes: contributors to CaP progression**

Somatic gain-of-function mutations allow neoplastic cells to acquire new properties that can aid the cancerous cells in finding new avenues for progression to more advanced disease. A multitude of AR gain-of-function attributes are likely to exist and most probably reflected in the composition of the AR interactome. As such, many proteins have been identified that interact with the AR and collaborate with it to execute its transcriptional program [87–89]. These observations suggest that the interplay between the AR, its associated interactors, and specific transcription factors can be selective and very dynamic [37, 58, 59, 87, 89]. All together, these findings also point to the complexity of the AR-interacting protein unit, suggesting that many functions of the AR are beyond our current understanding. Furthermore, the great functional diversity of the components of AR complexes exemplifies the intricate nature of protein–protein interactions associated with generating the appropriate AR biological output, and that mutant CaP ARs may have a their own unique ability to define new interactions. Therefore, the functional effect of AR needs to be investigated and show that certain AR properties, through protein–protein interactions can confer a growth advantage to cells. To do so, one would need to take into consideration a number of factors: (1) mutational status of the protein; (2) ligand status; (3) an amendable technology to assess protein–protein interactions; and (4) an encompassing process by which to analyze the data that would provide information on ontological function and most importantly clinical relevancy.

#### **3.1. AR protein isolation methodology**

To date, several techniques have been employed to isolate AR protein complexes including two-hybrid screens and GST pull-downs; however, several limitations have been an obstacle to isolating complexes in their natural cellular environment. First, previous approaches have either used yeast or bacterial systems [90–92]. One shortcoming of these systems is that fulllength AR cannot be expressed; therefore, only N- or C-terminal portions or specific AR domains have only been used. Second, within these systems, the use of a truncated AR, folding, and post-translationally modifications issues arise. Finally, the single most critical aspect of charactering any protein complex, for AR maintaining ligand binding, to the receptor during the isolation process, ensures an "active" complex is isolated. Therefore, our laboratory has developed a mammalian tissue cell culture expression and purification system that retains the ability of AR to maintain its ligand-binding activity [93, 94]. The purification method employed by the following methodology ensures up to 90% of labeled-androgen ligand is still bound to the AR following fractionation. We therefore have the ability to capture both cytoplasmic and nuclear ARs under physiological conditions, with excellent recovery, that demonstrate measurable hormone binding even in *in vitro* conditions. We then have undertaken the process of purifying a number of AR complexes: (1) 0CAG-AR, T877A-AR, WT-AR, in the presence or absence of the synthetic androgen mibolerone (MB) [59]; (2) T877A-AR, in the presence of a panel of hormone ligands (DHT, MB, testosterone, R1881, estradiol, dexamethasone, proges‐ terone, and cyproterone acetate) [58]; (3) AR-V7 and ARv567es (Paliouras and Trifiro, unpub‐ lished data). We have been able to confirm the purification of our complexes by assessing known AR interactors [59]. However, to truly define the spectrum of proteins in the AR complexes, a more robust methodology and platform was needed, and as such, mass spec‐ trometry approach was employed. Data generated by mass spectrometry were then analyzed using a sophisticated network analysis methodology.

#### **3.2. Proteomic-coupled network analysis**

Our ability to capture both liganded and unliganded AR complexes by affinity chromatogra‐ phy under physiological conditions allowed us to pursue a proteomics approach to charac‐ terize the components of AR complexes. This can be done by subjecting such complexes to tryptic digestion followed by MS to assign protein identification [95–97]. To our MS data, a label-free quantitative method was also applied for the comparison of peptide abundance across the different experimental paradigms [98, 99].

Therefore, to highlight potentially novel gain-of-function properties associated with mutant CaP ARs, comparative proteomic characterization studies of AR complexes were done in different experimental backgrounds. To do so, we performed network analysis on individual AR-interacting protein lists derived from our proteomic studies and pursued comparative studies to analyze changes in protein composition based on stimulation condition. We have compiled a human protein interaction data from diverse data resources and annotation databases, such as Biomolecular Interaction Network Database (BIND) [100], the Database of Interacting Proteins (DIP) [101], Human Protein Reference Database (HPRD) [102], IntAct [103], and Molecular INTeraction database (MINT) [104], most of which contain curated interaction data and high-throughput data, consisting of 4000 proteins and 22,000 signaling relations/protein interactions.

protein; (2) ligand status; (3) an amendable technology to assess protein–protein interactions; and (4) an encompassing process by which to analyze the data that would provide information

To date, several techniques have been employed to isolate AR protein complexes including two-hybrid screens and GST pull-downs; however, several limitations have been an obstacle to isolating complexes in their natural cellular environment. First, previous approaches have either used yeast or bacterial systems [90–92]. One shortcoming of these systems is that fulllength AR cannot be expressed; therefore, only N- or C-terminal portions or specific AR domains have only been used. Second, within these systems, the use of a truncated AR, folding, and post-translationally modifications issues arise. Finally, the single most critical aspect of charactering any protein complex, for AR maintaining ligand binding, to the receptor during the isolation process, ensures an "active" complex is isolated. Therefore, our laboratory has developed a mammalian tissue cell culture expression and purification system that retains the ability of AR to maintain its ligand-binding activity [93, 94]. The purification method employed by the following methodology ensures up to 90% of labeled-androgen ligand is still bound to the AR following fractionation. We therefore have the ability to capture both cytoplasmic and nuclear ARs under physiological conditions, with excellent recovery, that demonstrate measurable hormone binding even in *in vitro* conditions. We then have undertaken the process of purifying a number of AR complexes: (1) 0CAG-AR, T877A-AR, WT-AR, in the presence or absence of the synthetic androgen mibolerone (MB) [59]; (2) T877A-AR, in the presence of a panel of hormone ligands (DHT, MB, testosterone, R1881, estradiol, dexamethasone, proges‐ terone, and cyproterone acetate) [58]; (3) AR-V7 and ARv567es (Paliouras and Trifiro, unpub‐ lished data). We have been able to confirm the purification of our complexes by assessing known AR interactors [59]. However, to truly define the spectrum of proteins in the AR complexes, a more robust methodology and platform was needed, and as such, mass spec‐ trometry approach was employed. Data generated by mass spectrometry were then analyzed

Our ability to capture both liganded and unliganded AR complexes by affinity chromatogra‐ phy under physiological conditions allowed us to pursue a proteomics approach to charac‐ terize the components of AR complexes. This can be done by subjecting such complexes to tryptic digestion followed by MS to assign protein identification [95–97]. To our MS data, a label-free quantitative method was also applied for the comparison of peptide abundance

Therefore, to highlight potentially novel gain-of-function properties associated with mutant CaP ARs, comparative proteomic characterization studies of AR complexes were done in different experimental backgrounds. To do so, we performed network analysis on individual AR-interacting protein lists derived from our proteomic studies and pursued comparative studies to analyze changes in protein composition based on stimulation condition. We have

on ontological function and most importantly clinical relevancy.

using a sophisticated network analysis methodology.

across the different experimental paradigms [98, 99].

**3.2. Proteomic-coupled network analysis**

**3.1. AR protein isolation methodology**

100 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

Quantitative MS data, between stimulation conditions, were used to discern protein abun‐ dances. These values were then incorporated into the protein interaction network mapping, to represent a "strength of interaction" coefficient. Between the different experimental conditions, a comparative network analysis was applied [105–108], which was different between our stimulation-specific networks, that is, hierarchical clustering. Immediately what was clear that specific AR protein complexes can be distinguished by the presence or absence of androgen [59]. Analysis of the T877A-AR promiscuous mutant, under different hormone stimulations, showed that although each hormone is able to induce androgen-dependent gene activation [e.g., prostate-specific antigen (PSA)], the proteome profile of each hormone is different. Moreover, although four different androgens were used (DHT, testosterone, MB, and R1881), the proteomic profiles of these androgen ligands do not segregate together. In our hierarchical clustering, we observed that progesterone and dexamethasone AR complexes have proteomic profiles that look like R1881 and MB, respectively [58]. Most recently, analysis of ARv567es protein interactome is very different from androgen stimulated full-length AR (unpublished data), even though ARv567es variant has been characterized as a "constitutive‐ ly" active receptor [76, 77].

From the each AR variant protein interaction network, specific network modules (a set of interacting proteins constituting a subnetwork) are delineated by number of linked interacting proteins interactions and ontological function. The association of subnetwork modules based on biological processes may suggest pathways involved in either tumorigenesis or tumor metastasis. Therefore, to establish statistically significant biological functions, we also imple‐ mented the incorporation of Gene Ontological (GO) terms onto each protein the network. We extracted subnetworks in which GO-term-mapped-nodes were directly linked and highlighted subnetworks and pathways to identify gene enrichment of the proteins/genes from a set of clinical prostatic microarray datasets (http://www.ncbi.nlm.nih.gov/geo/) [109, 110] and RNA sequencing (https://tcga-data.nci.nih.gov/tcga/) [8, 111]. Results show that expression levels of the interacting partners/GO-terms were able to discern normal vs. cancer and correlated with patient survival. More intriguing, different AR protein interaction clusters could differentiate prostatic disease between White (non-Hispanic) vs. African-American males [58]. Nor could we find a gene set that was shared between the two diverse and genetically distinct groups of men. This would suggest that there are AR functional classes that can be used to predict prostatic disease between genetically diverse groups and presumably determine therapeutic modalities. However, the underlining mechanism for these results is not known at this time, although differential population-specific AR activity and disease susceptibility have been very well described clinically [112–115]. Although there have been numerous studies employing microarrays, and recent proteomic screens [116, 117], simple single gene or protein analysis is inadequate to the study of complexity of disease processes, if conclusions toward clinical outcomes wish to be made. Although several "single" genes and proteins have been identified in these studies that are involved with distinct tumor progression and survival profiles and are proposed as prognostic markers; however, once these genes begin to be analyzed as a combined "cluster" model, they do not to translate into statistically significant results related to clinical specimens. The lack of understanding how these genes and proteins act within their functional context and how these components are integrated into signaling pathways and exist as dynamic complexes to execute distinct programs may be responsible for their failure to predict disease progression.

#### **3.3. AR: More than a transcription factor**

The above-mentioned work now strongly suggests that the AR functionality extends beyond its classical role as a transcription factor and includes the novel properties of alternative RNA splicing, DNA methylation, proteasomal interaction, and RNA translation at polyribosomes [58, 59], with evidence now suggesting that the ARv567es variant may also participate in glucose metabolism (Paliouras and Trifiro, unpublished data). A number of novel ARinteracting partners have been characterized, with the majority having been identified in the proteomic screen. These proteins include, heat-shock protein 27 (HSP27) [118], DDX5 [119], SAM68 [120], deleted in breast cancer 1 (DBC1) [121], minichromosome maintenance 7 (MCM7) [122], α-actinin 4 (ACTN4) [116], peroxiredin 1 (PRDX1) [123], DEAD-box polypep‐ tide 17 (DDX17) [124], nucleophosim (NPM1) [125], and Ying Yang 1 (YY1) [126]. Furthermore, these findings point to the complexity of the AR-interacting protein unit and suggest it is involved in a number of different pathways that could function as part of a group of inter‐ connected pathways, whose individual compositions alter depending on AR mutational and stimulation status, to generate the appropriate AR biological output.

#### **4. Animal models for CaP**

The impact of animal models, especially mouse models, has contributed tremendously to our understanding of tumorigenesis, disease etiology, and drug development. However, one of the difficulties with animals is recapitulating the heterogeneousness of the human cancer. Although mice and other animals do share a high degree of genetic similarity and protein homology, there are still some stark differences in trying to mimic human disease. For use of genetically engineered mouse models (GEMMs), several outstanding issues have arisen for the study of CaP and include the following: animal life span and correlating disease onset and stages of disease progression to human counterparts; the dissimilarities in prostate organs; diet and nutrition; and assessing clinical relevancy to disease pathology, etiology, and outcomes. For CaP researchers, along with GEMMs, a number of other animal model ap‐ proaches can also be utilized, including a number of spontaneous non-murine CaP models, will also be discussed. Moreover, throughout the discussion of assessing CaP animal models, attempts will be made discuss the role AR continues to make.

#### **4.1. Spontaneous non-murine models**

inadequate to the study of complexity of disease processes, if conclusions toward clinical outcomes wish to be made. Although several "single" genes and proteins have been identified in these studies that are involved with distinct tumor progression and survival profiles and are proposed as prognostic markers; however, once these genes begin to be analyzed as a combined "cluster" model, they do not to translate into statistically significant results related to clinical specimens. The lack of understanding how these genes and proteins act within their functional context and how these components are integrated into signaling pathways and exist as dynamic complexes to execute distinct programs may be responsible for their failure to

The above-mentioned work now strongly suggests that the AR functionality extends beyond its classical role as a transcription factor and includes the novel properties of alternative RNA splicing, DNA methylation, proteasomal interaction, and RNA translation at polyribosomes [58, 59], with evidence now suggesting that the ARv567es variant may also participate in glucose metabolism (Paliouras and Trifiro, unpublished data). A number of novel ARinteracting partners have been characterized, with the majority having been identified in the proteomic screen. These proteins include, heat-shock protein 27 (HSP27) [118], DDX5 [119], SAM68 [120], deleted in breast cancer 1 (DBC1) [121], minichromosome maintenance 7 (MCM7) [122], α-actinin 4 (ACTN4) [116], peroxiredin 1 (PRDX1) [123], DEAD-box polypep‐ tide 17 (DDX17) [124], nucleophosim (NPM1) [125], and Ying Yang 1 (YY1) [126]. Furthermore, these findings point to the complexity of the AR-interacting protein unit and suggest it is involved in a number of different pathways that could function as part of a group of inter‐ connected pathways, whose individual compositions alter depending on AR mutational and

The impact of animal models, especially mouse models, has contributed tremendously to our understanding of tumorigenesis, disease etiology, and drug development. However, one of the difficulties with animals is recapitulating the heterogeneousness of the human cancer. Although mice and other animals do share a high degree of genetic similarity and protein homology, there are still some stark differences in trying to mimic human disease. For use of genetically engineered mouse models (GEMMs), several outstanding issues have arisen for the study of CaP and include the following: animal life span and correlating disease onset and stages of disease progression to human counterparts; the dissimilarities in prostate organs; diet and nutrition; and assessing clinical relevancy to disease pathology, etiology, and outcomes. For CaP researchers, along with GEMMs, a number of other animal model ap‐ proaches can also be utilized, including a number of spontaneous non-murine CaP models, will also be discussed. Moreover, throughout the discussion of assessing CaP animal models,

stimulation status, to generate the appropriate AR biological output.

attempts will be made discuss the role AR continues to make.

predict disease progression.

**4. Animal models for CaP**

**3.3. AR: More than a transcription factor**

102 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

One of the first animal models to study CaP was in rats. Rats are one of the few animals that develop spontaneous CaP disease [127, 128]. The best studied rat model is the Dunning rat model, which develops slow-growing, well-differentiated, and non-metastatic tumors. Some of the outstanding issues that arise are the rarity of tumors and the variability in the pheno‐ types. There is also a long latency period in tumor development and a lack of metastasis. However, tumors from Dunning rats are initially androgen dependent and eventually becoming androgen independent. Further refinement of Dunning rats has produced animals that are able to develop highly metastatic tumors that spread to lymph nodes and the lungs [129].

CaP also spontaneously occurs in dogs and most closely resembles humans in terms of disease characteristics [130]. CaP in dogs is age dependent, which ideally allows for the study of disease progression, and, in 24% of cases, is able to metastasize to bone. DPC-1, CaP cells derived from dogs, have also been observed to potentially display a number of molecular characteristics including androgen-dependent gene profiling with positive prostate-specific antigen (PSA) and prostate-specific membrane antigen (PMSA) expression [131, 132]. The expression of the progressive disease PMSA marker in DPC-1 cells have allowed for the development of directed radiolabeled-PMSA monoclonal antibodies for SPECT/CT imaging [133]. Another dog model, using cells derived from bone metastasis and injected into dogs, could similarly be used for PET imaging [134]. However, tumors do not regress in castrated dogs and thus are androgen independent. As with rats, there is also a relatively long period for tumor development in dogs. However, the high costs, the gestation period, and the difficulty to genetic manipulate the animals make dogs a very difficult model to use experimentally.

#### **4.2. Genetically engineered mouse models (GEMM) for prostate cancer**

Murine models are also not without their limitations, especially as there has not been a single reported case of mice spontaneously developing CaP [135]. Mice have the similar limitations as all other animal models that they are significantly thousands of time smaller and live 30–50 times shorter than humans [136]. As such, a great deal of time and effort has been put into genetically manipulating mice so that they do develop CaP and accurately represent the human disease. However, the human prostate is anatomical different from its mouse coun‐ terpart, as the mouse prostate has a lobular structure consisting of four lobes (anterior/ coagulating, ventral, dorsal, and lateral) [137], the human prostate organ is a single lobe divided into three zones (central, transitional, and peripheral), and whether the stroma cells surrounding the mouse lobes is similar in comparison with the human stroma cells. The majority of human CaP is also found in the peripheral zone. In mice, the dorsal/lateral lobes have been best described as most similar to the human peripheral zone [135, 138]. On closer assessment, human and mouse prostates become more similar, with stroma cells surrounding epithelial cells. The epithelial cell compartment is also comprised by two cell layers (basal and terminally differentiated luminal cells); also, there are populations of epithelial cell precursors and neuroendocrine cells. In mice, basal cells differentiate into luminal and neuroendocrine cells during prostate development [135, 139].

From the first GEMM for prostate cancer (CaP) developed by Greenberg et al., 1994 [140], to the most recent AR splice variant model by Liu et al. [141], no single model accurately encompasses the entire spectrum of human CaP progression. As CaP is late onset and slowly developing disease, it would be counterintuitive to experimental design. Thus, criteria need to be considered when using mouse models: (1) should reproducibly recapitulate one or more stages of disease progression; (2) should originate within epithelial cells of the prostate; (3) although ideally progression to invasive adenocarcinoma would be desired, but prostatic intraepithelial neoplasia (PIN) should be observed and display associated pathological criteria such as increased inflammation; (4) should display the molecular pathology observed in human CaP tumors, this would include gene and protein expression profile changes that are indicative of an androgen responsive tumor; (5) tumor should respond to ADT or castration. Often times in humans, failure to respond to ADT is linked with the emergence of CRPC and is usually associated with increased expression and nuclear localization of AR since CRPC remains dependent on AR signaling [142]; (6) tumors should achieve bone metastasis (common sites of metastasis observed in human patients). Although rare bone metastasis has been observed in some GEMM, visceral (lung and liver) metastasis appears to be most common.

#### *4.2.1. AR targeted models*

Several attempts have been undertaken to produce a GEMM that targets AR signaling and function. The mouse AR (mAR) shares over 90% homology with its human ortholog; however, mAR interestingly lacks an expanded CAG-polyglutamine tract, instead mice possess a mixed CAG/CAC-glutamine/histidine tract. One of the first AR-targeted mouse models was to target the overexpression of mAR to the prostate secretory epithelium, using the prostate-specific and androgen-responsive mouse probasin (Pb) promoter [143]. By 52 weeks, mice developed high-grade prostatic intraepithelial neoplasia (HGPIN) by 52 weeks. Mice also showed increased proliferation in dorsal/lateral and ventral lobes as marked by increased expression of Ki67 proliferation marker. Even with the increased expression/activity of the mAR, it was insufficient to progress prostatic pathology to CaP.

Another group of investigators opted to take into consideration the differences in the genetic polymorphism of the polyglutamine tract between mice and humans and replace exon 1 of the mAR with exon 1 of the human AR [144]. Three transgenic whole knock-in "humanized" AR mice expressing three different polyglutamine tract lengths (12Q, 21Q, and 48Q) were created. As the length of the polyglutamine tract is linked to AR activity and risk for CaP [34, 51], the reasoning behind the three mice was to differentiated disease progression with AR activity. All mice appear to maintain androgen-dependent gene expression, however, do not develop any prostatic pathology, even with the short 12Q tract mouse. However, when these mice were crossed to TRAMP mice (see below), the length of the polyglutamine tract was linked to the initiation of prostatic tumors, with shorter having higher incidence of tumors vs. longer tracts, which appear to offer a degree of protection in tumor initiation. Of note, researchers also assessed AR mutations of tumors from their 21Q humanized AR crossed to TRAMP mice under a number of different conditions (intact, intact/bicalutamide, intact/flutamide, and castrated). Along with assessing specific somatic mutations (missense, non-sense, small indolent inser‐ tion/deletions), they also assessed changes in the length of polyglutamine tract. They found an average mutation rate of 4.0/10,000 bp of AR coding sequence, with missense mutations accounting for 54.1% of putative mutations, with a majority of mutations identified in one or two clones per tumor [145]. Half of the mutations identified also were found in the LBD region, as has often been shown to be responsible for promiscuous ligand-binding gain-of-function properties of the receptor [68, 146]. Contraction of the polyglutamine tract was also assessed, as it is also commonly observed in disease initiation; however, it was not observed. Although this AR mutation rate is higher than reported in clinical samples [39], it does highlight the mutational sensitivity of AR correlated to disease progression.

Recently, a GEMM was created to study the role of the AR splice variant, ARv567es, in CaP development [141]. ARv567es clone was cloned downstream of androgen responsive Pb promoter, where endogenous mAR would initially drive expression of the ARv567es, then upon castration of the animals, an adequate expression of ARv567es would then continue to expand its own expression. Thus, the investigators would be able to study the influence of ARv567es on the progression of CaP in castrate-resistant state. The coordinate expression of full-length AR and ARv567es variants were able to illicit epithelial hyperplasia by 16 weeks and invasive adenocarcinoma by 52 weeks. Upon castration at 16 weeks, mice were able to maintain nuclear localization of ARv567es and able to develop more aggressive neoplasias than sham controls. Gene expression profiling of tumors from ARv567es castrated mice also suggested that there is an enrichment of oncogenic pathways, including Wnt/β-catenin, NFkB, and K-Ras signaling, that have been linked to aggressive CaP.

#### *4.2.2. TRAMP and LADY*

From the first GEMM for prostate cancer (CaP) developed by Greenberg et al., 1994 [140], to the most recent AR splice variant model by Liu et al. [141], no single model accurately encompasses the entire spectrum of human CaP progression. As CaP is late onset and slowly developing disease, it would be counterintuitive to experimental design. Thus, criteria need to be considered when using mouse models: (1) should reproducibly recapitulate one or more stages of disease progression; (2) should originate within epithelial cells of the prostate; (3) although ideally progression to invasive adenocarcinoma would be desired, but prostatic intraepithelial neoplasia (PIN) should be observed and display associated pathological criteria such as increased inflammation; (4) should display the molecular pathology observed in human CaP tumors, this would include gene and protein expression profile changes that are indicative of an androgen responsive tumor; (5) tumor should respond to ADT or castration. Often times in humans, failure to respond to ADT is linked with the emergence of CRPC and is usually associated with increased expression and nuclear localization of AR since CRPC remains dependent on AR signaling [142]; (6) tumors should achieve bone metastasis (common sites of metastasis observed in human patients). Although rare bone metastasis has been observed in some GEMM, visceral (lung and liver) metastasis appears to be most common.

Several attempts have been undertaken to produce a GEMM that targets AR signaling and function. The mouse AR (mAR) shares over 90% homology with its human ortholog; however, mAR interestingly lacks an expanded CAG-polyglutamine tract, instead mice possess a mixed CAG/CAC-glutamine/histidine tract. One of the first AR-targeted mouse models was to target the overexpression of mAR to the prostate secretory epithelium, using the prostate-specific and androgen-responsive mouse probasin (Pb) promoter [143]. By 52 weeks, mice developed high-grade prostatic intraepithelial neoplasia (HGPIN) by 52 weeks. Mice also showed increased proliferation in dorsal/lateral and ventral lobes as marked by increased expression of Ki67 proliferation marker. Even with the increased expression/activity of the mAR, it was

Another group of investigators opted to take into consideration the differences in the genetic polymorphism of the polyglutamine tract between mice and humans and replace exon 1 of the mAR with exon 1 of the human AR [144]. Three transgenic whole knock-in "humanized" AR mice expressing three different polyglutamine tract lengths (12Q, 21Q, and 48Q) were created. As the length of the polyglutamine tract is linked to AR activity and risk for CaP [34, 51], the reasoning behind the three mice was to differentiated disease progression with AR activity. All mice appear to maintain androgen-dependent gene expression, however, do not develop any prostatic pathology, even with the short 12Q tract mouse. However, when these mice were crossed to TRAMP mice (see below), the length of the polyglutamine tract was linked to the initiation of prostatic tumors, with shorter having higher incidence of tumors vs. longer tracts, which appear to offer a degree of protection in tumor initiation. Of note, researchers also assessed AR mutations of tumors from their 21Q humanized AR crossed to TRAMP mice under a number of different conditions (intact, intact/bicalutamide, intact/flutamide, and castrated). Along with assessing specific somatic mutations (missense, non-sense, small indolent inser‐

*4.2.1. AR targeted models*

insufficient to progress prostatic pathology to CaP.

104 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

The first murine prostate cancer models took advantage of some recent advances in the areas of oncogenetics and steroid hormone receptor functionality. As such, the viral SV40 early region, comprised of the large T antigen (Tag) and small t antigen, was cloned downstream of the androgen hormone responsive rat Pb promoter. After selection of lines of animals with higher expression of SV40 early region in the ventral and dorsal lobes, it yielded the transgenic adenocarcinoma mouse prostate (TRAMP) model [140, 147]. TRAMP mice develop progres‐ sive forms of CaP, even distant site metastasis. They are characterized with rapid development of PIN by 12 weeks with adenocarcinoma, predominantly in the dorsal/lateral lobes, arising by 24 weeks of age. The mice can also display castrate-resistant disease, where mice castrated at 12 weeks did not affect primary tumor development or metastasis in the majority of mice with 100% in the lymph and 67% lung metastasis [148].

The LADY CaP model is similar to the TRAMP model, in that it utilizes, rather than the entire SV40 early region, only the large T antigen under the control of the long 12-kb Pb promoter [149]. These mice also lead to the development of hyperplasia and PIN by 10 weeks, followed by high-grade epithelial dysplasia and adenocarcinoma by 20 weeks. By 33 weeks of age, the mice display metastatic disease to the liver, lung, and bone with a 90% penetrance [150]. The metastatic tumors are all neuroendocrine type cancers, similar to TRAMP metastatic tumors [151].

TRAMP and LADY models also have been used for a number of preclinical drug studies [152– 161]; however, questions arise Whether a model that develops localized primary prostatic disease between 20 and 24 weeks is a proper representation of human disease evolution? Furthermore, these models can be referred to as "brutish" with the utilization of the SV40 T antigen region; as such a genetic element has never been implicated in human CaP. However, the T antigen has been identified to bind and inhibit TP53 and RB tumor suppressors, the molecular chaperone DNAJ, and complement p300/CBP, while small t antigen has been shown to bind to the phosphatase PP2A and a number of proteins known to contribute to CaP and other neoplasias [162]. Loss-of-function/deletion mutations TP53 [163–166] and RB [167–172] have been linked to CaP progression, and together, DNAJ [173–175] and p300/CBP [87, 176] have also been describe as AR protein complex proteins and shown to be involved in mediating AR signaling [37, 59, 87]. However, even if the TRAMP/LADY models can be considered feedforward models, because of their dependency on AR signaling, to both drive expression of SV40 through the Pb promoter and simultaneously potentially contribute to a favorable cellular environment for AR function; the other questions to arise are Whether the molecular pathology of TRAMP/LADY mice share concordance with expression profiles (genes/proteins) found in clinical CaP specimens from representative disease stages? Currently, the analysis has not been performed.

#### *4.2.3. PTEN deficiency*

Phosphatase and tensin homolog (PTEN) is an important regulator of the PI3K/AKT signaling pathway and is frequently deleted/mutated in a number of human cancers [177–182]. In CaP, PTEN deletions occur in approximately 23% of HGPIN, 68% of localized primary tumors [183], and 86% of CRPC [184] and thus has become a candidate for developing into a mouse model. Although homozygous knock-out (KO) Pten mice are embryonic lethal, heterozygous Pten+/− mice develop a number of neoplasias, including lymphomas, dysplastic intestinal polyps, endometrial complex atypical hyperplasia, and thyroid neoplasia [185]. However, common human tumors, such as brain, breast, and skin, associated with PTEN deletion are absent from mice. Pten+/− mice also have a spectrum of prostatic phenotypes, with 70% of mice displaying hyperplasia and dysplasia between 6 and 30 weeks [186]. Using a reduced activity of hypo‐ morphic Pten allele, it has been shown that Pten+/hyp mice can promote progression from hyperplasia to PIN between 6 and 22 weeks of age between 25 and 37.5% of the time, however, with only a single case of adenocarcinoma observed [185, 187, 188].

Due to the latency of prostatic disease development in Pten+/− mice, researchers have under‐ taken to cross these mice with other genes associated with CaP with the objective to accelerate disease progression. This has included crosses to p27Kip1 and Nkx3.1 loss-of-function allele mouse strains. In 13–22 weeks, Pten+/−, p27Kip1-/- mice develop PIN with 100% penetrance and about 25% of mice develop invasive CaP [189]. Alone, Nkx3.1 loss-of-function mice do not develop PIN or CaP in mice; however, in combination with Pten+/− mice, they show an accelerated incidence and progression to HGPIN/early carcinoma at 26 weeks, with 100% penetrance of HGPIN at 52 weeks [190]. By allowing the Pten+/−; Nkx3.1+/− mice to age more than 52 weeks, allows HGPIN lesions to progress to invasive adenocarcinoma. Furthermore, surgical castration at 24 weeks, of these animals, resulted in partial regression of the prostatic lesions and decreased expression of AR [191].

TRAMP and LADY models also have been used for a number of preclinical drug studies [152– 161]; however, questions arise Whether a model that develops localized primary prostatic disease between 20 and 24 weeks is a proper representation of human disease evolution? Furthermore, these models can be referred to as "brutish" with the utilization of the SV40 T antigen region; as such a genetic element has never been implicated in human CaP. However, the T antigen has been identified to bind and inhibit TP53 and RB tumor suppressors, the molecular chaperone DNAJ, and complement p300/CBP, while small t antigen has been shown to bind to the phosphatase PP2A and a number of proteins known to contribute to CaP and other neoplasias [162]. Loss-of-function/deletion mutations TP53 [163–166] and RB [167–172] have been linked to CaP progression, and together, DNAJ [173–175] and p300/CBP [87, 176] have also been describe as AR protein complex proteins and shown to be involved in mediating AR signaling [37, 59, 87]. However, even if the TRAMP/LADY models can be considered feedforward models, because of their dependency on AR signaling, to both drive expression of SV40 through the Pb promoter and simultaneously potentially contribute to a favorable cellular environment for AR function; the other questions to arise are Whether the molecular pathology of TRAMP/LADY mice share concordance with expression profiles (genes/proteins) found in clinical CaP specimens from representative disease stages? Currently, the analysis

106 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

Phosphatase and tensin homolog (PTEN) is an important regulator of the PI3K/AKT signaling pathway and is frequently deleted/mutated in a number of human cancers [177–182]. In CaP, PTEN deletions occur in approximately 23% of HGPIN, 68% of localized primary tumors [183], and 86% of CRPC [184] and thus has become a candidate for developing into a mouse model. Although homozygous knock-out (KO) Pten mice are embryonic lethal, heterozygous Pten+/− mice develop a number of neoplasias, including lymphomas, dysplastic intestinal polyps, endometrial complex atypical hyperplasia, and thyroid neoplasia [185]. However, common human tumors, such as brain, breast, and skin, associated with PTEN deletion are absent from mice. Pten+/− mice also have a spectrum of prostatic phenotypes, with 70% of mice displaying hyperplasia and dysplasia between 6 and 30 weeks [186]. Using a reduced activity of hypo‐ morphic Pten allele, it has been shown that Pten+/hyp mice can promote progression from hyperplasia to PIN between 6 and 22 weeks of age between 25 and 37.5% of the time, however,

Due to the latency of prostatic disease development in Pten+/− mice, researchers have under‐ taken to cross these mice with other genes associated with CaP with the objective to accelerate disease progression. This has included crosses to p27Kip1 and Nkx3.1 loss-of-function allele mouse strains. In 13–22 weeks, Pten+/−, p27Kip1-/- mice develop PIN with 100% penetrance and about 25% of mice develop invasive CaP [189]. Alone, Nkx3.1 loss-of-function mice do not develop PIN or CaP in mice; however, in combination with Pten+/− mice, they show an accelerated incidence and progression to HGPIN/early carcinoma at 26 weeks, with 100% penetrance of HGPIN at 52 weeks [190]. By allowing the Pten+/−; Nkx3.1+/− mice to age more than 52 weeks, allows HGPIN lesions to progress to invasive adenocarcinoma. Furthermore,

with only a single case of adenocarcinoma observed [185, 187, 188].

has not been performed.

*4.2.3. PTEN deficiency*

In 2003, Wang et al., generated a mouse model that specifically deleted exon 5 of Pten in the prostate [192]. These mice developed hyperplasia in 4 weeks, PIN at 6 weeks, and frank adenocarcinoma with 100% penetrance between 9 and 24 weeks. The mice also respond to surgical castration with an observed increase in apoptosis and extended survival time vs. noncastrated animals. However, castrated animals still maintained prostates 5- to 10-fold larger than WT counterparts and reduced AR expression. Although reduced AR expression is consistent with other Pten deficiency mice, this is not what is observable in human CaP [193]. Additionally, metastasis to the lymph nodes and lungs at 12–29 weeks was observed in 45% of animals.

Currently, there are a number of prostate-specific conditional Pten KO mice that have been developed that employ alternative promoters. A PSA-promoter-driven Pten KO resulted in 100% penetrance of adenocarcinoma and carcinoma by 56 weeks [194]. However, by simulta‐ neously knocking out, Pten and Nkx3.1, coupled with tamoxifen inductions, slowly developed HGPIN with microinvasion [195]. Tumors regress in castrated mice, but then continue to progress to microinvasive adenocarcinoma while maintaining nuclear AR expression, suggesting that AR signaling remains active in the mice following castration. Combinatorial ADT and inhibition of AKT (MK2206) and mTOR (MK8669) function significantly reduced tumor burden [196].

#### **5. Cell metabolism, ROS, DNA damage, and the AR**

The AR has long been known to have dramatic effects on the prostate gland. The acute withdrawal of androgens lead to severe atrophy of the prostate gland in short time frames originally referred to as involution, which in currently acknowledged as a programmed cell death event [197]. The AR also has significant effects on the overall anabolic and intermediary metabolism, promoting glucose uptake, and pursuing through both the glycolytic, TCA cycle and fatty acid metabolism.

A number of non-genomic influences have been associated with specific risks to the develop‐ ment of CaP, one of these risk factors has been nutrition and diet, especially Western (highfat/low-carbohydrate) vs. non-Western (low fat/high carbohydrate) has been extensively reviewed [198–200]. Likewise, GEMM also have been shown to be influenced by high-fat diets. TRAMP mice given a Western-type diet containing 21.2% fat and 0.2% cholesterol vs. regular chow diet (4.5% fat and 0.002% cholesterol), with 33% of mice showing large and very pronounced tumors at 28 weeks, with increased tumor size and weight and hyperplasia [201]. Western-type fed TRAMP mice also showed increased expression of cell cycle-related (cyclin D1) and proliferation (proliferating cell nuclear antigen—PCNA) markers. There was also an increase in lung metastasis with an average of 3 ± 1.04 foci vs. 0.43 ± 0.2 foci, in Western-type vs. regular chow-fed mice. Another group also observed similar results with a high-fat diet fed TRAMP mouse [202]. Along with seeing an increase in tumor size and increase prostatic hyperplasia, they also observed a decrease in the expression of glutathione peroxidase 3 (GPx3). GPx3 is an important antioxidant enzyme responsible for detoxifying cells of reactive oxygen species (ROS). Increased ROS levels in one of the consequences on high-fat diets and has been shown to interfere with a number of cellular processes, including damaging DNA [203]. GPx3 levels have been shown to be downregulated in CaP [204, 205]. The combinatorial observation that high-fat fed TRAMP mice have larger tumors with cellular changes (increased ROS levels, reduced GPx3 expression) suggests a potential mechanism for a role of cellular metabolism in CaP progression. Increased cellular metabolism and downstream effects of increased ROS levels and DNA damage create the scenario for tumor cells to incur more mutations that may lead to more aggressive tumor growth and drug resistance.

The AR thus has an intrinsic ingrained property of promoting prostatic cellular metabolism. It is not unreasonable that in CaP initiation and evolution, alterations in AR allowing further enhanced metabolism may be the fundamental mechanisms allowing for new mutations to be created. Heightened metabolism has a direct effect on reactive oxygen species generation (ROS) as hypermetabolism can result in exaggerated mitochondria fluxes [206–211]. It is now well appreciated that cancer metabolism is unique many times demonstrating heightened glucose uptake and abnormal mitochondrial pathways including glutamine lysis and reverse carboxylation. These metabolic properties are not reflective of energy needs and can be considered in conjunction with fatty acid oxidations as a metabolic phenotype-supporting ROS leading to DNA alterations, in essence a powerful mutator phenotype.

#### **Author details**

Miltiadis Paliouras1,2\*, Carlos Alvarado1,2 and Mark Trifiro1,2

\*Address all correspondence to: miltiadis.paliouras@mcgill.ca

1 Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, QC, Canada

2 Department of Medicine, McGill University, Montreal, QC, Canada

#### **References**


[3] Forbes SA, Tang G, Bindal N, et al. COSMIC (the Catalogue of Somatic Mutations in Cancer): a resource to investigate acquired mutations in human cancer. Nucleic Acids Res 2010;38:D652–7.

hyperplasia, they also observed a decrease in the expression of glutathione peroxidase 3 (GPx3). GPx3 is an important antioxidant enzyme responsible for detoxifying cells of reactive oxygen species (ROS). Increased ROS levels in one of the consequences on high-fat diets and has been shown to interfere with a number of cellular processes, including damaging DNA [203]. GPx3 levels have been shown to be downregulated in CaP [204, 205]. The combinatorial observation that high-fat fed TRAMP mice have larger tumors with cellular changes (increased ROS levels, reduced GPx3 expression) suggests a potential mechanism for a role of cellular metabolism in CaP progression. Increased cellular metabolism and downstream effects of increased ROS levels and DNA damage create the scenario for tumor cells to incur more

The AR thus has an intrinsic ingrained property of promoting prostatic cellular metabolism. It is not unreasonable that in CaP initiation and evolution, alterations in AR allowing further enhanced metabolism may be the fundamental mechanisms allowing for new mutations to be created. Heightened metabolism has a direct effect on reactive oxygen species generation (ROS) as hypermetabolism can result in exaggerated mitochondria fluxes [206–211]. It is now well appreciated that cancer metabolism is unique many times demonstrating heightened glucose uptake and abnormal mitochondrial pathways including glutamine lysis and reverse carboxylation. These metabolic properties are not reflective of energy needs and can be considered in conjunction with fatty acid oxidations as a metabolic phenotype-supporting ROS

1 Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, QC, Canada

[1] Forbes SA, Beare D, Gunasekaran P, et al. COSMIC: exploring the world's knowledge of somatic mutations in human cancer. Nucleic Acids Res 2015;43:D805–11.

[2] Forbes SA, Bindal N, Bamford S, et al. COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic Acids Res 2011;39:D945–50.

mutations that may lead to more aggressive tumor growth and drug resistance.

108 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

leading to DNA alterations, in essence a powerful mutator phenotype.

Miltiadis Paliouras1,2\*, Carlos Alvarado1,2 and Mark Trifiro1,2

\*Address all correspondence to: miltiadis.paliouras@mcgill.ca

2 Department of Medicine, McGill University, Montreal, QC, Canada

**Author details**

**References**


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11.


### **Reconsideration of Hormonal Therapy in the Era of Next‐ Generation Hormonal Therapy**

Yasuyoshi Miyata, Yohei Shida, Tomohiro Matsuo, Tomoaki Hakariya and Hideki Sakai

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63282

#### **Abstract**

Hormonal therapy is a major and effective tool in the treatment of prostate cancer patients. This is especially true for patients in the advanced stages of disease. Unfortunately, almost all prostate cancer cells will develop into castration‐resistant prostate cancer (CRPC) despite continued therapy and suppression of testosterone levels. Up until 5–6 years ago, there was little effective therapy for the treatment of CRPC patients. However, recently, a variety of methodologies and drugs such as cabazitaxel and sipuleucel‐T have been approved globally for the treatment of CRPC. Two novel drugs, abiraterone acetate and enzartamide, have also become available as potential treatment options. However, the anticancer effects of these two drugs are not always satisfactory in terms of prolonging survival. These drugs are also associated with adverse events and are expensive when compared with the costs of previously used anticancer drugs. In this section, we pay particular attention to hormonal therapies that do not include the use of abiraterone acetate or enzartamide. We believe that a detailed understanding of the range of currently available hormonal therapies, including their associated benefits and limitations, is important for supporting the prolongation of survival in patients with advanced prostate cancer. Therefore, this section offers a valuable discussion on the treatment strategies for prostate cancer including CRPC.

**Keywords:** steroidal antiandrogens, nonsteroidal anti‐androgens, estrogens, steroids, castration‐resistant prostate 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**

Prostate cancer is one of the most common malignancies diagnosed worldwide in men. At present, prostate cancer patients with organ‐confined disease can obtain an excellent oncolog‐ ical outcome through radical operation and radiotherapy. On the other hand, a variety of hormonal therapies are often necessitated for patients with advanced prostate cancer. Be‐ cause the malignant aggressiveness of prostate cancer has been known to be suppressed by orchiectomy since the 1940s, androgen deprivation therapy (ADT) has been commonly administered. This is associated with the fact that the prostate is an androgen‐dependent organ and that androgen receptor (AR) signaling plays an important role in the growth and progres‐ sion of prostate cancer cells. In fact, as a first‐line therapy, surgical castration, chemical castration, and anti‐androgen treatment are usually used in the treatment of patients with metastatic prostate cancer [1]. Anti‐androgens are broadly divided into two chemical types, steroidal and nonsteroidal. As a part of hormone therapy, estrogen and estrogen‐containing agents can also be administered to patients with prostate cancer. A variety of glucocorticoids is also common‐ ly used.

However, most hormone‐naive prostate cancer patients will develop castration‐resistant prostate cancer (CRPC) despite therapeutic suppression of testosterone levels and even though continued therapy can proceed without adverse effects. Furthermore, CRPC has a high malignant potential and aggressiveness. This is due to the number of heterogeneous types of cancer cells that develop a variety of abnormal signal pathways as a means to survive in the castration environment. In fact, the prognosis of patients with CRPC is often poor and no therapy that had high efficacy and high compliance had been available until the middle of the 2000s. In 2004, two clinical trials with large study populations and sophisticated methodologies demonstrated that the anticancer effects of docetaxel‐based chemotherapy are superior to those of mitoxantrone and prednisolone treatments [2, 3]. Based on these facts, docetaxel has become a standard therapy for the treatment of CRPC. However, prolongation of survival by docetaxel chemotherapy is less than six months [4]. Additionally, docetaxel has major problems related to multiple and severe adverse events [3, 5]. Consequently, many urologists, physicians, and investigators have focused on the development of new therapeutic strategies to prolong survival in CRPC patients.

During the past 5–6 years, a variety of treatment methods and drugs for CRPC have been approved in many countries. Approved drugs include cabazitaxel and sipuleucel‐T [6, 7]. In addition, ADT options for patients with CRPC have also changed during the past few years as the novel drugs, abiraterone acetate and enzartamide (termed as next‐generation anti‐ androgens) have become available [8, 9]. Abiraterone is a specific steroidogenic inhibitor that irreversibly inhibits CYP17A1 [10]. Enzalutamide is an anti‐androgen‐receptor inhibitor that has been shown to improve prognosis in patients with CRPC [11]. Thus, many urologists and medical oncologists agree that treatment strategies for patients with CRPC are developing remarkably well [12]. However, the anticancer effects, including prolongation of patient survival, associated with these new treatments are not always adequate. For example, CRPC develops resistance to these second‐generation agents quickly and thus the anticancer effects of abiraterone acetate and enzartamide can be decreased [13, 14].

Thus, although a variety of new anticancer agents have been developed, their anticancer effects in CRPC patients are not always satisfactory, particularly in terms of prolonging patient survival. In addition, CRPC patients often have many comorbidities due to past treatments and aging. Therefore, in discussions on treatment strategies for prostate cancer patients, it is essential to assess information regarding drug adverse reactions and safety. In addition, special attention must be paid to the cost of therapies as treatment periods are usually lengthy, and recently developed anticancer agents, including next‐generation anti‐androgens, are expen‐ sive. Based on these facts, we present in this chapter, the clinical benefits, safety, and caution points of hormonal therapies that do not involve next‐generation antiandrogens. In other words, we will re‐evaluate hormonal therapy for treatment of prostate cancer patients in the era of next‐generation antiandrogen agents.

#### **2. Antiandrogen agents**

**1. Introduction**

126 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

ly used.

to prolong survival in CRPC patients.

Prostate cancer is one of the most common malignancies diagnosed worldwide in men. At present, prostate cancer patients with organ‐confined disease can obtain an excellent oncolog‐ ical outcome through radical operation and radiotherapy. On the other hand, a variety of hormonal therapies are often necessitated for patients with advanced prostate cancer. Be‐ cause the malignant aggressiveness of prostate cancer has been known to be suppressed by orchiectomy since the 1940s, androgen deprivation therapy (ADT) has been commonly administered. This is associated with the fact that the prostate is an androgen‐dependent organ and that androgen receptor (AR) signaling plays an important role in the growth and progres‐ sion of prostate cancer cells. In fact, as a first‐line therapy, surgical castration, chemical castration, and anti‐androgen treatment are usually used in the treatment of patients with metastatic prostate cancer [1]. Anti‐androgens are broadly divided into two chemical types, steroidal and nonsteroidal. As a part of hormone therapy, estrogen and estrogen‐containing agents can also be administered to patients with prostate cancer. A variety of glucocorticoids is also common‐

However, most hormone‐naive prostate cancer patients will develop castration‐resistant prostate cancer (CRPC) despite therapeutic suppression of testosterone levels and even though continued therapy can proceed without adverse effects. Furthermore, CRPC has a high malignant potential and aggressiveness. This is due to the number of heterogeneous types of cancer cells that develop a variety of abnormal signal pathways as a means to survive in the castration environment. In fact, the prognosis of patients with CRPC is often poor and no therapy that had high efficacy and high compliance had been available until the middle of the 2000s. In 2004, two clinical trials with large study populations and sophisticated methodologies demonstrated that the anticancer effects of docetaxel‐based chemotherapy are superior to those of mitoxantrone and prednisolone treatments [2, 3]. Based on these facts, docetaxel has become a standard therapy for the treatment of CRPC. However, prolongation of survival by docetaxel chemotherapy is less than six months [4]. Additionally, docetaxel has major problems related to multiple and severe adverse events [3, 5]. Consequently, many urologists, physicians, and investigators have focused on the development of new therapeutic strategies

During the past 5–6 years, a variety of treatment methods and drugs for CRPC have been approved in many countries. Approved drugs include cabazitaxel and sipuleucel‐T [6, 7]. In addition, ADT options for patients with CRPC have also changed during the past few years as the novel drugs, abiraterone acetate and enzartamide (termed as next‐generation anti‐ androgens) have become available [8, 9]. Abiraterone is a specific steroidogenic inhibitor that irreversibly inhibits CYP17A1 [10]. Enzalutamide is an anti‐androgen‐receptor inhibitor that has been shown to improve prognosis in patients with CRPC [11]. Thus, many urologists and medical oncologists agree that treatment strategies for patients with CRPC are developing remarkably well [12]. However, the anticancer effects, including prolongation of patient survival, associated with these new treatments are not always adequate. For example, CRPC As mentioned earlier, prostate cancer cells are, in the majority of cases, androgen‐dependent. Testosterone, of testicular origin, comprises 95% of androgen content. Androgens stimulate cell proliferation and tumor growth by binding to AR in the cancer cells. Therefore, the first‐ line of treatment for advanced prostate cancer has been androgen deprivation by medical castration through administration of luteinizing hormone‐releasing hormone agonist/antag‐ onists or by surgical castration with bilateral orchiectomy. Antiandrogens are AR receptor antagonists that compete with dihydroteststerone for AR binding. Upon binding, the antian‐ drogens act to inhibit the tumor growth in patients with prostate cancer. This section reviews first‐generation antiandrogens used in the treatment of prostate cancer including CRPC.

#### **2.1. Steroidal antiandrogens**

Antiandrogens can be classified based on their chemical structure into two types, steroidal and nonsteroidal. The steroidal antiandrogens, cyproterone acetate, spironolactone, synthetic progestins, and chlormadinone acetate are well known in oncology. We first discuss the steroidal antiandrogens, progesterone analogue mifepristone (RU‐486), cyproterone acetate, and the mineralocorticoid analogue, spironolactone. However, it should be noted that these agents are rarely used in clinical situations because of their partial androgenic agonistic– antagonistic activity.

RU‐486 is a progesterone analogue best known as a progesterone receptor (PR) antagonist. It was developed in France as a medical approach to terminating pregnancy [15]. RU‐486 also inhibits glucocorticoid receptor (GR) function and has been used for treating Cushing syn‐ drome [16]. More importantly, RU‐486 is also an AR antagonist. Binding studies indicate that it has a higher affinity for AR than either hydroxyflutamide or bicalutamide [17]. However, a Phase II study of RU‐486 demonstrated limited activity in patients with CRPC. In that study, RU‐486 also stimulated a marked increase in the levels of adrenal androgens, testosterone, and dihydrotestosterone (DHT) [18].

Cyproterone acetate is nonspecific and can activate the mineralocorticoid receptor (MR), GR, and PR. A meta‐analysis of randomized trials showed that combined androgen blockade (CAB) with nonsteroidal antiandrogens, including nilutamide and flutamide, appeared slightly favorable to cancer patient survival when compared with the effects of androgen suppression (AS) monotherapy. On the other hand, CAB with cyproterone acetate was associated with an inferior survival rate [19].

Spironolactone is an MR antagonist that is used to treat side effects related to mineralocorticoid excess. Richards et al. [20] showed that spironolactone significantly activates both wild type and mutant AR and that it should be avoided in the treatment of all patients with CRPC. As a result, CAB with steroidal antiandrogens has been recognized as an inferior treatment to the use of nonsteroidal antiandrogens and is considered unsuitable for prostate cancer treatment.

#### *2.1.1. Steroidal antiandrogens in Japan*

Cyproterone acetate, RU‐486, and nilutamide are not approved in Japan for use in the treatment of patients with prostate cancer. On the other hand, chlormadinone acetate is approved in Japan but is not used in other countries. Evaluation of the relative efficacy and safety of these agents globally is therefore difficult. However, chlormadinone acetate has been reported to have advantages in terms of causing fewer adverse events. We therefore present information regarding chlormadinone acetate in this section.

Several Japanese groups have reported on the efficacy of chlormadinone acetate as an alter‐ native antiandrogen therapy in the treatment of men with relapsed prostate cancer following first‐ or second‐line hormonal therapy [21–23]. Steroidal antiandrogens including cyproterone acetate and chlormadinone acetate have also proven efficacious in the treatment of prostate cancer patients who suffer from hot flushes [24, 25]. However, Igawa et al. [26] reported that CAB therapy using chlormadinone acetate led to a significantly poorer survival outcome versus the use of bicalutamide. Nevertheless, because this survival trend was not observed in M0 cases, they concluded that chlormadinone acetate might still be an option for CAB therapy, depending on the clinical stage and the severity of adverse effects including hot flushes [26].

#### *2.1.2. Clinical study and basic research*

Ongoing clinical studies and basic research using chlormadinone are presented in **Table 1**. There has been at least one evaluation on whether low‐dose chlormadinone has an effect on continued active surveillance (**Table 1**, No.1; UMIN000012284). Another study has evaluated whether chlormadinone has a more favorable effect on lipid and bone metabolism than that of bicalutamide (No.2; UMIN000018478). In terms of basic research, Koike et al. [27] have reported on the effects of chlormadinone acetate on the development and progression of prostate cancer in their *PTEN*‐deficient mouse model (**Table 1**, No. 3). They demonstrated that chlormadinone acetate treatment suppressed the proliferation of cancer cells but did not decrease the development of prostatic intraepithelial neoplasia (PIN). This means that chlormadinone did not act to prevent prostate cancer onset. The findings from this study suggest that inhibiting androgen signaling is effective in preventing the proliferation of prostate cancer caused by PTEN dysfunction. It has also been suggested that androgen plays an important role at the early stages of prostate cancer development in this mouse model [27].


**Table 1.** Clinical studies and basic research on chlorm.

#### **2.2. Nonsteroidal antiandrogens**

RU‐486 also stimulated a marked increase in the levels of adrenal androgens, testosterone, and

Cyproterone acetate is nonspecific and can activate the mineralocorticoid receptor (MR), GR, and PR. A meta‐analysis of randomized trials showed that combined androgen blockade (CAB) with nonsteroidal antiandrogens, including nilutamide and flutamide, appeared slightly favorable to cancer patient survival when compared with the effects of androgen suppression (AS) monotherapy. On the other hand, CAB with cyproterone acetate was

Spironolactone is an MR antagonist that is used to treat side effects related to mineralocorticoid excess. Richards et al. [20] showed that spironolactone significantly activates both wild type and mutant AR and that it should be avoided in the treatment of all patients with CRPC. As a result, CAB with steroidal antiandrogens has been recognized as an inferior treatment to the use of nonsteroidal antiandrogens and is considered unsuitable for prostate cancer treatment.

Cyproterone acetate, RU‐486, and nilutamide are not approved in Japan for use in the treatment of patients with prostate cancer. On the other hand, chlormadinone acetate is approved in Japan but is not used in other countries. Evaluation of the relative efficacy and safety of these agents globally is therefore difficult. However, chlormadinone acetate has been reported to have advantages in terms of causing fewer adverse events. We therefore present information

Several Japanese groups have reported on the efficacy of chlormadinone acetate as an alter‐ native antiandrogen therapy in the treatment of men with relapsed prostate cancer following first‐ or second‐line hormonal therapy [21–23]. Steroidal antiandrogens including cyproterone acetate and chlormadinone acetate have also proven efficacious in the treatment of prostate cancer patients who suffer from hot flushes [24, 25]. However, Igawa et al. [26] reported that CAB therapy using chlormadinone acetate led to a significantly poorer survival outcome versus the use of bicalutamide. Nevertheless, because this survival trend was not observed in M0 cases, they concluded that chlormadinone acetate might still be an option for CAB therapy, depending on the clinical stage and the severity of adverse effects including hot flushes [26].

Ongoing clinical studies and basic research using chlormadinone are presented in **Table 1**. There has been at least one evaluation on whether low‐dose chlormadinone has an effect on continued active surveillance (**Table 1**, No.1; UMIN000012284). Another study has evaluated whether chlormadinone has a more favorable effect on lipid and bone metabolism than that of bicalutamide (No.2; UMIN000018478). In terms of basic research, Koike et al. [27] have reported on the effects of chlormadinone acetate on the development and progression of prostate cancer in their *PTEN*‐deficient mouse model (**Table 1**, No. 3). They demonstrated that chlormadinone acetate treatment suppressed the proliferation of cancer cells but did not decrease the development of prostatic intraepithelial neoplasia (PIN). This means that

dihydrotestosterone (DHT) [18].

associated with an inferior survival rate [19].

128 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

*2.1.1. Steroidal antiandrogens in Japan*

*2.1.2. Clinical study and basic research*

regarding chlormadinone acetate in this section.

The nonsteroidal antiandrogens, flutamide and bicalutamide, are well known in oncology and are major therapeutic tools in the treatment of prostate cancer patients. Therefore, information on their anticancer effects, including causation of decreases in serum prostate‐specific antigen (PSA) levels and enhanced survival rates, has been presented in numerous reviews. There is also a corresponding amount of literature on their adverse effects. Here, we introduce the clinical benefits and limitations of nonsteroidal antiandrogens in the treatment of CRPC patients.

#### *2.2.1. Optional treatment for castration‐resistant prostate cancer*

Bicalutamide is the most common used steroidal antiandrogen due to its good curative effects and limited adverse effects. It is combined with luteinizing hormone‐releasing hormone agonist/antagonist therapy to treat CRPC. Some researchers have indicated that dose elevation of antiandrogen agents may enhance their efficacy. For example, the routine dose of bicaluta‐ mide is 50 mg/day but evidence suggests that higher doses might be more effective [28]. Klotz et al. [29] reported that 22% of the patients showed a ≥50% decline in serum PSA levels following an increase in the dose of bicalutamide from 50 mg/day to 150 mg/day. Lodde et al. [30] reported the palliative benefits of 150 mg/day bicalutamide therapy in 44.7% of 38 CRPC nonmetastasis patients. These studies therefore demonstrate that a high proportion of CRPC patients could benefit from treatments involving elevated doses of steroidal antiandrogens. Available data also indicate that bicalutamide at a dose of much greater than 50 mg (at least 150 mg) daily in combination with castration may increase efficacy against CRPC progression. However, in all these studies earlier, either the median response duration is brief or the evaluation indices are limited.

Meanwhile, potential predictive factors for improved responses to high‐dose (150 mg) bicalutamide therapy have been discussed. Qian et al. [28] suggested that secondary hormonal therapy with 150 mg bicalutamide daily was effective in patients with CRPC. Patients with a lower Gleason score, lower serum PSA concentrations, and who were using flutamide as a first‐line nonsteroidal antiandrogen achieved more benefits when treated with bicalutamide 150 mg therapy. Patients with PSA decreases ≥85% had improved times of response to bicalutamide 150 mg therapy. Moreover, when compared with the common side effects of androgen‐deprivation therapy, the adverse effects of bicalutamide 150 mg therapy were well‐ tolerated.

#### *2.2.2. Combination with molecular target drugs*

Angiogenesis, mediated by the vascular endothelial growth factor receptor (VEGFR) pathway, may be a good target for treatment of prostate cancer; it has been implicated in both the development and progression of the disease [31–33]. Studies have found that median levels of plasma VEGF are significantly higher in patients with metastatic prostate cancer when compared with those with localized cancer and that elevated plasma and urine levels of VEGF may be independent negative prognostic factors [34–36]. These findings suggest that inhibiting the VEGFR pathway might be an effective approach in prostate cancer.

In addition, the mammalian target‐of‐rapamycin (mTOR) is a critical molecule in controlling the proliferation of tumor cells. It can be activated by mutation or activation of signaling molecules such as PI3K or Akt. Alterations in the PI3K/Akt/mTOR pathway play an important role in prostate cancer, and it is estimated that upregulation occurs in 30–50% of prostate cancer cases [37]. Recently, clinical trials on the efficacy and tolerability of antiandrogen and molec‐ ular target combination therapy have been conducted (**Table 2**), but these have included only a small number of patients. Results have shown that neither the PSA‐response rate nor the PSA‐progression free survival is fully satisfactory. In addition, peculiar adverse events have been associated with molecular target agents. Hence, these therapies are not currently in practical use.


N, number of patients; PSA, prostate specific antigen; RR, response rates; Ref, references; wks, weeks; mos, months.

\* PSA response was defined as ≥ 50% decline.

also a corresponding amount of literature on their adverse effects. Here, we introduce the clinical benefits and limitations of nonsteroidal antiandrogens in the treatment of CRPC

Bicalutamide is the most common used steroidal antiandrogen due to its good curative effects and limited adverse effects. It is combined with luteinizing hormone‐releasing hormone agonist/antagonist therapy to treat CRPC. Some researchers have indicated that dose elevation of antiandrogen agents may enhance their efficacy. For example, the routine dose of bicaluta‐ mide is 50 mg/day but evidence suggests that higher doses might be more effective [28]. Klotz et al. [29] reported that 22% of the patients showed a ≥50% decline in serum PSA levels following an increase in the dose of bicalutamide from 50 mg/day to 150 mg/day. Lodde et al. [30] reported the palliative benefits of 150 mg/day bicalutamide therapy in 44.7% of 38 CRPC nonmetastasis patients. These studies therefore demonstrate that a high proportion of CRPC patients could benefit from treatments involving elevated doses of steroidal antiandrogens. Available data also indicate that bicalutamide at a dose of much greater than 50 mg (at least 150 mg) daily in combination with castration may increase efficacy against CRPC progression. However, in all these studies earlier, either the median response duration is brief or the

Meanwhile, potential predictive factors for improved responses to high‐dose (150 mg) bicalutamide therapy have been discussed. Qian et al. [28] suggested that secondary hormonal therapy with 150 mg bicalutamide daily was effective in patients with CRPC. Patients with a lower Gleason score, lower serum PSA concentrations, and who were using flutamide as a first‐line nonsteroidal antiandrogen achieved more benefits when treated with bicalutamide 150 mg therapy. Patients with PSA decreases ≥85% had improved times of response to bicalutamide 150 mg therapy. Moreover, when compared with the common side effects of androgen‐deprivation therapy, the adverse effects of bicalutamide 150 mg therapy were well‐

Angiogenesis, mediated by the vascular endothelial growth factor receptor (VEGFR) pathway, may be a good target for treatment of prostate cancer; it has been implicated in both the development and progression of the disease [31–33]. Studies have found that median levels of plasma VEGF are significantly higher in patients with metastatic prostate cancer when compared with those with localized cancer and that elevated plasma and urine levels of VEGF may be independent negative prognostic factors [34–36]. These findings suggest that inhibiting

In addition, the mammalian target‐of‐rapamycin (mTOR) is a critical molecule in controlling the proliferation of tumor cells. It can be activated by mutation or activation of signaling molecules such as PI3K or Akt. Alterations in the PI3K/Akt/mTOR pathway play an important role in prostate cancer, and it is estimated that upregulation occurs in 30–50% of prostate cancer

the VEGFR pathway might be an effective approach in prostate cancer.

*2.2.1. Optional treatment for castration‐resistant prostate cancer*

130 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

patients.

evaluation indices are limited.

*2.2.2. Combination with molecular target drugs*

tolerated.

\*\*PSA response was defined as ≥ 30% decline.

**Table 2.** Combination therapy of bicalutamide and molecular target therapy for castration‐resistant prostate cancer.

#### **3. Estrogens and estrogen‐based therapy**

It is well‐known that estrogens carry a significant risk for cardiovascular events. Androgen deprivation therapy is therefore the first choice as primary therapy for advanced prostate cancer. However, many authors have now investigated the anticancer effects of diethylstil‐ bestrol [42], transdermal estradiol [43], and more recently, oral ethinylestradiol [44, 45] in the treatment of CRPC as well as in the treatment of hormone‐naive prostate cancer. These authors have concluded that estrogen therapy is still relevant and can induce PSA response (50% PSA decline) rates as high as 69.6% in CRPC patients. Moreover, estrogen therapy is associated with fewer of the toxicities associated with ADT. It can maintain bone mineral density, suppress hot flushes, and improve cognitive function and lipo‐metabolism in castrated men.

Estrogens inhibit the hypothalamic–pituitary–testicular axis through negative feedback mechanisms. Moreover, estrogen administration can induce a decrease in the levels of adrenal androgens and testosterone produced by the Leydig cells of the testes [46]. A direct cytotoxic effect of estrogens on prostate cancer cells has not been fully investigated. However, there are some reports on their cytotoxic effects on *in vivo* and *in vitro* castrate xenograft models [47, 48].

#### **3.1. Diethylstilbestrol**

Diethylstilbestrol (DES) is a synthetic estrogen. In the 1940s, Huggins and Hodges [49] re‐ ported that DES suppressed the progression of prostate cancer. DES was then used as a first‐ line hormonal therapy for over 10 years in the treatment of prostate cancer patients. However, in 1970, increased mortality from cardiovascular and thromboembolic events as‐ sociated with DES was reported in a prospective randomized controlled trial (VACURG study) [50]. Briefly, of the 1103 patients treated with DES (5 mg/daily) with no anticoagula‐ tion therapy, 17% of the patients died due to cardiovascular events (the mortality associated with cardiovascular event in the placebo group was 11.7%). Therefore, to suppress the risk of adverse events including cardiovascular disease, administration of lower doses of DES was investigated in clinical trials. However, although this approach significantly reduced cardiovascular morbidity, it was still higher than that associated with castration. For exam‐ ple, in the EORTC 30805 study, the cardiovascular mortality of the DES (1 mg/daily) and or‐ chidectomy‐alone groups was 14.8% and 8.3%, respectively [51]. Meanwhile, the cardiovascular toxicity of DES was significantly decreased by the use of anticoagulants such as warfarin or aspirin [52, 53]. From these facts, we note that DES treatment has a relatively high risk of cardiovascular events and that it must be used with anticoagulation therapy. On the other hand, we also understand that DES is one of the most effective and useful thera‐ peutic options for prostate cancer patients as it can suppress cancer progression.

In terms of PSA response (>50% decline from baseline) to DES treatment in CRPC patients, approximately 40% patients were reported to be PSA responders [42, 54]. More recently, Wilkins et al. [55] disclosed results from a larger CRPC cohort (231 patients) treated with DES at a dose of 1–3 mg daily and with aspirin at 75 mg. They reported that the PSA response rate was 28.9% and that the median time to PSA progression was 4.6 months. These results cannot be considered satisfactory in terms of cancer control. However, interestingly, Wilkins et al. also reported that 18% of the patients showed an improvement in bone pain. Other investigators have also observed an improvement in certain types of pain in CRPC patients treated with DES [56]. Overall, DES remains a reasonable palliative option for patients with symptomatic CRPC even though its survival benefits may be limited. However, careful informed consent from the patients who are fully apprised of the cardiovascular risks associated with DES should be required.

#### **3.2. Transdermal estradiol**

As mentioned earlier, cardiovascular disease and thrombosis are the most dangerous adverse events associated with estrogen‐based agents. In contrast to oral estrogens, parenterally administered estrogens do not increase liver protein synthesis and may be less prothrombotic [43]. In fact, in women who receive estrogen replacement therapy, the risk of cardiovascular events is increased with oral, but not transdermal, estradiol. There have been several infor‐ mative studies about safety, potential side effects, and efficacy in the use of transdermal estradiol in the treatment of CRPC patients [43, 57]. In a Phase II study, CRPC patients suffering continued disease progression after primary hormonal therapy were treated with transdermal estradiol (0.6 mg per 24 h) [43]. Toxicity associated with the transdermal estradiol application was modest and no cardiovascular events occurred. In terms of cancer control, three of the 24 patients (12.5%) showed a PSA response. Another study analyzed the safety and efficacy of transdermal estradiol patch in the treatment of CRPC patients after ADT and chemotherapy [57]. This study showed a PSA response rate of 10%. No cardiovascular events were observed. Thus, transdermal estrogen therapy was well‐tolerated in the CRPC patients, and there were no significant cardiovascular complications. However, as with oral estradiol, the anticancer effects of transdermal estradiol appear to offer very limited survival benefit.

#### **3.3. Ethinylestradiol**

line hormonal therapy for over 10 years in the treatment of prostate cancer patients. However, in 1970, increased mortality from cardiovascular and thromboembolic events as‐ sociated with DES was reported in a prospective randomized controlled trial (VACURG study) [50]. Briefly, of the 1103 patients treated with DES (5 mg/daily) with no anticoagula‐ tion therapy, 17% of the patients died due to cardiovascular events (the mortality associated with cardiovascular event in the placebo group was 11.7%). Therefore, to suppress the risk of adverse events including cardiovascular disease, administration of lower doses of DES was investigated in clinical trials. However, although this approach significantly reduced cardiovascular morbidity, it was still higher than that associated with castration. For exam‐ ple, in the EORTC 30805 study, the cardiovascular mortality of the DES (1 mg/daily) and or‐ chidectomy‐alone groups was 14.8% and 8.3%, respectively [51]. Meanwhile, the cardiovascular toxicity of DES was significantly decreased by the use of anticoagulants such as warfarin or aspirin [52, 53]. From these facts, we note that DES treatment has a relatively high risk of cardiovascular events and that it must be used with anticoagulation therapy. On the other hand, we also understand that DES is one of the most effective and useful thera‐

132 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

peutic options for prostate cancer patients as it can suppress cancer progression.

be required.

**3.2. Transdermal estradiol**

In terms of PSA response (>50% decline from baseline) to DES treatment in CRPC patients, approximately 40% patients were reported to be PSA responders [42, 54]. More recently, Wilkins et al. [55] disclosed results from a larger CRPC cohort (231 patients) treated with DES at a dose of 1–3 mg daily and with aspirin at 75 mg. They reported that the PSA response rate was 28.9% and that the median time to PSA progression was 4.6 months. These results cannot be considered satisfactory in terms of cancer control. However, interestingly, Wilkins et al. also reported that 18% of the patients showed an improvement in bone pain. Other investigators have also observed an improvement in certain types of pain in CRPC patients treated with DES [56]. Overall, DES remains a reasonable palliative option for patients with symptomatic CRPC even though its survival benefits may be limited. However, careful informed consent from the patients who are fully apprised of the cardiovascular risks associated with DES should

As mentioned earlier, cardiovascular disease and thrombosis are the most dangerous adverse events associated with estrogen‐based agents. In contrast to oral estrogens, parenterally administered estrogens do not increase liver protein synthesis and may be less prothrombotic [43]. In fact, in women who receive estrogen replacement therapy, the risk of cardiovascular events is increased with oral, but not transdermal, estradiol. There have been several infor‐ mative studies about safety, potential side effects, and efficacy in the use of transdermal estradiol in the treatment of CRPC patients [43, 57]. In a Phase II study, CRPC patients suffering continued disease progression after primary hormonal therapy were treated with transdermal estradiol (0.6 mg per 24 h) [43]. Toxicity associated with the transdermal estradiol application was modest and no cardiovascular events occurred. In terms of cancer control, three of the 24 patients (12.5%) showed a PSA response. Another study analyzed the safety and efficacy of transdermal estradiol patch in the treatment of CRPC patients after ADT and chemotherapy

Treatment with ethinylestradiol was used in the 1980s as palliative therapy in patients with advanced prostate cancer. However, ethinylestradiol treatment has become less common since the development of newer treatment forms such as ADT [58]. On the other hand, one advantage of ethinylestradiol is that it is inexpensive. This is an important consideration in assessing treatment strategies. At present, ethinylestradiol is not used as a first‐line hormonal therapy. However, it is still used as a second‐line or a later option for CRPC patients [59]. Several investigators have reported on the efficacy and adverse events of ethinylestradiol in treatments of CRPC patients. For example, Onita et al. [60] reported that a decrease in serum PSA levels was seen in all 15 tested CRPC; 11 patients (73.3%) showed decreases of more than 50% without severe side effects. Other investigators have performed ethinylestradiol monotherapy at a dose of 1.5 mg/day for CRPC patients for whom more than one salvage therapy had not been effective [45]. In this retrospective study, the PSA response rate was 69.6%, and the median progression‐free survival was estimated as 300 days. On the other hand, adverse events occurred in 3 of the 23 patients (13%). These adverse effects included elevation of liver enzymes, anorexia, and heart failure. Recently, results of a larger prospective study of ethinylestradiol monotherapy were reported [44]. In this study, 116 patients with metastatic CRPC were administered ethinylestradiol at a daily dose of 1 mg and with aspirin at a daily dose of 100 mg. A PSA response was observed in 79 patients (70.5%). PSA levels lower than 4 ng/mL in serum were observed in 24 patients (21.4%). Toxic adverse effects that required


N, number of patients; PSA, prostate specific antigen; RR, response rates; PFS, progression‐free survival; Ref, references; mos, months; NA, not available; wks., weeks; Trans, transdermal.

\*PSA response was defined as ≥50% decline.

**Table 3.** Summary of the anticancer effects in the studies of estrogens.

treatment cessation were described for 26 patients (23.2%). The main adverse effect requiring treatment cessation was thromboembolism (18 patients). Overall, however, no patient died as a result of treatment toxicity.

In addition to monotherapy, several clinical studies on combination therapies that include ethinylestradiol have been conducted. For example, in one small study, administration of 1 mg oral ethinylestradiol combined with lanreotide acetate (somatostatin analog) resulted in a decline in serum PSA levels of >50% in 9 out of the 10 CRPC patients (90%) [59]. Overall, administration of ethinylestradiol in CRPC cases resulted in a high percentage of PSA responses. The potential for cardiovascular toxicity could be managed through appropriate patient selection and concomitant anticoagulation therapy. A summary of the anticancer effects of estrogens is shown in **Table 3**.

#### **4. Corticosteroids**

Corticosteroids suppress the production of androgens from the adrenal gland through the regulation of the pituitary–adrenal axis. Consequently, corticosteroids can inhibit the malig‐ nant behavior and survival of prostate cancer cells. In addition to this indirect role, they are known to inhibit the growth of prostate cancer cells by interfering directly with a variety of cancer‐related factors [61]. Recognizing this, corticosteroids have been used in cancer therapy for decades. They have been administered to prostate cancer patients both as a mono‐therapy and in combination with other anticancer agents. Unfortunately, its anticancer effects, including prolongation of survival, are limited when used as a single agent [62]. In this section, we discuss corticosteroids in terms of their efficacy when used in combination therapy for the treatment of CRPC.

#### **4.1. Which types of glucocorticoids are better?**

A variety of glucocorticoids, including prednisone, prednisolone, hydrocortisone, and dexa‐ methasone, have anticancer effects and associated clinical benefits for prostate cancer pa‐ tients [63–65]. However, many investigators have suggested that their anticancer effects differ from each other. For example, the PSA response rates of prednisolone (5 mg × 2 =10 mg daily) and hydrocortisone (40 mg daily) administered to CRPC patients were reported to be 26% and 22%, respectively [66, 67]. In the case of prednisone, 34% of the patients had a decrease in PSA levels of more than 50% [64]. In the previous reports, PSA response rates associated with prednisone, prednisolone, and hydrocortisone treatments have ranged from 9 to 33% [68]. On the other hand, in the case of dexamethasone, decreases in PSA levels of ≥50% were detected in 50 of 102 (49%) of the CRPC patients treated at a dose of 0.5 mg daily [68]. Other investigators also reported a similar decrease in PSA levels in 61% of CRPC pa‐ tients treated with dexamethasone at a dose of 1.5 mg or 2.25 mg daily [69]. Based on these reports, dexamethasone appears to have a significantly greater anticancer effect than other glucocorticoids [70]. However, one report indicated that dexamethasone at a dose of 1.5 mg daily showed a reduction of PSA levels of ≥50% in only 28% of the CRPC patients [70]. Thus, there is no general agreement on what specific glucocorticoid should be recommended for the treatment of CRPC patients. Recently, the first head‐to‐head clinical comparison of pre‐ dnisolone versus dexamethasone as monotherapies in the treatment of CRPC patients was conducted [68]. In this study, patients were randomized, in a 1:1:1 ratio, between adminis‐ tration of intermittent dexamethasone (8 mg twice daily for 3 days every 3 weeks), daily dexamethasone (0.5 mg once daily), and prednisolone (5 mg twice daily). The intermittent dexamethasone treatment was terminated mid‐study due to a lack of observed antitumor activity. Thus, comparisons of anticancer effects were conducted only between the daily dexamethasone and prednisolone treatments. A decrease in PSA levels of ≥50% was detect‐ ed in 16 of 39 (41%) of the dexamethasone‐treated patients and in 8 of 36 (22%) predniso‐ lone‐treated patients. Although this difference did not approach statistical significance, the investigators concluded that dexamethasone might be a more effective treatment than pre‐ dnisolone. Other investigators have supported this conclusion [70].

#### **4.2. Combination with next‐generation antiandrogens**

treatment cessation were described for 26 patients (23.2%). The main adverse effect requiring treatment cessation was thromboembolism (18 patients). Overall, however, no patient died as

In addition to monotherapy, several clinical studies on combination therapies that include ethinylestradiol have been conducted. For example, in one small study, administration of 1 mg oral ethinylestradiol combined with lanreotide acetate (somatostatin analog) resulted in a decline in serum PSA levels of >50% in 9 out of the 10 CRPC patients (90%) [59]. Overall, administration of ethinylestradiol in CRPC cases resulted in a high percentage of PSA responses. The potential for cardiovascular toxicity could be managed through appropriate patient selection and concomitant anticoagulation therapy. A summary of the anticancer

Corticosteroids suppress the production of androgens from the adrenal gland through the regulation of the pituitary–adrenal axis. Consequently, corticosteroids can inhibit the malig‐ nant behavior and survival of prostate cancer cells. In addition to this indirect role, they are known to inhibit the growth of prostate cancer cells by interfering directly with a variety of cancer‐related factors [61]. Recognizing this, corticosteroids have been used in cancer therapy for decades. They have been administered to prostate cancer patients both as a mono‐therapy and in combination with other anticancer agents. Unfortunately, its anticancer effects, including prolongation of survival, are limited when used as a single agent [62]. In this section, we discuss corticosteroids in terms of their efficacy when used in combination therapy for the

A variety of glucocorticoids, including prednisone, prednisolone, hydrocortisone, and dexa‐ methasone, have anticancer effects and associated clinical benefits for prostate cancer pa‐ tients [63–65]. However, many investigators have suggested that their anticancer effects differ from each other. For example, the PSA response rates of prednisolone (5 mg × 2 =10 mg daily) and hydrocortisone (40 mg daily) administered to CRPC patients were reported to be 26% and 22%, respectively [66, 67]. In the case of prednisone, 34% of the patients had a decrease in PSA levels of more than 50% [64]. In the previous reports, PSA response rates associated with prednisone, prednisolone, and hydrocortisone treatments have ranged from 9 to 33% [68]. On the other hand, in the case of dexamethasone, decreases in PSA levels of ≥50% were detected in 50 of 102 (49%) of the CRPC patients treated at a dose of 0.5 mg daily [68]. Other investigators also reported a similar decrease in PSA levels in 61% of CRPC pa‐ tients treated with dexamethasone at a dose of 1.5 mg or 2.25 mg daily [69]. Based on these reports, dexamethasone appears to have a significantly greater anticancer effect than other glucocorticoids [70]. However, one report indicated that dexamethasone at a dose of 1.5 mg daily showed a reduction of PSA levels of ≥50% in only 28% of the CRPC patients [70]. Thus,

a result of treatment toxicity.

**4. Corticosteroids**

treatment of CRPC.

effects of estrogens is shown in **Table 3**.

134 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

**4.1. Which types of glucocorticoids are better?**

One clinical study evaluated the anticancer effects and safety profile of abiraterone acetate when used in combination with prednisone as a means to suppress secondary mineralocorti‐ coid excess [71]. Another study reported a reduction in the PSA levels >50% in CRPC patients treated with dexamethasone in addition to abiraterone acetate [71]. This report also demon‐ strated that the anticancer effects of combination therapy of abiraterone acetate and prednisone were detected regardless of prior dexamethasone exposure [71]. Furthermore, the PSA response rates, defined as a 50% or more reduction in PSA levels associated with dexametha‐ sone and prednisolone were reported to be 47% and 24% (P = 0.05), respectively, in CRPC patients during a randomized Phase II trial [67]. Based on this, a hypothesis that a "steroid switch" from prednisone to dexamethasone would be effective in the treatment of CRPC patients with disease progression under abiraterone and prednisone treatment has been suggested [72]. In fact, one retrospective study of 30 CRPC patients who underwent such a "steroid switch" while abiraterone was administered, showed that durable PSA responses occurred in up to 40% of the patients [72]. In this study, the dosage of prednisolone or dexamethasone was 5 mg b.i.d and 0.5–1.0 mg daily.

#### **4.3. Combination with immunotherapy**

Sipuleucel‐T is the recognized leading immunotherapeutic cancer vaccine (dendritic cell vaccine therapy). A variety of additional immunotherapeutic agents that can be used singly or combination therapy is currently under development. In fact, based on results from clinical trials, personalized peptide vaccination strategies that use multiple anticancer peptides has been reported to be effective and safe in CRPC patients [73, 74]. Several reports have also demonstrated that low‐dose dexamethasone is a useful partner for personalized peptide vaccination in the treatment of CRPC. This is because dexamethasone does not suppress the immune system. Dexamethasone also exerts its anticancer effects in a direct manner as well as by reducing AR signaling [5, 75]. Recently, Phase II randomized controlled trials have demonstrated that immunotherapy that comprised personalized peptide vaccination and low‐ dose dexamethasone was well‐tolerated in chemotherapy‐naive CRPC patients and yielded a better outcome when compared to the effects of dexamethasone alone [76]. In short, progres‐ sion‐free survival periods, as evaluated by serum PSA responses, with peptide vaccine + dexamethasone (n = 37) and dexamethasone alone (n = 35) were 22.0 and 7.0 months (P = 0.076), respectively. In addition, the median overall survival in patients treated with peptide vaccine + dexamethasone (73.9 months) was significantly longer (P = 0.00084) than those treated with dexamethasone alone (34.9 month)

#### **4.4. Modulatory approach for castration‐resistant prostate cancer**

Dexamethasone is often used as a component of modular therapy approaches in the treat‐ ment of CRPC. The aim of the modulatory approach is to inhibit the malignant activities of cancer and stroma cells through regulation of a variety of different pathological features in‐ cluding cancer‐related molecules, angiogenesis, inflammation, and altered immune respons‐ es.

The effect of a modular therapy consisting of capecitabine, pioglitazone (PPARα/γ receptor agonist), refecoxib (or etoricoxib, a cyclooxygenase (COX)‐2 inhibitor), and dexamethasone on 36 patients with metastatic CRPC was analyzed [77]. One half of treated patients (n = 18) showed a biochemical response defined as a ≥25% PSA decrease. Median periods of progres‐ sion‐free and overall survival were 4 and 14.4 months, respectively [77]. Results from a study on a modulatory therapy comprising imatinib (a platelet derived growth factor receptor (PDGFR) inhibitor), pioglitazone, etoricoxib, dexamethasone, and low‐dose treosulfan have also been reported [78]. In that Phase II study, the anticancer effects and adverse events of the modular therapy were assessed in 61 CRPC patients. A total of the 23 patients (37.7%) were reported as PSA responders. Median progression‐free survival period was approximately 15 months. However, all the patients experienced one or more adverse events and 27 patients (41.5%) had serious events. The most frequent adverse event was peripheral edema (56.9%). Nausea (38.5%), fatigue (35.4%), and dyspnea (35.4%) were also common occurrences. One of key characteristics of CRPC is its heterogeneity. Therefore, a variety of different approaches is essential in controlling tumor growth and progression. Based on this, modulatory therapy would appear to be a useful strategy. However, in general, this approach has been associated with a relatively high frequency of adverse events. In this section, we emphasized the impor‐ tance of dexamethasone because of its anticancer effects as a GR agonist and its suppression of a variety of adverse events.

#### **Author details**

Yasuyoshi Miyata\* , Yohei Shida, Tomohiro Matsuo, Tomoaki Hakariya and Hideki Sakai

\*Address all correspondence to: int.doc.miya@m3.dion.ne.jp

Department of Urology, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto, Nagasaki, Japan

#### **References**

dose dexamethasone was well‐tolerated in chemotherapy‐naive CRPC patients and yielded a better outcome when compared to the effects of dexamethasone alone [76]. In short, progres‐ sion‐free survival periods, as evaluated by serum PSA responses, with peptide vaccine + dexamethasone (n = 37) and dexamethasone alone (n = 35) were 22.0 and 7.0 months (P = 0.076), respectively. In addition, the median overall survival in patients treated with peptide vaccine + dexamethasone (73.9 months) was significantly longer (P = 0.00084) than those treated with

Dexamethasone is often used as a component of modular therapy approaches in the treat‐ ment of CRPC. The aim of the modulatory approach is to inhibit the malignant activities of cancer and stroma cells through regulation of a variety of different pathological features in‐ cluding cancer‐related molecules, angiogenesis, inflammation, and altered immune respons‐

The effect of a modular therapy consisting of capecitabine, pioglitazone (PPARα/γ receptor agonist), refecoxib (or etoricoxib, a cyclooxygenase (COX)‐2 inhibitor), and dexamethasone on 36 patients with metastatic CRPC was analyzed [77]. One half of treated patients (n = 18) showed a biochemical response defined as a ≥25% PSA decrease. Median periods of progres‐ sion‐free and overall survival were 4 and 14.4 months, respectively [77]. Results from a study on a modulatory therapy comprising imatinib (a platelet derived growth factor receptor (PDGFR) inhibitor), pioglitazone, etoricoxib, dexamethasone, and low‐dose treosulfan have also been reported [78]. In that Phase II study, the anticancer effects and adverse events of the modular therapy were assessed in 61 CRPC patients. A total of the 23 patients (37.7%) were reported as PSA responders. Median progression‐free survival period was approximately 15 months. However, all the patients experienced one or more adverse events and 27 patients (41.5%) had serious events. The most frequent adverse event was peripheral edema (56.9%). Nausea (38.5%), fatigue (35.4%), and dyspnea (35.4%) were also common occurrences. One of key characteristics of CRPC is its heterogeneity. Therefore, a variety of different approaches is essential in controlling tumor growth and progression. Based on this, modulatory therapy would appear to be a useful strategy. However, in general, this approach has been associated with a relatively high frequency of adverse events. In this section, we emphasized the impor‐ tance of dexamethasone because of its anticancer effects as a GR agonist and its suppression

, Yohei Shida, Tomohiro Matsuo, Tomoaki Hakariya and Hideki Sakai

Department of Urology, Nagasaki University Graduate School of Biomedical Sciences,

dexamethasone alone (34.9 month)

136 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

of a variety of adverse events.

\*Address all correspondence to: int.doc.miya@m3.dion.ne.jp

**Author details**

Yasuyoshi Miyata\*

Sakamoto, Nagasaki, Japan

es.

**4.4. Modulatory approach for castration‐resistant prostate cancer**


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### **Genetic Association Studies on Prostate Cancer**

Zorana Nikolić, Dušanka Savić Pavićević and Goran Brajušković

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63280

#### **Abstract**

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144 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

The modern research on molecular basis of prostate cancer (PCa) development includes studies aiming to identify potential genetic markers which could be used in diagnostics and/or monitoring of PCa. Genome-wide association studies (GWASs) have identified over 75 variants associated with PCa risk. One of the major PCarelated regions identified through GWASs is found to be a segment of 8q24. Other important PCa-susceptibility regions are 17q12, 17q24, 10q11, and 19q13. Candidategene based approach has also provided evidence of association between PCa risk and genetic variants located in functionally significant genes (both protein-coding and noncoding RNA genes) involved in normal prostatic cell growth, malignant transformation, or in the development of metastases. Nevertheless, the success of these studies is questionable, since numerous candidates for PCa-susceptibility variants were identified, but these results failed to replicate. The main aim of both types of genetic association studies on PCa is the identification of potential PCa genetic markers which could be used for constructing reliable algorithms for evaluating the risk for PCa development and/or PCa progression.

**Keywords:** prostate cancer, association study, GWAS, candidate gene, validation study, replication study

#### **1. Introduction**

Alarming statistics on prostate cancer (PCa) incidence and mortality, as well as the results of epidemiological studies, have led to focusing research efforts on discovering molecular mechanisms underlying its onset and progression [1]. Still, molecular basis of PCa pathogene‐ sis remains largely unknown, while the results of studies in this area of research suggest that

© 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.

PCa is one of the most genetically and molecularly heterogeneous malignant tumors [2]. Among PCa cases, most are sporadic, while a significantly smaller percent represents familial type, including hereditary cases. High-penetrability PCa-related loci are not common in popula‐ tions and are found to be associated with hereditary PCa. Since PCa represents a multifactori‐ al disease with polygenic basis, and sporadic cases are much more frequently diagnosed, most of the research in the area of PCa molecular genetics has focused on genetic variants with low penetrability [3].

The modern research on molecular basis of PCa development includes studies aiming to identify potential genetic markers which could be used in diagnostics and/or monitoring of PCa [1]. This is of utmost importance, since one of the major issues in clinical practice related to PCa is a large percent of latent PCa among newly diagnosed [4]. The overdiagnosis of PCa in early diagnosed cases, due to indolent forms, leads to unnecessary morbidity because of application of invasive therapeutic procedures [5]. This led to focusing the research efforts on discovering genetic markers that could be used for assessing the biological potential of early diagnosed PCa. Therefore, the use of these genetic markers, together with standard prognostic parameters of PCa progression, which include initial serum PSA level, Gleason score, and clinical stage, could greatly improve the current clinical protocols by being implemented in algorithms for evaluating the patient's risk of PCa and/or PCa aggressiveness [1].

Studies aiming to identify potential PCa-related loci are designed as case-control or case-only studies, which evaluate the differences in genotype distributions between cases and controls, as well as between different groups of patients, classified according to clinical characteristics. The most validated loci associated with PCa risk were identified through Genome-wide association studies (GWAS) [6]. Nevertheless, numerous PCa-related genetic variants were found in studies based on selected candidate genes [7].

#### **2. Linkage analyses and high-penetrability loci**

Linkage analyses have led to identification of the first high-penetrability PCa susceptibility loci [1]. These studies were based on analyses in hereditary PCa, which is a less frequent type of PCa, and yielded high or moderate-penetrability loci, such as HPC1 (eng. *Hereditary Prostate Cancer 1*, HPC1) mapped in chromosomal region 1q24-25 [8], PCAP (eng. *Predisposing for Cancer Prostate*, PCAP) mapped in 1q42.2-43 [9] and HPCX (eng. *Hereditary Prostate Cancer on X Chromosome*, HPCX) located in 1q42.2-43 [10]. Later on, additional linkage studies identified several other loci primarily associated with familial PCa and rarely found to be altered in sporadic type [1, 3]. Fine-mapping of these regions has led to identifying several candidate genes, such as *RNASEL* or *ELAC2* [11, 12]. Nevertheless, since the major percent of PCa is sporadic types, numerous studies have focused on identifying low-penetrability loci associ‐ ated with not only sporadic PCa, but also potentially contributing to the risk of developing familial type of disease [3]. These studies were not designed as linkage, but instead as genetic association studies with case–control matched groups.

#### **3. Genome-wide association studies**

PCa is one of the most genetically and molecularly heterogeneous malignant tumors [2]. Among PCa cases, most are sporadic, while a significantly smaller percent represents familial type, including hereditary cases. High-penetrability PCa-related loci are not common in popula‐ tions and are found to be associated with hereditary PCa. Since PCa represents a multifactori‐ al disease with polygenic basis, and sporadic cases are much more frequently diagnosed, most of the research in the area of PCa molecular genetics has focused on genetic variants with low

The modern research on molecular basis of PCa development includes studies aiming to identify potential genetic markers which could be used in diagnostics and/or monitoring of PCa [1]. This is of utmost importance, since one of the major issues in clinical practice related to PCa is a large percent of latent PCa among newly diagnosed [4]. The overdiagnosis of PCa in early diagnosed cases, due to indolent forms, leads to unnecessary morbidity because of application of invasive therapeutic procedures [5]. This led to focusing the research efforts on discovering genetic markers that could be used for assessing the biological potential of early diagnosed PCa. Therefore, the use of these genetic markers, together with standard prognostic parameters of PCa progression, which include initial serum PSA level, Gleason score, and clinical stage, could greatly improve the current clinical protocols by being implemented in

algorithms for evaluating the patient's risk of PCa and/or PCa aggressiveness [1].

found in studies based on selected candidate genes [7].

146 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

**2. Linkage analyses and high-penetrability loci**

association studies with case–control matched groups.

Studies aiming to identify potential PCa-related loci are designed as case-control or case-only studies, which evaluate the differences in genotype distributions between cases and controls, as well as between different groups of patients, classified according to clinical characteristics. The most validated loci associated with PCa risk were identified through Genome-wide association studies (GWAS) [6]. Nevertheless, numerous PCa-related genetic variants were

Linkage analyses have led to identification of the first high-penetrability PCa susceptibility loci [1]. These studies were based on analyses in hereditary PCa, which is a less frequent type of PCa, and yielded high or moderate-penetrability loci, such as HPC1 (eng. *Hereditary Prostate Cancer 1*, HPC1) mapped in chromosomal region 1q24-25 [8], PCAP (eng. *Predisposing for Cancer Prostate*, PCAP) mapped in 1q42.2-43 [9] and HPCX (eng. *Hereditary Prostate Cancer on X Chromosome*, HPCX) located in 1q42.2-43 [10]. Later on, additional linkage studies identified several other loci primarily associated with familial PCa and rarely found to be altered in sporadic type [1, 3]. Fine-mapping of these regions has led to identifying several candidate genes, such as *RNASEL* or *ELAC2* [11, 12]. Nevertheless, since the major percent of PCa is sporadic types, numerous studies have focused on identifying low-penetrability loci associ‐ ated with not only sporadic PCa, but also potentially contributing to the risk of developing familial type of disease [3]. These studies were not designed as linkage, but instead as genetic

penetrability [3].

The *Human genome project* was critical for making high-throughput genome-wide analyses possible. Not only that this project yielded DNA sequence information but also provided basis for development of methodology, including high-throughput genotyping assays, as well as software tools for analyzing large amount of genetic data [13]. Therefore, sequencing of human DNA provided basis for GWASs, including those on PCa [14].

To date (February 2016), GWASs have identified over 75 variants associated with prostate cancer risk, predominantly in populations of European ancestry (**Figure 1**) [15, 16]. The first GWASs were conducted in 2007, for which a large collection of samples were obtained from PCa patients and healthy controls, as well as databases that included clinical data of patients were constructed [17–19]. The necessity of a large number of subjects for this type of study was obvious even in this early period of conducting GWASs.

**Figure 1.** Ideograms of human chromosomes with marked PCa susceptibility loci identified through GWASs. Ideo‐ grams were obtained from NCBI Map Viewer, while GWAS hits were found in NHGRI-EBI GWAS catalog.

As in other complex diseases, PCa GWASs are usually designed in a multistage manner, with the whole set of tag-single nucleotide polymorphisms (tag-SNPs) being evaluated in the first phase, and only subsets of the most significant SNP being replicated in much larger groups of patients and controls in next phases [20, 21]. Thus, repeating the tests yields the most significant results [20].

The results of initial GWASs showed that most of the PCa-associated genetic variants are located in so-called "gene-deserts". The lack of protein-coding genes in these regions was explained by the supposed presence of regulatory sequences of major proto-oncogenes and tumor-suppressive genes [22, 23]. Today, another explanation is also the presence of genes encoding regulatory RNA molecules within PCa-risk regions [24].

#### **3.1. 8q24 region**

One of the major PCa-related regions was found to be 8q24. Within approximately 1 million base pairs segment of 8q24 reside multiple variants associated with PCa [25]. This region was first identified as associated with PCa susceptibility in a genome-wide linkage study conducted in Icelandic population [26]. Later on, the association of genetic variants within this region with PCa risk was shown in initial GWASs from 2007. Gudmunsson et al., Haiman et al. and Yeager et al. have shown the association between previously reported rs1447295 and PCa risk [17–19]. Also, these first GWASs identified other PCa susceptibility variants within 8q24, rs6983267, and rs16901979. Afterward, GWASs have provided evidence for association of other single-nucleotide genetic variants (SNVs) from 8q24 with PCa risk, such as rs4242382, rs7017300, and rs7837688 [27, 28]. In the recent years, by implementing clinical data and by using case-only design, both GWASs and validation studies have provided evidence for an association of several loci within 8q24 with PCa aggressiveness or survival [29–32].

PCa-susceptibility region within 8q24 was defined as *gene desert*, since no known proteincoding genes were located within it. Nevertheless, the possible biological explanation for the effect of genetic variants located in 8q24 on PCa risk was their influence on the regulation of the expression of nearby genes, mainly *C-MYC*. It was suggested that regulatory sequences controlling the transcription rate of *C-MYC* gene were located in 8q24, and that functional genetic variants which are in strong linkage disequilibrium (LD) with PCa susceptibility locus or several loci effect the sequence and therefore the function of regulatory elements [23]. Previous studies on molecular mechanisms of PCa pathogenesis have shown the functional significance of *C-MYC*, both by analyzing mutational signatures of malignant prostate tissue and by conducting functional analyses in cell cultures, which included stimulation or silencing of *C-MYC* expression [33]. Other than prostate cancer, several other malignancies were associated with 8q24, including breast and colorectal cancer. Some of the subregions of 8q24 associated with these cancers are found to overlap with those related to PCa, while others differ (**Figure 2**) [34].

**Figure 2.** PCa risk-associated regions within 8q24. Lower part of the figure represents Haploview output for a segment of 8q24 (ch8:127500000..129000000) with marked subregions associated with PCa in GWASs. The upper part of the fig‐ ure is a representation of genes located in the region of interest obtained from Ensembl genome browser (GRCh37).

#### **3.2. 17q12**

**3.1. 8q24 region**

148 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

(**Figure 2**) [34].

One of the major PCa-related regions was found to be 8q24. Within approximately 1 million base pairs segment of 8q24 reside multiple variants associated with PCa [25]. This region was first identified as associated with PCa susceptibility in a genome-wide linkage study conducted in Icelandic population [26]. Later on, the association of genetic variants within this region with PCa risk was shown in initial GWASs from 2007. Gudmunsson et al., Haiman et al. and Yeager et al. have shown the association between previously reported rs1447295 and PCa risk [17–19]. Also, these first GWASs identified other PCa susceptibility variants within 8q24, rs6983267, and rs16901979. Afterward, GWASs have provided evidence for association of other single-nucleotide genetic variants (SNVs) from 8q24 with PCa risk, such as rs4242382, rs7017300, and rs7837688 [27, 28]. In the recent years, by implementing clinical data and by using case-only design, both GWASs and validation studies have provided evidence for an

association of several loci within 8q24 with PCa aggressiveness or survival [29–32].

PCa-susceptibility region within 8q24 was defined as *gene desert*, since no known proteincoding genes were located within it. Nevertheless, the possible biological explanation for the effect of genetic variants located in 8q24 on PCa risk was their influence on the regulation of the expression of nearby genes, mainly *C-MYC*. It was suggested that regulatory sequences controlling the transcription rate of *C-MYC* gene were located in 8q24, and that functional genetic variants which are in strong linkage disequilibrium (LD) with PCa susceptibility locus or several loci effect the sequence and therefore the function of regulatory elements [23]. Previous studies on molecular mechanisms of PCa pathogenesis have shown the functional significance of *C-MYC*, both by analyzing mutational signatures of malignant prostate tissue and by conducting functional analyses in cell cultures, which included stimulation or silencing of *C-MYC* expression [33]. Other than prostate cancer, several other malignancies were associated with 8q24, including breast and colorectal cancer. Some of the subregions of 8q24 associated with these cancers are found to overlap with those related to PCa, while others differ

**Figure 2.** PCa risk-associated regions within 8q24. Lower part of the figure represents Haploview output for a segment of 8q24 (ch8:127500000..129000000) with marked subregions associated with PCa in GWASs. The upper part of the fig‐ ure is a representation of genes located in the region of interest obtained from Ensembl genome browser (GRCh37).

17q12 is another PCa susceptibility region identified through initial GWAS. Two of the genetic variants located in 17q12, rs7501939 and rs3760511, were found to be associated with the risk of developing PCa in the study by Gudmundsson et al. conducted in 2007 [35]. In this GWAS, minor alleles of these two single nucleotide genetic variants were found to confer the increased risk of PCa in cohorts of participants from Iceland, Netherlands, and the USA, while in the group of Hispanics this genetic association was not shown [35]. The results of this GWAS were further validated in multiple populations, mostly of European origin [36–43]. Validation studies were even conducted in Africans in which genetic association studies on PCa are scarce [37, 44–47]. The most recent meta-analysis of both GWASs and validation studies has also shown the association of these genetic variants with PCa risk [43].

SNVs rs7501939 and rs3760511 are located in the first intron of the hepatocyte nuclear factor 1 β(*HNF1β* ) or transcription factor 2 (*TCF2*) which is a transcription factor showing tissuespecific expression pattern. Therefore, the association of genetic variants located in 17q12 with PCa risk could be explained by the effect of functional genetic variants on *HNF1β* function or expression [41].

#### **3.3. 17q24**

Another PCa-susceptibility region on chromosome 17 is 17q24. Genetic variants located within this region which were found to be associated with PCa are intergenic variants. Similar to 8q24 genetic variants, those located in 17q24 are found in a gene desert, probably harboring multiple regulatory sequences controlling the expression of surrounding genes [48]. One of the most proximal genes is *SOX9*, which is an important proto-oncogene in prostatic tissue. Recent findings have shown the location of PCa-associated genetic variants in an enhancer looping to *SOX9* gene [48]. Among these genetic variants is a tag-SNP previously identified through GWAS, as well as potentially functional genetic variants found by deep sequencing of PCasusceptibility region [35, 48].

#### **3.4. 10q11**

Two out of the three GWASs, which were published in 2008 in the same issue of *Nature Genetics*, have identified PCa-associated genetic variants in the region 10q11 [27, 36]. After‐ ward, other studies have provided additional evidence to support the association between 10q11 and PCa susceptibility, including both GWASs and validation studies [49–55]. One of these PCa risk-associated genetic variants was located in the close proximity of the transcrip‐ tion start site of the gene Microseminoprotein B (*MSMB* ) which encodes a tumor-suppressor, and was, therefore, even considered as potentially functional. For the risk allele of this genetic variant, it was further shown to affect the expression of *MSMB* gene in a negative manner [56, 57]. The other gene in proximity to this genetic variant is *Nuclear receptor coactivator 4* (*NCOA4*). NCOA4 protein interacts with androgen receptor (AR) and acts as corepressor of androgenresponding genes. Therefore, functional genetic variants in LD with GWAS hits could potentially contribute to PCa risk by affecting the expression of these two genes, or others in proximity [58].

#### **3.5. 19q13**

Region 19q13 harboring kallikrein genes *KLK2* and *KLK3* was found to be associated with PCa susceptibility through GWASs [59]. Several genetic variants associated with PCa risk were located in *KLK3* gene, such as missense SNV rs17632542 identified by fine-mapping of PCaassociated subregion 19q13.33. These genes encode serine-proteases, one of which is PSA, used for PCa diagnosis and disease monitoring. Therefore, the association of PCa-risk genetic variants with serum PSA level was evaluated, yielding statistically significant results for potentially functional SNV rs17632542 [15, 59].

Another subregion associated with PCa risk is 19q13.4 in which a GWASs hit is in strong LD in Chinese population with germline deletion affecting *LILRA3* gene, involved in inflamma‐ tory pathways [60].

#### **4. Candidate gene-based approaches**

Even before GWASs, the necessity of conducting association studies in order to identify low and moderate penetrability genetic variants that contribute to PCa risk was obvious. Therefore, numerous candidate genes were analyzed for genetic variants associated with PCa, with questionable success due to false discoveries and the lack of replication [61]. Candidates were selected based on their potential functional significance in normal prostatic cell growth, malignant transformation, or in the development of metastases. Therefore, among these candidate genes are those encoding proteins involved in androgen signaling, cell-cycle control mechanisms, major tumor-suppressors, or proto-oncogenes, as well as those involved in cellular adhesion or communication with surrounding cellular or matrix components of prostate epithelium [62, 63]. This implies the need for previous knowledge when designing case-control studies using candidate gene approach [64].

Even though these studies were common before GWASs, they are still conducted in numerous populations, aiming to confirm previously found associations, or to identify new ones by analyzing other candidates, selected by using modern research results, such as those involved in regulatory functions of non-coding RNAs [65].

#### **4.1. Protein-coding genes**

#### *4.1.1. Androgen signaling*

Since androgen signaling is essential for growth and survival of prostate epithelial cells, genes involved in androgen biosynthesis, signal reception and transduction, as well as in androgen metabolism have emerged as candidates for case–control studies [63]. Most of these studies involved Androgen receptor (AR), as the major component of androgen signaling and regulation of expression of androgen-responding genes. Among these studies, major percent‐ age relied on analyzing the potential association of the length of CAG repeat string with exon 1 which encodes a poly-glutamine tract of AR with PCa risk [66]. This homopolymeric tract is located in N-terminal domain of AR, which possesses transactivational properties and its length is inversely correlated with transactivation function [67]. Even though initial results were promising, the supposed association was not confirmed in a large percentage of later studies, and the effect sizes were not large enough to support the substantial biological role. Therefore, the association of this genetic variant with PCa risk remains controversial [68–70].

Another three-nucleotide (GGN) repeat string, encoding polyglycine tract in AR, was analyzed for potential association between its length and PCa risk. This repeat string is also located in exon 1, but less studied than the CAG repeat tract, possibly due to technical problems in amplifying GC-rich DNA regions [71]. The effect of the length of GGN repeat string on transactivational properties of AR is still unclear, and the other proposed mechanism of potential functional significance is the effect on AR translation [72]. Studies on the potential association of this microsatellite on PCa risk and progression yielded contrasting results [73– 79].

Mixed results were also found for *SRD5A2* (type II steroid 5α-reductase), which is the major enzyme converting testosterone to dihydrotestosterone. Similarly, studies analyzing genetic variants within *CYP17*, *CYP19A*, *HSD17B*, and *HSD3B* have shown initial promising results, lacking consistent validation [63, 80].

#### *4.1.2. Carcinogen metabolism*

potentially contribute to PCa risk by affecting the expression of these two genes, or others in

Region 19q13 harboring kallikrein genes *KLK2* and *KLK3* was found to be associated with PCa susceptibility through GWASs [59]. Several genetic variants associated with PCa risk were located in *KLK3* gene, such as missense SNV rs17632542 identified by fine-mapping of PCaassociated subregion 19q13.33. These genes encode serine-proteases, one of which is PSA, used for PCa diagnosis and disease monitoring. Therefore, the association of PCa-risk genetic variants with serum PSA level was evaluated, yielding statistically significant results for

Another subregion associated with PCa risk is 19q13.4 in which a GWASs hit is in strong LD in Chinese population with germline deletion affecting *LILRA3* gene, involved in inflamma‐

Even before GWASs, the necessity of conducting association studies in order to identify low and moderate penetrability genetic variants that contribute to PCa risk was obvious. Therefore, numerous candidate genes were analyzed for genetic variants associated with PCa, with questionable success due to false discoveries and the lack of replication [61]. Candidates were selected based on their potential functional significance in normal prostatic cell growth, malignant transformation, or in the development of metastases. Therefore, among these candidate genes are those encoding proteins involved in androgen signaling, cell-cycle control mechanisms, major tumor-suppressors, or proto-oncogenes, as well as those involved in cellular adhesion or communication with surrounding cellular or matrix components of prostate epithelium [62, 63]. This implies the need for previous knowledge when designing

Even though these studies were common before GWASs, they are still conducted in numerous populations, aiming to confirm previously found associations, or to identify new ones by analyzing other candidates, selected by using modern research results, such as those involved

Since androgen signaling is essential for growth and survival of prostate epithelial cells, genes involved in androgen biosynthesis, signal reception and transduction, as well as in androgen metabolism have emerged as candidates for case–control studies [63]. Most of these studies involved Androgen receptor (AR), as the major component of androgen signaling and

proximity [58].

tory pathways [60].

potentially functional SNV rs17632542 [15, 59].

150 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

**4. Candidate gene-based approaches**

case-control studies using candidate gene approach [64].

in regulatory functions of non-coding RNAs [65].

**4.1. Protein-coding genes**

*4.1.1. Androgen signaling*

**3.5. 19q13**

Among genes involved in cell detoxification, those encoding glutathione-S-transferases have been mostly analyzed. Nevertheless, most of these studies yielded insignificant results on association with PCa risk [81]. Other frequently analyzed genes involved in metabolism of carcinogens are *PON1*, *CYP1A1, CYP1B1*, and *CYP3A4* [80, 82–85].

Two genetic variants within *PON1* have been analyzes in multiple populations, L55M and Q129R. The results to date are inconclusive, but the meta-analysis conducted in 2012 suggested the association of L55M missense variant with PCa risk [82]. Also, a recent meta-analysis on only three PCa studies and Q129R showed statistically significant association for several genetic models of association [83].

The most commonly analyzed SNVs in *CYP1A1* are missense variants rs1048943 (p.Ile462Val) and rs4646903, which are also called MspI polymorphisms, since they alter the recognition site for MspI restriction enzyme. Numerous studies and also the recent meta-analyses showed the association between these SNVs and PCa risk [84–86].

The results obtained for genetic variants in *CYP1B1* and *CYP3A4* are controversial, with the recent meta-analyses suggesting the association of L432V, N453S, and A119S polymorphisms of *CYP1B1* and A392G in *CYP3A4* with PCa susceptibility [87, 88].

#### *4.1.3. DNA repair, cell cycle control, and apoptosis*

Dysfunctions of DNA repair pathway, apoptosis regulation, and cell cycle control mechanisms alter the cells response to DNA damage and lead to uncontrolled proliferation, progression and metastasis of malignant diseases. Also, genetic variants in genes involved in these processes could potentially attribute to cancer susceptibility and/or progression risk [62].

Among the genes analyzed for association between genetic variants and prostate cancer risk or aggressiveness are *XRCC1* and *XRCC3* (X-ray repair cross-complementing proteins 1 and 3), *ERCC1* and *ERCC2* (Excision repair cross-complementing rodent repair deficiency, complementation group 1 and 2), *LIG4* (Ligase IV), *ATM* (Ataxia telangiectasia mutated), *XPD* (Xeroderma pigmentosum group D), *MDM2* (Human mouse double-minute 2 protein), *CDKN1A*, and *CDKN1B* (Cyclin-Dependent Kinase Inhibitors 1A, and 1B), *CCND1* (Cyclin D1) as well as *BCL2* (B-cell lymphoma 2) and *TP53* (tumor protein p53) [89–102]. Genetic variants within most of these genes were found to be associated with PCa aggressiveness or response to therapy. Nevertheless, these results were seldom replicated in multiple populations.

The most common SNVs in *XRCC1* studied in case–control studies on cancer risk are rs1799782 (p.Arg194Trp), rs25489 (p.Arg280His), and rs25487 (p.Arg399Gln) [89, 103]. These genetic variants were also analyzed for their potential association with PCa risk in numerous studies, but the obtained results were inconsistent [89, 90]. For rs25489, association with radiationinduced late toxicity in PCa patients was also shown [104]. Similarly, rs861539 (p.Thr241Met) in *XRCC3* was found to be associated with early adverse effects induced by radiotherapy, based on quantitative data synthesis of 6 studies [105].

A recent study conducted in Spain showed the association of rs11615 in *ERCC1* and rs17503908 in *ATM* with PCa aggressiveness [93]. Genetic variants in the same chromosomal region as *ERCC1* were previously analyzed in a large study that provided opposing results. Neverthe‐ less, this previous study was designed as to include subjects from multiple populations, and its results could therefore be influenced by genetic backgrounds of study participants [93, 106].

Among genetic variants located in *MDM2*, missense variant SNP309 in the promoter region was most frequently analyzed. This SNV was found to be associated with both PCa risk and aggressiveness in multiple studies [107, 108]. The first study on this subject yielded no evidence of the supposed association [109]. Nevertheless, results obtained in several later studies suggested the association of SNP309 with the risk of PCa progression to the more advanced stage, or the statistical trend of significance was reached [108].

Numerous studies conducted on a potential association between *CCND1* genetic variant rs603965 (p.Ala870Gly) and PCa risk, yielding inconsistent results [99]. This SNV was found to affect alternative splicing and thus alter the C-terminal domain. Other genetic variants within this gene were shown to be associated with the risk of PCa biochemical reoccurrence after radical prostatectomy [110].

The most extensively analyzed SNV located in *TP53* gene is rs1042522 (p.Arg72Pro). This genetic variant was found to be associated with PCa risk, especially among Caucasians [102]. When it comes to *BCL2*, encoding the founding member of apoptosis regulatory proteins, promoter SNV c.-938C > A was associated with PCa risk, although lacking replication, as well as with disease-free survival and biochemical recurrence of PCa after radical prostatectomy [100, 101, 111].

#### *4.1.4. Vitamin D signaling*

*4.1.3. DNA repair, cell cycle control, and apoptosis*

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on quantitative data synthesis of 6 studies [105].

stage, or the statistical trend of significance was reached [108].

after radical prostatectomy [110].

Dysfunctions of DNA repair pathway, apoptosis regulation, and cell cycle control mechanisms alter the cells response to DNA damage and lead to uncontrolled proliferation, progression and metastasis of malignant diseases. Also, genetic variants in genes involved in these processes could potentially attribute to cancer susceptibility and/or progression risk [62].

Among the genes analyzed for association between genetic variants and prostate cancer risk or aggressiveness are *XRCC1* and *XRCC3* (X-ray repair cross-complementing proteins 1 and 3), *ERCC1* and *ERCC2* (Excision repair cross-complementing rodent repair deficiency, complementation group 1 and 2), *LIG4* (Ligase IV), *ATM* (Ataxia telangiectasia mutated), *XPD* (Xeroderma pigmentosum group D), *MDM2* (Human mouse double-minute 2 protein), *CDKN1A*, and *CDKN1B* (Cyclin-Dependent Kinase Inhibitors 1A, and 1B), *CCND1* (Cyclin D1) as well as *BCL2* (B-cell lymphoma 2) and *TP53* (tumor protein p53) [89–102]. Genetic variants within most of these genes were found to be associated with PCa aggressiveness or response to therapy. Nevertheless, these results were seldom replicated in multiple populations.

The most common SNVs in *XRCC1* studied in case–control studies on cancer risk are rs1799782 (p.Arg194Trp), rs25489 (p.Arg280His), and rs25487 (p.Arg399Gln) [89, 103]. These genetic variants were also analyzed for their potential association with PCa risk in numerous studies, but the obtained results were inconsistent [89, 90]. For rs25489, association with radiationinduced late toxicity in PCa patients was also shown [104]. Similarly, rs861539 (p.Thr241Met) in *XRCC3* was found to be associated with early adverse effects induced by radiotherapy, based

A recent study conducted in Spain showed the association of rs11615 in *ERCC1* and rs17503908 in *ATM* with PCa aggressiveness [93]. Genetic variants in the same chromosomal region as *ERCC1* were previously analyzed in a large study that provided opposing results. Neverthe‐ less, this previous study was designed as to include subjects from multiple populations, and its results could therefore be influenced by genetic backgrounds of study participants [93, 106].

Among genetic variants located in *MDM2*, missense variant SNP309 in the promoter region was most frequently analyzed. This SNV was found to be associated with both PCa risk and aggressiveness in multiple studies [107, 108]. The first study on this subject yielded no evidence of the supposed association [109]. Nevertheless, results obtained in several later studies suggested the association of SNP309 with the risk of PCa progression to the more advanced

Numerous studies conducted on a potential association between *CCND1* genetic variant rs603965 (p.Ala870Gly) and PCa risk, yielding inconsistent results [99]. This SNV was found to affect alternative splicing and thus alter the C-terminal domain. Other genetic variants within this gene were shown to be associated with the risk of PCa biochemical reoccurrence

The most extensively analyzed SNV located in *TP53* gene is rs1042522 (p.Arg72Pro). This genetic variant was found to be associated with PCa risk, especially among Caucasians [102]. When it comes to *BCL2*, encoding the founding member of apoptosis regulatory proteins, Vitamin D signaling in PCa has stimulatory effect on apoptosis, as well as inhibitory effect on the progression of cell cycle. Therefore, multiple genetic variants within the gene encoding the receptor for vitamin D (*VDR*) were analyzed for their potential association with PCa risk and/ or progression. Most of them are loci named FokI, BsmI, ApaI, and T I, according to restriction enzyme used for genotyping, Cdx2 in promoter region and polyA microsatellite, which were most frequently tested [112–114].

Even though the initial results on these loci were promising, in multiple populations, they were not replicated [113, 115]. The association of these genetic variants with PCa progression parameters and the disease outcome also remains inconclusive [113, 116].

#### *4.1.5. Chronic inflammation and angiogenesis*

Numerous genes involved in chronic inflammation have been studies for association of genetic variants that reside within them with PCa risk and/or progression [117]. Also, the importance of vascular support to cancer growth stimulated the association studies on PCa analyzing genetic variants located in angiogenesis-related genes [62]. Since these processes are code‐ pendent, numerous genes primarily found to be involved in chronic inflammation are also discussed as angiogenesis-related genes, and vice versa.

Among the most important factors of chronic inflammation are *TGF-β*, *COX2*, *TNF-α*, and *IL-1 β*, as well as *PPAR-γ*. To date, several SNVs in *TGF-β1* have been identified as PCa-suscepti‐ bility variants, some of them also associated with PCa aggressiveness [118–123]. The studies on the most of the chronic inflammation-related genes provided conflicting results [117].

There have been various PCa case–control studies involving *Vascular endothelial growth factor* (*VEGF*) gene, encoding the important proangiogenic growth factor, as well as genes encoding Interleukin 8 (IL-8 ) and Interleukin 10 (IL-10) [96, 124–128] for genetic variant rs1570360 [c.-1154G > A] located in the promoter region of *VEGF*, statistically significant association with PCa risk was shown in several studies [126]. Most other *VEGF* genetic variants analyzed for potential association with PCa risk and/or progression are also located in the promoter region [126, 129–131]. These SNVs could be associated with transcription rate of *VEGF* [132], which is positively correlated with tumor stage, Gleason score, as well as with shorter period of disease-free survival [133].

Candidates for this type of studies were also genes encoding transcription factors which regulate the expression of *VEGF*, such as Hypoxia inducible factor 1 (*HIF1A* ), Epidermal growth factor (*EGF* ), and Lymphotoxin α (*LTA*). Nevertheless, except for *HIF1A*, association of genetic variants within these genes with PCa risk was not shown, or was mostly found in small sample studies and poorly replicated [62, 125, 126, 134].

Some of the key regulators of angiogenesis are also fibroblast growth factors (*FGF*s). Therefore, receptor *FGFR4* gene has been analyzed for genetic variants associated with PCa risk and/or progression. The most commonly tested SNV is a missense variant rs351855 (p.Gly388Arg), found to be associated with PCa risk and aggressiveness in a relatively small number of studies [135].

Among the most extensively analyzed candidate genes in PCa-related case-control studies are *NOS3* and *NOS2A*, encoding nitric oxide synthases [136]. Both endothelial and inducible nitric oxide synthases, encoded by these genes, are enzymes that catalyze the production of NO from L-arginine and L-citrulline amino acids [137]. Being the major producer of NO in endothelial cells, eNOS, encoded by *NOS3*, is involved in the control of vascular tone and angiogenesis, which is essential for tumor growth and the development of metastases. Yet, the synthesis of NO is associated with apoptosis, which has the opposing effect on carcinogenesis [138]. Numerous genetic variants within these genes, especially *NOS3*, have been analyzed for potential association with PCa risk and/or progression [136]. Most commonly analyzed SNVs are -786 T > C (rs2070744) and 894G > T (rs1799983) [139–147], while several studies included insertion-deletion polymorphism 4a4b located in intron 4 of *NOS3* [140, 146, 148, 149]. For rs1799983, which is a missense genetic variant, it was hypothesized to affect NOS3 stability [150]. The other common SNV, rs2070744, affects promoter activity by allele C creating a binding site with validation protein 1A (RPA1) [151].

Angiogenesis process and tumor invasion also require degradation of extracellular matrix and basal membranes, which are catalyzed by matrix metalloproteinases. Among the genes encoding this class of enzymes, *MMP2* and *MMP9* are analyzed for genetic variants associated with PCa risk, and also for disease aggressiveness, due to their functional significance in tumor invasiveness [139, 152–156]. Commonly analyzed genetic variant in *MMP2* promoter is rs243865. For minor allele of this SNV it was shown to be associated with reduced transcription rate of *MMP2* [157].

#### *4.1.6. Cellular adhesion*

Among genes involved in cellular adhesion, *CDH1* encoding E-cadherin was the candidate gene for the most case-control studies on PCa. Since aberrant expression of this gene is correlated with the increased metastatic potential of PCa, genetic variants in its promoter region were analyzed for potential association with PCa risk and progression [158, 159]. Most extensively studied SNV−160C > A was found to affect *CDH1* expression and was identified as PCa susceptibility genetic variant in multiple populations [158, 160].

Only few studies also included genetic variants in genes encoding intercellular adhesion molecules (ICAMs), proteins involved in cellular adhesion and signaling. The analyzed genetic variants are those located in *ICAM-1*, *ICAM-4*, and *ICAM-5* genes and need a further evaluation for potential association with PCa risk and/or progression [161, 162].

#### **4.2. Long noncoding RNA genes**

Some of the key regulators of angiogenesis are also fibroblast growth factors (*FGF*s). Therefore, receptor *FGFR4* gene has been analyzed for genetic variants associated with PCa risk and/or progression. The most commonly tested SNV is a missense variant rs351855 (p.Gly388Arg), found to be associated with PCa risk and aggressiveness in a relatively small number of studies

Among the most extensively analyzed candidate genes in PCa-related case-control studies are *NOS3* and *NOS2A*, encoding nitric oxide synthases [136]. Both endothelial and inducible nitric oxide synthases, encoded by these genes, are enzymes that catalyze the production of NO from L-arginine and L-citrulline amino acids [137]. Being the major producer of NO in endothelial cells, eNOS, encoded by *NOS3*, is involved in the control of vascular tone and angiogenesis, which is essential for tumor growth and the development of metastases. Yet, the synthesis of NO is associated with apoptosis, which has the opposing effect on carcinogenesis [138]. Numerous genetic variants within these genes, especially *NOS3*, have been analyzed for potential association with PCa risk and/or progression [136]. Most commonly analyzed SNVs are -786 T > C (rs2070744) and 894G > T (rs1799983) [139–147], while several studies included insertion-deletion polymorphism 4a4b located in intron 4 of *NOS3* [140, 146, 148, 149]. For rs1799983, which is a missense genetic variant, it was hypothesized to affect NOS3 stability [150]. The other common SNV, rs2070744, affects promoter activity by allele C creating a

Angiogenesis process and tumor invasion also require degradation of extracellular matrix and basal membranes, which are catalyzed by matrix metalloproteinases. Among the genes encoding this class of enzymes, *MMP2* and *MMP9* are analyzed for genetic variants associated with PCa risk, and also for disease aggressiveness, due to their functional significance in tumor invasiveness [139, 152–156]. Commonly analyzed genetic variant in *MMP2* promoter is rs243865. For minor allele of this SNV it was shown to be associated with reduced transcription

Among genes involved in cellular adhesion, *CDH1* encoding E-cadherin was the candidate gene for the most case-control studies on PCa. Since aberrant expression of this gene is correlated with the increased metastatic potential of PCa, genetic variants in its promoter region were analyzed for potential association with PCa risk and progression [158, 159]. Most extensively studied SNV−160C > A was found to affect *CDH1* expression and was identified

Only few studies also included genetic variants in genes encoding intercellular adhesion molecules (ICAMs), proteins involved in cellular adhesion and signaling. The analyzed genetic variants are those located in *ICAM-1*, *ICAM-4*, and *ICAM-5* genes and need a further evaluation

as PCa susceptibility genetic variant in multiple populations [158, 160].

for potential association with PCa risk and/or progression [161, 162].

binding site with validation protein 1A (RPA1) [151].

154 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

rate of *MMP2* [157].

*4.1.6. Cellular adhesion*

[135].

The potential involvement of long noncoding RNAs (lncRNAs) in prostate carcinogenesis was suggested not only by the results of expression analyses that showed several known oncogenic and/or tumor-suppressive lcnRNAs to be aberrantly expressed in malignant prostatic tissue or plasma samples from patients with PCa but also by the identification of several PCa-specific lncRNAs [163, 164].

Several SNVs in lncRNA genes were identified as PCa susceptibility variants in case–control studies on PCa. In their study published in 2011, Jin et al. have stated that eight SNVs identified to that time through GWAS are located in lncRNA intervals [165]. They also identified a SNV in a putative lncRNA which was not later experimentally confirmed as a PCa-susceptibility variants [165]. In a study published in 2013, Xue et al. have shown the association between two tag-SNPs in Prostate cancer gene expression marker 1 (*PCGEM1* ) and PCa risk in Chinese population [166]. Genetic variant in another PCa-specific gene, prostate cancer associated 3 (*PCA3*), was analyzed for the length of a TAAA repeat string in the promoter region. This genetic variant was also found to be associated with PCa risk [167]. In a GWAS published in 2014, Cook et al. have identified rs7918885 in *RP11-543 F8.2* gene as a PCa-susceptibility SNV in West African men, although GWAS statistical significance threshold was not reached [168]. Also, by using fine-mapping and resequencing of PCa-susceptibility subregion of 8q24, *lncRNA* gene prostate cancer noncoding RNA 1 (*PRNCR1*) was found to be located between the most significantly associated genetic variant [169].

#### **4.3.** *MicroRNA* **genes**

Dysregulation of diverse regulatory mechanisms based on microRNA activity has been implicated in prostate carcinogenesis. Therefore, possibly functional genetic variants located in *microRNA* genes emerged as potential PCa-associated loci. Among these genetic variants are those that potentially influence microRNA biogenesis, stability of mature microRNAs, efficiency of target gene regulation, as well as target specificity. By affecting these features of microRNA regulatory mechanisms, microRNA SNVs could be associated with aberrant expression of various important PCa-related oncogenes or tumor-suppressive genes [170–172].

MicroRNA genetic variants have been analyzed for their potential association with PCa in only a few studies conducted in Asian populations and in a single population of European origin. These studies have provided discordant results on the effects of genetic variants in rs2910164 in *hsa-miR-146a* [173–176], *hsa-miR-196a2* [174, 176, 177], and rs3746444 in *hsa-miR-499* gene on PCa risk [174, 177]. In a recent study, rs4705342 located in *hsa-miR-143* gene promoter was found to be associated with the risk of developing PCa [178]. Since the number of conducted studies is small, additional findings from multiple populations are needed in order to make further conclusions.

Another SNV, rs895819 located in a gene encoding miR-27a, which is androgen-regulated and stimulates the androgen signalization in a positive feedback loop, was found to be associated with PCa risk, as well as with the development of distant metastases. Nevertheless, these results are derived from a single study on PCa risk and rs895819 conducted relatively recent and needs further validation [177].

#### **5. Replication, validation studies, and Meta-analyses**

Differences in genetic backgrounds are an important issue in genetic association studies. Therefore, interpretation of data requires discussing the potential differences between populations. Therefore, in order to analyze such differences, multiple validation analyses are conducted in various population and ethnicities. These studies are designed so that they resemble as much as possible to the original study that yielded genetic associations, or the lack of it. The ratio for conducting such studies is the possible lack of association between identified PCa-susceptibility variants with PCa risk in certain populations, or the differences in effect sizes [179]. Replication studies, conducted in confirmation group of participants from the same population in which the initial results were found, is a method of checking reproducibility and evaluating possible false positives and effect overestimation [179, 180].

Currently, replications and validations are conducted for both GWASs results, as well as for results from candidate gene-based studies. Of utmost importance is conducting replication and validation analyses of hits from studies with relatively small sample sizes, as well as with poorly clinically characterized cases with the lack of data on possible confounders, or ques‐ tionable recruitment of controls [180]. Also, an important issue in case–control studies on PCa is the type of control group, which is in some cases healthy controls, while in others group of patients with benign prostatic hyperplasia (BPH). Furthermore, classification systems for patients with PCa which are used for evaluating potential genetic associations with PCa progression differ between studies, which together with small sizes of patient groups, calls for replication of acquired statistically significant data.

All of these issues are a potential reason for the opposing results on the association of the most of genetic variants analyzed in multiple studies with PCa risk and progression. Therefore, in order to elucidate the effect of these genetic variants, meta-analyses of eligible studies are frequently conducted. Combining the results from smaller studies through data synthesis in meta-analysis could result in increased statistical power [181]. Therefore, meta-analyses could provide more precise estimations, as well as the insight in the potential effect of confounders [182], such as ethnicity, participant recruitment strategy, or study size.

#### **6. Future perspectives**

The main aim of genetic association studies on PCa is the identification of potential PCa genetic markers which could be used for constructing reliable algorithms for evaluating the risk for PCa development and/or PCa progression [1]. Therefore, it is important not only to identify these PCa-related genetic variants, but also to precisely characterize their effect sizes. In order to do that, ethnic differences need to be taken into account [179]. Other important issues in interpreting results of association studies are gene–gene and gene-environment interactions. Therefore, future research and designing such algorithms require integration of knowledge on genetic associations, cellular pathways, and statistical epistasis in which real biological interaction could be reflected.

Since the major problem in clinical practice related to PCa is the overdiagnosis and monitoring of patients [4], additional studies on PCa aggressiveness with clinically well characterized groups of PCa patients are needed to identify genetic variants associated with PCa progression risk. The later implementation of algorithms based on these genetic variants could greatly improve clinical protocols in monitoring and treating PCa.

#### **7. Conclusion**

results are derived from a single study on PCa risk and rs895819 conducted relatively recent

Differences in genetic backgrounds are an important issue in genetic association studies. Therefore, interpretation of data requires discussing the potential differences between populations. Therefore, in order to analyze such differences, multiple validation analyses are conducted in various population and ethnicities. These studies are designed so that they resemble as much as possible to the original study that yielded genetic associations, or the lack of it. The ratio for conducting such studies is the possible lack of association between identified PCa-susceptibility variants with PCa risk in certain populations, or the differences in effect sizes [179]. Replication studies, conducted in confirmation group of participants from the same population in which the initial results were found, is a method of checking reproducibility and

Currently, replications and validations are conducted for both GWASs results, as well as for results from candidate gene-based studies. Of utmost importance is conducting replication and validation analyses of hits from studies with relatively small sample sizes, as well as with poorly clinically characterized cases with the lack of data on possible confounders, or ques‐ tionable recruitment of controls [180]. Also, an important issue in case–control studies on PCa is the type of control group, which is in some cases healthy controls, while in others group of patients with benign prostatic hyperplasia (BPH). Furthermore, classification systems for patients with PCa which are used for evaluating potential genetic associations with PCa progression differ between studies, which together with small sizes of patient groups, calls for

All of these issues are a potential reason for the opposing results on the association of the most of genetic variants analyzed in multiple studies with PCa risk and progression. Therefore, in order to elucidate the effect of these genetic variants, meta-analyses of eligible studies are frequently conducted. Combining the results from smaller studies through data synthesis in meta-analysis could result in increased statistical power [181]. Therefore, meta-analyses could provide more precise estimations, as well as the insight in the potential effect of confounders

The main aim of genetic association studies on PCa is the identification of potential PCa genetic markers which could be used for constructing reliable algorithms for evaluating the risk for PCa development and/or PCa progression [1]. Therefore, it is important not only to identify these PCa-related genetic variants, but also to precisely characterize their effect sizes. In order to do that, ethnic differences need to be taken into account [179]. Other important issues in

**5. Replication, validation studies, and Meta-analyses**

evaluating possible false positives and effect overestimation [179, 180].

[182], such as ethnicity, participant recruitment strategy, or study size.

replication of acquired statistically significant data.

**6. Future perspectives**

and needs further validation [177].

156 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

The efforts for improving clinical protocols in PCa diagnostics, monitoring and treatment resulted in conducting genetic association studies on PCa. These studies aim to identify potential PCa genetic markers and characterize their association with PCa risk and/or pro‐ gression through measuring effect sizes. The identified and validated genetic markers could then be used for constructing reliable algorithms for evaluating the risk for PCa development and, more importantly, for PCa progression. Implementing such algorithms in clinical practice is expected to improve the distinction between early diagnosed PCa cases that require aggressive treatment and latent PCa cases which remain indolent during patient's lifetime.

#### **Author details**

Zorana Nikolić, Dušanka Savić Pavićević and Goran Brajušković\*

\*Address all correspondence to: brajuskovic@bio.bg.ac.rs

Faculty of Biology, Centre for Human Molecular Genetics, University of Belgrade, Belgrade, Serbia

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### **Key Genes in Prostate Cancer Progression: Role of MDM2, PTEN, and TMPRSS2-ERG Fusions**

Appu Rathinavelu and Arkene Levy

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64337

#### **Abstract**

In recent years, multiple genes or their protein products have been linked to initiation and progression of prostate cancer. Such genes include TMPRSS2, ERG, PTEN, and MDM2. This chapter discusses the pathological roles as well as the potential diagnos‐ tic and therapeutic applications of these genes that are highly expressed in prostate cancer when compared to other cancer types. The presence of these genes and related defects are linked to growth, progression, metastasis, invasiveness and resistance in prostate cancers. While knowledge related to TMPRSS2, ERG, and PTEN have been accumulating in the last two decades, the prometastatic role of MDM2 has been emerging in the last few years and revealing important functions related to prostate cancer progression.

**Keywords:** prostate cancer, TMPRSS2-ERG, PTEN, MDM2

#### **1. Introduction**

Prostate cancer (PCa) is a long latency tumor that occurs in males that are typically aged 50 years and older. Globally, more than 1.1 million cases of prostate cancer were recorded in 2012, accounting for around 8% of all new cancer cases and 15% in men [1]. In 2015, an estimated 220,800 men will be diagnosed with PCa in the United States and an estimated 27,540 men will die due to the disease making this malignancy the second leading cause of cancerrelated death in men [2]. In addition, African-American (AA) men have the highest inci‐ dence and mortality from PCa when compared to other races [2]. The pathophysiology of prostate cancer is not fully elucidated, but it is well established that this dreadful disease is primarily initiated by cellular proliferation within pre-existing ducts and glands, which is

© 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.

referred to as Prostatic intraepithelial neoplasia (PIN). The PIN eventually progresses to invasive prostate cancer [3]. Clinical manifestations of the disease are variable and based on the transport by blood or the lymphatic system to metastatic sites and the effects of local‐ ized tumor growth. Localized prostate cancer is typically curable with targeted local thera‐ py such as radical prostatectomy or radiation therapy. In metastatic prostate cancer, one of the successful strategies of treatment is surgical or chemical castration leading to androgen deprivation therapy (ADT) [4]. Unfortunately, approximately 33% of patients develop resistance to these treatments with the eventual increases in the number of androgens, prostate specific antigen (PSA), and circulating tumor cells (CTCs), leading to the more progressive and metastatic castration resistant prostate cancer (CRPC) [5]. The poor prognosis associat‐ ed with metastatic prostate cancers is attributable in part to the highly heterogeneous nature of the cancer cells, which provides a significant hurdle fortreatment of the disease [6]. Multiple genomic alterations underlie the clinical heterogeneity of prostate cancer and such aberra‐ tions include, point mutations, microsatellite variations, and chromosomal alterations such as translocations, insertions, duplications, fusions, and deletions [6, 7]. Therefore, there is a heightened interest in understanding the role of these genetic changes in prostate cancer development and progression.

#### **2. Key genes in prostate cancer progression**

In the past decade, several genes associated with prostate cancer have been identified. Four such genes: the ETS-related gene (ERG), The Transmembrane Protease Serine 2 (TMPRSS2), Mouse double minute 2 homolog (MDM2), and Phosphatase and tensin homolog (PTEN) have gained recognition for their high specificity of expression in prostatic carcinomas.

#### **2.1. Prostate cancer and PTEN**

PTEN is a protein coding gene that encodes for phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase. It contains a tensin-like domain in addition to a catalytic domain similar to that of the dual specificity protein tyrosine phosphatases. PTEN is one of the most commonly mutated tumor suppressor genes in human prostate cancer. Interestingly, many aspects of PTEN expression and function, including transcriptional and posttranscriptional regulation, post-translational modifications, and protein-protein interac‐ tions have been shown to be altered in human prostate cancer. PTEN is a nonredundant phosphatase that directly interferes with the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway and thereby controls several processes that are impor‐ tant in the homeostasis of cell survival and a multitude of cellular functions, which includes growth, proliferation, metabolism, migration, and cellular architecture [8]. PTEN removes the phosphate from the D3 position of phosphatidylinositol-3,4,5-triphosphate (PIP3), a product of PI3K, thus, can lead to inhibition of downstream AKT activation in normal conditions. However, when PTEN is mutated there is sustained activation of AKT that can lead to cell proliferation, angiogenesis and other related events. AKT exists in three isoforms, namely AKT1, AKT2, and AKT3, which are typically activated by the phosphorylation at two specific sites: Thr308 by PDK1 [9] and Ser473 by the mammali‐ an target of rapamycin complex 2 (mTORC2) [10]. Activated AKT can drive cell survival, proliferation, growth, angiogenesis, and metabolism by phosphorylating downstream signaling proteins, which include inhibitory phosphorylation of GSK3, FOXO, BAD, p21, p27, and PGC I and activating phosphorylation of mTORC I mammalian target of rapamycin complex I (mTORC I), IKK-β, MDM2, ENTPD5, SREBP1C, AS160, and SKP2, which eventually leads to cell cycle progression and proliferation [10, 11]. Inhibition of GSK3β has been shown to specifically prevent the degradation of cyclin D1 and βcatenin, which can further support G1 to S phase transition in different types of cancers including prostate cancers [11, 12]. Activation of AKT also helps to evade apoptosis directly by phosphorylation of the pro-apoptotic protein BAD [13]. Hence, re-expres‐ sion of wild-type *PTEN* in *PTEN* null prostate cancer cell lines can lead to the initia‐ tion of apoptosis and regression of tumors [14]. In addition, AKT directly activates the mTOR pathway by phosphorylating TSC2, which dismantles the TSC1/TSC2 complex that keeps the Rheb in an inhibited state. Once released from the TSC1/TSC2 inhibi‐ tion, the Rheb can stimulate the phosphotransferase activity of mTORC1 and phosphor‐ ylate the S6 kinase (S6K) and 4E-binding protein (4EBP1), which in turn initiates capdependent protein translation [15, 16]. Therefore, as a consequence of PTEN loss in prostate cancers, PI3K/AKT/mTOR pathway activation can strongly lead to enhanced translation of mRNAs involved in cell growth and proliferation.

referred to as Prostatic intraepithelial neoplasia (PIN). The PIN eventually progresses to invasive prostate cancer [3]. Clinical manifestations of the disease are variable and based on the transport by blood or the lymphatic system to metastatic sites and the effects of local‐ ized tumor growth. Localized prostate cancer is typically curable with targeted local thera‐ py such as radical prostatectomy or radiation therapy. In metastatic prostate cancer, one of the successful strategies of treatment is surgical or chemical castration leading to androgen deprivation therapy (ADT) [4]. Unfortunately, approximately 33% of patients develop resistance to these treatments with the eventual increases in the number of androgens, prostate specific antigen (PSA), and circulating tumor cells (CTCs), leading to the more progressive and metastatic castration resistant prostate cancer (CRPC) [5]. The poor prognosis associat‐ ed with metastatic prostate cancers is attributable in part to the highly heterogeneous nature of the cancer cells, which provides a significant hurdle fortreatment of the disease [6]. Multiple genomic alterations underlie the clinical heterogeneity of prostate cancer and such aberra‐ tions include, point mutations, microsatellite variations, and chromosomal alterations such as translocations, insertions, duplications, fusions, and deletions [6, 7]. Therefore, there is a heightened interest in understanding the role of these genetic changes in prostate cancer

In the past decade, several genes associated with prostate cancer have been identified. Four such genes: the ETS-related gene (ERG), The Transmembrane Protease Serine 2 (TMPRSS2), Mouse double minute 2 homolog (MDM2), and Phosphatase and tensin homolog (PTEN) have

PTEN is a protein coding gene that encodes for phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase. It contains a tensin-like domain in addition to a catalytic domain similar to that of the dual specificity protein tyrosine phosphatases. PTEN is one of the most commonly mutated tumor suppressor genes in human prostate cancer. Interestingly, many aspects of PTEN expression and function, including transcriptional and posttranscriptional regulation, post-translational modifications, and protein-protein interac‐ tions have been shown to be altered in human prostate cancer. PTEN is a nonredundant phosphatase that directly interferes with the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway and thereby controls several processes that are impor‐ tant in the homeostasis of cell survival and a multitude of cellular functions, which includes growth, proliferation, metabolism, migration, and cellular architecture [8]. PTEN removes the phosphate from the D3 position of phosphatidylinositol-3,4,5-triphosphate (PIP3), a product of PI3K, thus, can lead to inhibition of downstream AKT activation in normal conditions. However, when PTEN is mutated there is sustained activation of AKT that can lead to cell proliferation, angiogenesis and other related events. AKT exists in three isoforms, namely AKT1, AKT2, and AKT3, which are typically activated by the

gained recognition for their high specificity of expression in prostatic carcinomas.

development and progression.

**2.1. Prostate cancer and PTEN**

**2. Key genes in prostate cancer progression**

180 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

The *PTEN* gene is comprised of nine exons and totally codes for 403 amino acids [17]. The substrate binding site of PTEN is in the C2 domain, which can bind to the phospholipid membranes. The C2 domain also contains a signature motif HCXXGXXRthatis typically found in the protein tyrosine phosphateses (PTPs) and in the dual specific protein phosphatases (DPPs). In addition, there is a short phosphatidylinositol-4,5-bisphosphate (PIP2) binding domain (PDB) on the N terminus, a motif on the C-terminal tail that interacts with PDZ-BD domain-containing proteins, and regulates protein stability and two PEST domains contain‐ ing proline (P), glutamic acid (E), serine (S), and threonine (T) amino acids, which acts as a signal peptide that is also involved in the stability and degradation of PTEN [18]. When PIP2 binds to the PDB domain of PTEN it produces a conformational change in the protein leading to allosteric activation of substrate binding site for attracting the substrates for de-phosphor‐ ylation [19]. In addition to the allosteric activation, the positive charge of the substrate binding pocket of PTEN's is also essential for accommodating larger substrates such as phosphoino‐ sitides. The phosphatase domain of PTEN is a evolutionarily conserved domain that harbors nearly 40% of its cancer-associated mutations, and the most common mutations are Cl24S mutation, which abolishes both lipid binding and protein phosphatase activity, and the G129E mutation that destroys the lipid phosphatase activity [20–22]. However, some of the impor‐ tant PTEN tumorigenic mutations occur on the C2 domain also, confirm the importance of the structural integrity of the C terminus in maintaining PTEN activity and protein stability [23, 24] (**Figure 1**). In prostate cancer, PTEN loss most commonly results from a somatic muta‐ tion generated through copy number loss rather than point mutation [25, 26], however, recent exome sequencing has identified several recurrent mutations also in the PTEN gene [27, 28].

**Figure 1.** Different domains of PTEN and the phosphorylation sites. (Obtained from: Cell Res. 2008; 18: 807–816.)

#### *2.1.1. PTEN loss combined with alterations in inflammatory pathway regulators*

Various lines of evidence suggest that chronic inflammation is a closely associated event in the tumorigenic mechanisms of prostate cancer [29, 30] and to the several mutations that are causing this disease. A cytokine that is most commonly associated with tumor growth, proliferation, and angiogenesis in many cancers and also the most frequently found inflam‐ matory mediator in prostate cancer is IL-6 [31]. When expressed at high levels, in addition to imposing the inflammatory functions, a strong correlation between the circulating levels of IL-6 and advancement in the stages of prostate cancer, therapeutic resistance, and as a result an overall poor prognosis has been well established until now [32]. Although one of the most important consequences of IL-6 expression is the stimulation of the JAK/STAT3 pathway [33], phosphorylation of STAT3 at Scr727 and activation of its function by the PI3K-AKT pathway cannot be ruled out completely because of the impact PTEN mutations can produce on this pathway [34]. Such activation of STAT3 can also lead to metastatic behavior of prostate cancer cells in both *in vitro* and *in vivo* conditions, through stimulation of angiogenesis and suppres‐ sion of antitumor immune responses [35]. Many inflammatory cytokines and chemokines promote tumor progression by converging on and stimulating the IKK2/NF-κB signaling axis [36]. In addition to the above-mentioned mechanisms, constitutive activation of NF-κB has been correlated well with disease progression in prostate cancer [37], and therefore inhibition of NF-κB activity in prostate cancers can suppress angiogenesis and subsequent tumor invasion and metastasis by downregulating downstream targets such as VEGF and MMP9 [38]. In this context, it was determined using a mouse model that a constitutively active version of IKK2 alone is insufficient for promoting prostate tumorigenesis; however, in combination with even heterozygous loss of *PTEN*, IKK2 activation can lead to an increase in tumor size, accompanied by increased inflammation [39]. Thus, earlier studies clearly demonstrate that the inflammatory cytokines secreted from the stromal microenvironment of the prostate cells can cooperate with PTEN loss to drive epithelial prostate tumor towards an invasive disease. Interestingly, recent studies have clearly indicated a greater role for the MDM2 oncogene in the progression of prostate cancer by impacting PI3K/AKT and NF-κB pathways [40, 41].

#### **2.2. MDM2 and prostate cancer**

**Figure 1.** Different domains of PTEN and the phosphorylation sites. (Obtained from: Cell Res. 2008; 18: 807–816.)

Various lines of evidence suggest that chronic inflammation is a closely associated event in the tumorigenic mechanisms of prostate cancer [29, 30] and to the several mutations that are causing this disease. A cytokine that is most commonly associated with tumor growth, proliferation, and angiogenesis in many cancers and also the most frequently found inflam‐ matory mediator in prostate cancer is IL-6 [31]. When expressed at high levels, in addition to imposing the inflammatory functions, a strong correlation between the circulating levels of IL-6 and advancement in the stages of prostate cancer, therapeutic resistance, and as a result an overall poor prognosis has been well established until now [32]. Although one of the most important consequences of IL-6 expression is the stimulation of the JAK/STAT3 pathway [33], phosphorylation of STAT3 at Scr727 and activation of its function by the PI3K-AKT pathway cannot be ruled out completely because of the impact PTEN mutations can produce on this pathway [34]. Such activation of STAT3 can also lead to metastatic behavior of prostate cancer cells in both *in vitro* and *in vivo* conditions, through stimulation of angiogenesis and suppres‐ sion of antitumor immune responses [35]. Many inflammatory cytokines and chemokines promote tumor progression by converging on and stimulating the IKK2/NF-κB signaling axis [36]. In addition to the above-mentioned mechanisms, constitutive activation of NF-κB has been correlated well with disease progression in prostate cancer [37], and therefore inhibition of NF-κB activity in prostate cancers can suppress angiogenesis and subsequent tumor invasion and metastasis by downregulating downstream targets such as VEGF and MMP9 [38]. In this context, it was determined using a mouse model that a constitutively active version of IKK2 alone is insufficient for promoting prostate tumorigenesis; however, in combination

*2.1.1. PTEN loss combined with alterations in inflammatory pathway regulators*

182 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

Alterations in the *TP53* gene is one of the most commonly detected gene defects in a wide range of cancers; however, alterations of this gene is believed to be of low frequency in prostate cancer [42], and their clinical significance is also not fully investigated. On the contrary, the *MDM2* gene seems to be amplified in a significant fraction of prostate cancers, and overexpression of MDM2 protein without amplification is also observed as an alternate mechanism of p53 inactivation in these cancers [43, 44]. It has been widely reported that *p21/WAF1* gene expres‐ sion could very well serve as an indicator of p53 activity because *p21/WAF1* is under the transcriptional control of p53 and therefore can be severely impacted when MDM2 is overex‐ pressed. However, the *MDM2* gene itself is under the transcriptional control of p53, which creates an auto-regulatory feedback loop in many cancer types (**Figure 2**) [45]. An interesting fact that was revealed through mutation analysis of various cancer samples is that, in prostate cancers, alterations in the *TP53* gene seem to be uncommon, and therefore the clinical signif‐ icance of *TP53* gene mutation has not been fully investigated for prostate cancers. Another important limitation of studies related to *TP53* gene defect in prostate cancer is that, in many cases their focus was confined to the analysis of p53 gene alterations without exploring other

**Figure 2.** The pro-angiogenesis, apoptosis, cytokine release, and cell cycle pathways that are impacted by MDM2 ex‐ pression.

possible mechanisms that might regulate its functions. For example, though the *MDM2* gene is amplified in a variety of tumors, MDM2 overexpression without amplification seems to be a common mechanism of p53 inactivation in certain cancers. As it was mentioned earlier in this section, it has been well established that *p21/WAF I* gene expression can serve as a good indicator of p53 activity, because *p21/WAFI* expression is under the transcriptional control of p53, and consequently indicate any related abnormality. However, several studies have analyzed the patterns of p53 expression and identified a correlation with MDM2 and p21 in prostate cancer patients. Results have confirmed a close association between levels of these markers and clinico-pathological parameters of poor outcome, including time to relapse and proliferative index. In addition, overexpression of MDM2 has been found to be associated with lack of response to chemoradiotherapy in oesophageal cancer and has been shown to exhibit androgen independence in prostate cancer cell lines [46, 47]. Thus, MDM2 overexpression was significantly associated with advanced stage prostate cancer (PCa) [48], a finding confirmed by several investigators [49, 50] validating the importance of MDM2 expression in prostate cancers. Recent studies have also shown that MDM2 expression enhances the angiogenic potential and proliferative capacity of PCa cells [51] and negatively impacts the effects of radiation and chemotherapy [52]. Thus, it is predictable that expression of MDM2 may play an important role, at least in part, in stimulating the aggressive nature of PCa in African-American (AA) patients. Recently, a single nucleotide polymorphism (SNP) referred as SNP309 was found at position 309 in the P2 promoter region of *MDM2* gene. This T > G polymorphism (rs22789744) which is located in the intronic portion of the promoter was shown to increase the binding affinity of the transcriptional activator Sp1, and increase the expression of MDM2 protein levels [53]. During the transcriptional activation of MDM2 gene, both the androgen receptor (AR) and estrogen receptors (ER) have been shown to form complexes with Sp1 and act as co-regulators and cause increase in protein expression [54, 55]. In addition, studies in ER-positive tumors such as breast and ovarian cancer have shown strong correlation between younger age of disease onset and the presence of *MDM2* SNP309 G allele [56, 57]. Interestingly, in the ovarian cancer patients, the age of onset in women with high level expression of ER and the presence of SNP309 G allele was 8 years earlier than those without the SNP309 G allele. Similarly, in a cohort of breast cancer patients with the G/G SNP309 genotype the age of onset was 7 years earlier than the patients with the T/T genotype. Fur‐ thermore, *MDM2* SNP309 G allele displayed early-onset of soft-tissue sarcoma, diffuse large B-cell lymphoma, colorectal cancer, and non-small cell lung cancer in premenopausal women with active estrogen signaling than the cohorts without the SNP309 polymorphism [58–61]. Hence, it is believed that SNP309 G allele found at the *MDM2* promoter region in AA patients may be responsible for the aggressive phenotype and early onset of their prostate cancers (48). Indeed, this appears to be one of the first studies of *MDM2* SNP309 showing the implication of this particular polymorphism to the racial differences in the clinic-pathologic presentation of the prostate cancer. Additionally, the above mentioned study is the first report that is closely correlating SNP309 genotype to MDM2 protein expression in a group of prostate cancer patients and showing its close correlation with tumor progression. Thus, several aspects of MDM2 expression and the gene polymorphisms seem to specifically impact the nature and progression of prostate cancers.

#### *2.2.1. MDM2 and cytokine expression*

possible mechanisms that might regulate its functions. For example, though the *MDM2* gene is amplified in a variety of tumors, MDM2 overexpression without amplification seems to be a common mechanism of p53 inactivation in certain cancers. As it was mentioned earlier in this section, it has been well established that *p21/WAF I* gene expression can serve as a good indicator of p53 activity, because *p21/WAFI* expression is under the transcriptional control of p53, and consequently indicate any related abnormality. However, several studies have analyzed the patterns of p53 expression and identified a correlation with MDM2 and p21 in prostate cancer patients. Results have confirmed a close association between levels of these markers and clinico-pathological parameters of poor outcome, including time to relapse and proliferative index. In addition, overexpression of MDM2 has been found to be associated with lack of response to chemoradiotherapy in oesophageal cancer and has been shown to exhibit androgen independence in prostate cancer cell lines [46, 47]. Thus, MDM2 overexpression was significantly associated with advanced stage prostate cancer (PCa) [48], a finding confirmed by several investigators [49, 50] validating the importance of MDM2 expression in prostate cancers. Recent studies have also shown that MDM2 expression enhances the angiogenic potential and proliferative capacity of PCa cells [51] and negatively impacts the effects of radiation and chemotherapy [52]. Thus, it is predictable that expression of MDM2 may play an important role, at least in part, in stimulating the aggressive nature of PCa in African-American (AA) patients. Recently, a single nucleotide polymorphism (SNP) referred as SNP309 was found at position 309 in the P2 promoter region of *MDM2* gene. This T > G polymorphism (rs22789744) which is located in the intronic portion of the promoter was shown to increase the binding affinity of the transcriptional activator Sp1, and increase the expression of MDM2 protein levels [53]. During the transcriptional activation of MDM2 gene, both the androgen receptor (AR) and estrogen receptors (ER) have been shown to form complexes with Sp1 and act as co-regulators and cause increase in protein expression [54, 55]. In addition, studies in ER-positive tumors such as breast and ovarian cancer have shown strong correlation between younger age of disease onset and the presence of *MDM2* SNP309 G allele [56, 57]. Interestingly, in the ovarian cancer patients, the age of onset in women with high level expression of ER and the presence of SNP309 G allele was 8 years earlier than those without the SNP309 G allele. Similarly, in a cohort of breast cancer patients with the G/G SNP309 genotype the age of onset was 7 years earlier than the patients with the T/T genotype. Fur‐ thermore, *MDM2* SNP309 G allele displayed early-onset of soft-tissue sarcoma, diffuse large B-cell lymphoma, colorectal cancer, and non-small cell lung cancer in premenopausal women with active estrogen signaling than the cohorts without the SNP309 polymorphism [58–61]. Hence, it is believed that SNP309 G allele found at the *MDM2* promoter region in AA patients may be responsible for the aggressive phenotype and early onset of their prostate cancers (48). Indeed, this appears to be one of the first studies of *MDM2* SNP309 showing the implication of this particular polymorphism to the racial differences in the clinic-pathologic presentation of the prostate cancer. Additionally, the above mentioned study is the first report that is closely correlating SNP309 genotype to MDM2 protein expression in a group of prostate cancer patients and showing its close correlation with tumor progression. Thus, several aspects of MDM2 expression and the gene polymorphisms seem to specifically impact the nature and

184 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

progression of prostate cancers.

In addition to being the trigger for developing cancers, MDM2 expression seems to be responsible for several events that promote cancer aggressiveness [48]. Increased expression of VEGF in cancer cells, which are positive for MDM2, is a well-established phenomenon that occurs through elevation of HIF-1alpha even during the absence of hypoxia in the tumor microenvironment [51]. In addition, many reports in the litera‐ ture confirm that MDM2 overexpression could lead to activation of STAT3 and NF-κB pathways and cause elevation of cytokines that in-turn can stimulate cancer progres‐ sion. One of the unique biological functions of MDM2 is its ability to induce sterile tissue inflammation, which is a major element of non-infectious tissue injury that occurs following exposure to toxins or reperfusion following ischemia. For example, an acute post-ischemic kidney injury that started as a sterile inflammatory response was re‐ versed using the MDM2 blockade with nutlin-3 [62]. This effect was found to be totally independent of p53 that was observed in a p53-deficient mice. Also, MDM2 blockade effectively suppressed the post-ischemic induction of pro-inflammatory cytokines and chemokines as well as the infiltration of leukocytes to the site of injury. Following these observations, the mechanism underlying MDM2-mediated inflammation was identified under *in vitro* conditions showing that MDM2 could act as a co-factor for NF-NF-κB binding to its gene promoter binding sites [62]. This was actually confirmed by the electromobility shift assay in p53-deficient mouse embryonic fibroblasts using lipopoly‐ saccharide (LPS) stimulation [62]. This observation is similar to several other reports which confirm that MDM2 blockade with nutlin-3 could effectively suppresses LPSinduced lung inflammation through interference of NF-NF-κB DNA binding in nutro‐ phils; however this effect of nutlin-3 was dependent on the presence of intact p53 [62]. Similar to the activation of NF-κB pathway, MDM2 might release other cytokines like Interleukins (IL's) and support growth and progression of cancer.

#### **2.3. TMPRSS2 and ERG fusions in prostate cancer**

TMPRSS2 is an androgen regulated prostate-specific protein that is encoded in humans by the TMPRSS2 gene [63]. It is a 492 amino acid type II transmembrane serine protease (70 KDa) that is expressed at the cell surface in order to regulate cell-cell and cell-matrix in‐ teractions [64]. The serine protease gene family, play crucial roles in different physiological and pathological processes such as digestion, blood coagulation, remodeling of tissues, inva‐ sion of tumor cells, inflammatory responses, and apoptosis. The TMPRSS2 protein contains a Serine protease domain (aa 255-492) with three catalytic residues of histidine, aspartate, and serine, respectively, a Scavenger receptor cysteine-rich domain (SRDR, aa 149-242), an LDL receptor class A (LDLRA, aa 113-148) domain and a predicted transmembrane domain (aa 84-106) [65].

ERG is a member of the erythroblastosis virus E26 (ETS) oncogene family. There are over 20 ETS transcription factor family members, but ERG is the ETS transcription factor primarily involved in prostate cancer gene fusions [66]. The ERG protein interacts with ETS members as well as other transcription factors through its protein-protein interacting domain to regulate transcriptional activity of several downstream target genes that are crucial for DNA damage, cell invasion and proliferation, epithelial to mesenchymal transformation (EMT) as well as cellular differentiation and epigenetic control [66–68].

TMPRSS2 is expressed in normal and neoplastic prostate tissue and is strongly induced by androgens in androgen-sensitive prostate cell lines [65]. A major milestone in PCa research was the identification of recurrent fusions between TMPRSS2 and ERG [63]. TMPRSS2-ERG is fused in PCa through deletion of genomic DNA via a homogeneous deletion site between ERG and TMPRSS2 on chromosome 21q22.2 or through translocation or both [69–71]. These rearrangements (**Figure 1**) result in the formation of a TMPRSS2-ERG fusion transcript and the overexpression of ERG [63]. The TMPRSS2 and ERG genes are both located on the same chromosome (21q) and the distance between the TMPRSS2 and ERG oncogene is relatively short at 3 mega bases (MB) (**Figure 3**). This short distance has been suggested to account for the higher frequency of TMPRSS2: ERG fusions in prostate cancer [69, 73].

TMPRSS2-ERG fusion occurs early in prostate carcinogenesis at the transition between benign and prostatic intraepithelial neoplasia (PIN). Approximately 50% of PCas from prostatespecific antigen (PSA) screened surgical cohorts are TMPRSS2-ERG fusion-positive, and >90% of PCas over-expressing ERG harbor TMPRSS2-ERG fusions [74]. Over eight isoforms of the TMPRSS2-ERG fusion transcript have been identified with varying levels of expression in different PCa samples [75]. The most frequently found TMPRSS2-ERG fusion in PCa is the deletion between the 5 UTR end of TMPRSS2 exon 1 and 5 end of ERG exon 4 [76].

**Figure 3.** Mechanism of TMPRSS2-ERG fusion (chromosome 21). (1) Large deletion of intervening genetic region be‐ tween ERG and TMPRSS2 genes (most common). (2) Translocation of TMPRSS2 and ERG genes. Reproduced with per‐ mission from the copyright holder: Hossain [72].

#### *2.3.1. Consequences of TMPRSS2-ERG fusion in prostate cancer*

TMPRSS2 is an androgen-responsive gene and AR regulated expression of the TMPRSS2- ERG fusion gene plays an early role in prostate cancer development and progression as its presence is required for prostate cancer initiation in ETS positive tumors [74]. The fusion results in the modulation of transcriptional patterns and cellular pathways causing the development of prostatic intraepithelial neoplasia (PIN) [77]. In particular, gene expression profiling has linked a deregulation of WNT and TGF-β/BMP signaling in

fusion-positive prostate tumors [78]. It has also been shown in transgenic mice that overexpression of ERG as a result of TMPRSS2: ERG fusion leads to the formation of murine PIN (mPIN) by 5–6 months of age [74, 79]. Several studies have also con‐ firmed that the overexpression of ERG leads to prostate cell migration and invasion that correlates with increased tumor metastasis and negative patient outcome [79, 80]. The most prominent role of ERG that has been consistently shown is its ability to increase cell migration and invasion via abrogating prostate epithelial differentiation and inducing epithelial to mesenchymal transition and motility-associated genes such as MMPs [81].

PCa specimens containing the TMPRSS2-ERG rearrangement are also significantly enriched for the loss of tumor suppressor gene phosphatase and tensin homologue PTEN [77], and it is already well established that aberrant PTEN activity is associated with poor prognosis in PCa [82]. Further studies have confirmed that TMPRSS2-ERG rearrangement cooperates with PTEN loss to promote prostate cancer progression from high-grade prostatic intraepithelial neoplasia (PIN) to invasive adenocarcinoma [77, 83].

#### *2.3.2. TMPRSS2-ERG fusions and ethnicity*

transcriptional activity of several downstream target genes that are crucial for DNA damage, cell invasion and proliferation, epithelial to mesenchymal transformation (EMT) as well as

TMPRSS2 is expressed in normal and neoplastic prostate tissue and is strongly induced by androgens in androgen-sensitive prostate cell lines [65]. A major milestone in PCa research was the identification of recurrent fusions between TMPRSS2 and ERG [63]. TMPRSS2-ERG is fused in PCa through deletion of genomic DNA via a homogeneous deletion site between ERG and TMPRSS2 on chromosome 21q22.2 or through translocation or both [69–71]. These rearrangements (**Figure 1**) result in the formation of a TMPRSS2-ERG fusion transcript and the overexpression of ERG [63]. The TMPRSS2 and ERG genes are both located on the same chromosome (21q) and the distance between the TMPRSS2 and ERG oncogene is relatively short at 3 mega bases (MB) (**Figure 3**). This short distance has been suggested to account for

TMPRSS2-ERG fusion occurs early in prostate carcinogenesis at the transition between benign and prostatic intraepithelial neoplasia (PIN). Approximately 50% of PCas from prostatespecific antigen (PSA) screened surgical cohorts are TMPRSS2-ERG fusion-positive, and >90% of PCas over-expressing ERG harbor TMPRSS2-ERG fusions [74]. Over eight isoforms of the TMPRSS2-ERG fusion transcript have been identified with varying levels of expression in different PCa samples [75]. The most frequently found TMPRSS2-ERG fusion in PCa is the

**Figure 3.** Mechanism of TMPRSS2-ERG fusion (chromosome 21). (1) Large deletion of intervening genetic region be‐ tween ERG and TMPRSS2 genes (most common). (2) Translocation of TMPRSS2 and ERG genes. Reproduced with per‐

TMPRSS2 is an androgen-responsive gene and AR regulated expression of the TMPRSS2- ERG fusion gene plays an early role in prostate cancer development and progression as its presence is required for prostate cancer initiation in ETS positive tumors [74]. The fusion results in the modulation of transcriptional patterns and cellular pathways causing the development of prostatic intraepithelial neoplasia (PIN) [77]. In particular, gene expression profiling has linked a deregulation of WNT and TGF-β/BMP signaling in

deletion between the 5 UTR end of TMPRSS2 exon 1 and 5 end of ERG exon 4 [76].

the higher frequency of TMPRSS2: ERG fusions in prostate cancer [69, 73].

cellular differentiation and epigenetic control [66–68].

186 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

mission from the copyright holder: Hossain [72].

*2.3.1. Consequences of TMPRSS2-ERG fusion in prostate cancer*

There are several studies evaluating the relationship between ethnicity and TMPRSS2-ERG expression in PCa. TMPRSS2-ERG gene fusion correlated with ethnicity in a multivariate analysis involving Caucasians [71], African-Americans, and Japanese men with PCa [71]. TMPRSS2-ERG gene fusion was present in 50% (21/42) of Caucasians, 31.3% (20/64) of African-Americans, and 15.9% (7/44) of Japanese patients. A subsequent study found that TMPRSS2- ERG gene fusions were identified in 48/112 tumors (42.9%) from a group of Caucasian men, while 28/105 tumors (26.7%; p = 0.015) from African-American men were positive for the gene fusion [84]. Interestingly, Mosquera and colleagues recognized that the TMPRSS2-ERG fusion through deletion, which has been associated with worse prognosis, is more common in PCa of African-American patients [73].

#### *2.3.3. Prognostic value of the TMPRSS2-ERG fusion gene*

The prognostic potential of TMPRSS2-ERG gene fusion is promising as it can be detected in urine, blood, and tissue using quantitative polymerase chain reaction [85, 86], Fluorescence in situ hybridization (FISH) [87], DNA sequencing, and Genechip [88]. This has significant applications toward understanding its role in PCa pathogenesis and developing novel diagnostics and targeted therapeutics. TMPRSS2 and TMPRSS2-ERG expression is decreased in response to ADT in primary PCa [89]. Interestingly, the ERG levels in TMPRSS2-ERG fusionpositive castration resistant prostate cancer CRPC are comparable with the levels in fusion gene-positive primary PC, and this confirms that TMPRSS2-ERG expression is reactivated by AR in CRPC [70]. These findings prove that restored AR receptor signaling contributes to the progression to CRPC in part through the TMPRSS2-ERG axis and highlights a therapeutic platform that can be explored in the management of CRPC. More recently, the TMPRSS2-ERG fusion has been linked to taxane resistance in preclinical models of castration-resistant prostate cancer, and TMPRSS2-ERG expression detection in the peripheral blood of metastatic castra‐ tion-resistant prostate cancer patients correlates with docetaxel resistance [90]. Therefore, its presence predicts resistance to docetaxel, and it may be useful to select treatment and to avoid possible toxicities in refractory patients.

#### **Acknowledgements**

The author Appu Rathinavelu, Ph.D., would like to thank the Fulbright Scholar Program of the United States Department of State Bureau of Educational and Cultural Affairs and the USIEF (United States International Educational Foundation) for the Nehru—Fulbright Scholar Award during the completion of this book chapter. The author would also like to Thank Nova Southeastern University and the Royal Dames of Ft. Lauderdale Inc, Florida, USA for their support.

#### **Author details**

Appu Rathinavelu1\* and Arkene Levy2

\*Address all correspondence to: appu@nova.edu

1 Rumbaugh Goodwin Institute for Cancer Research, College of Pharmacy, Nova Southeastern University, Fort Lauderdale, Florida, USA

2 Pharmacology Section, Department of Basic Medical Sciences, University of the West In‐ dies, Mona Campus, Jamaica

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1 Rumbaugh Goodwin Institute for Cancer Research, College of Pharmacy, Nova Southeastern

2 Pharmacology Section, Department of Basic Medical Sciences, University of the West In‐

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### **Maspin Expression and its Metastasis Suppressing Function in Prostate Cancer**

Eswar Shankar, Mario Candamo, Gregory T. MacLennan and Sanjay Gupta

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64336

#### **Abstract**

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Cancer Genet. 2013; 206: 1–11. doi:10.016/j.cancergen.2012.12.004

Int. 2013; 111: 836–842. doi:10.1111/bju.12119

196 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

5033–5041. doi:10.1158/0008-5472.can-06-3332

(16): 00212–00218. doi:10.1016/j.eururo.2016.02.034

*Ma*mmary *S*erine *P*rotease *In*hibitor (Maspin) is a unique member of the serpin family with tumor suppressive properties. Maspin is a secreted protein encoded by a class II tumor suppressor gene, expressed in normal prostate luminal and basal cells but reduced or absent in prostate cancer. Currently, there is a consensus that maspin expression in prostate cancer is an indicator of a better prognosis and is a predictive marker for therapeutic response in prostate cancer. Experimental evidence consistent‐ ly indicates that maspin suppresses tumor growth, invasion, and metastasis and promotes apoptosis in cancer cells. In this chapter, we discuss regulation of maspin expression, binding partners of maspin, and pathways through which maspin exerts its tumor suppressive properties. In addition, we summarize the progress that investiga‐ tors have made in clarifying the role of maspin in prostate cancer biology and in assessing its role as a diagnostic marker and therapeutic agent.

**Keywords:** tumor suppressor, prognostic marker, SERPINB5, prostate cancer, maspin

#### **1. Introduction**

*Ma*mmary *S*erine *P*rotease *In*hibitor or Maspin (SERPINB5 or PI5 *Homo sapiens*) is a 42 kDa, nonclassical, non-inhibitory member of the ovalbumin clade of serine protease inhibitors (ser‐ pins), encoded by the SERPIN5 gene [1]. Chromosome 18 encodes maspin along with gene cluster of other serpins in humans comprising squamous cell carcinoma antigens (SCCAs) 1 and 2 and plasminogen activator inhibitor type 2 (PAI-2) [1, 2]. Maspin has been characterized as a class II tumor suppressor gene, first recognized in 1994, in normal mammary tissue and breast

© 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.

cancer cell lines through subtractive hybridization, comparing genes expressed in different stages of a biological or pathological process [2]. Maspin has been shown to be downregulat‐ ed in many metastatic tumor cell lines, without evidence of an underlying mutation [3]. Maspin contains a reactive center loop (RCL), which is used to trap the target protease and inhibit its activity, a common characteristic ofinhibitory serpins [1, 2].Recent studies suggestthat serpins are involvedincell adhesionandplaya role inextracellularremodeling [2, 3]. However,maspin has been found to be more closely related to the non-inhibitory clade B serpins. As maspin's RCL is shorter than those of inhibitory serpins and unlike multiple other serpins, maspin does not undergo a stressed-relaxed conformational change to inhibit protease activity. Despite the reported activity as a serine protease inhibitor, several studies argue that the tumor suppres‐ sor activity of maspin is due to its ability to inhibit proteolysis. Some studies demonstrate the efficacy of maspin in the inhibition of activity of tissue-type plasminogen activator [4]. Further‐ more,maspinhasbeenshowntomediatetheinhibitionofurokinase-typeplasminogenactivator (uPA)onthe surfaceofprostate cancer cells [5].Althoughmaspinmightlacktheabilitytoinhibit serine proteases, its biological function can be attributed to RCL, which can be derived from its crystal structure [6]. Recent studies of maspin have provided evidence for its ability to regu‐ late cell adhesion, motility, apoptosis, and angiogenesis, which has been of utmost interest in medical field attempting to use maspin as a method of therapeutic intervention for prostate cancer and other forms of malignancies [3–7].

#### **2. Expression of maspin in normal prostate and cancer**

Maspin has been localized to the cell surface, nucleus, cytoplasm, and extracellular matrix of epithelial cells of different tissues [8]. In normal breast and prostate epithelial cells, maspin is highly expressed and found to be localized mostly in the cytoplasm but has also been detected in the nucleus, secretory vesicles, and occasionally at the cell surface [6–8]. Maspin expression is almost completely suppressed in the human prostate cancer LNCaP, DU145, and PC-3 cell lines [9]. At the tissue level, maspin's function seems to be directly correlated with its locali‐ zation. In benign prostate epithelium, maspin expression is uniformly noted in basal cells at high levels. In contrast, maspin expression is predominantly absent in benign secretory cells and elevated in secretory cells at the transition site between benign prostatic hyperplasia and high-grade PIN lesions [9, 10]. Pierson et al. noted higher expression of maspin in HGPIN lesions, particularly within secretory cells [11]. Elevated maspin immunoreactivity in the secretory cells appeared at the transition area between benign prostate tissue and HGPIN, whereas less intense maspin staining was observed in neoplastic cells adjacent to HGPIN. No change in maspin expression was observed in the basal cells near HGPIN, compared to normal basal cells [12]. Moreover, an inverse relationship between maspin expression and tumor progression was noted in clinical specimens with gradual disappearance in primary prostate cancer. Due to the loss of basal layer during prostate cancer progression, strong immunohis‐ tochemical staining of maspin was lost in the basal cells [12, 13]. A progressive decrease in maspin expression was noted with increase in Gleason grade, with complete loss of maspin in high grade and metastatic tumors (**Figure 1**). Maspin expression was significantly higher in tumor specimens of patients treated with neoadjuvant androgen ablation therapy before radical prostatectomy [14]. Prostate cancer patients whose tumors expressed maspin had a significantly longer recurrence-free survival [6, 12].

**Figure 1.** Expression of maspin in various representative human prostate specimens. Paraffin-embedded (4.0 μm) sec‐ tions from benign *A*, and prostate cancer of various Gleason grades *B*–*D*, were used for maspin expression by immu‐ nohistochemistry. A strong nuclear and cytoplasmic staining was observed in benign tissue where the basal cell cytoplasm and nuclei were strongly and uniformly immunoreactive for maspin. Less intense cytoplasmic staining was noted in secretory cells. In low-grade cancer (Gleason grade 3), loss of nuclear maspin staining was observed and tu‐ mors progressively exhibited reduced maspin expression where the majority of high-grade tumors exhibit little or no cytoplasmic immunoreactivity. Magnified at ×20 and inset ×40.

#### **3. Regulation of maspin expression**

cancer cell lines through subtractive hybridization, comparing genes expressed in different stages of a biological or pathological process [2]. Maspin has been shown to be downregulat‐ ed in many metastatic tumor cell lines, without evidence of an underlying mutation [3]. Maspin contains a reactive center loop (RCL), which is used to trap the target protease and inhibit its activity, a common characteristic ofinhibitory serpins [1, 2].Recent studies suggestthat serpins are involvedincell adhesionandplay a role inextracellularremodeling [2, 3]. However,maspin has been found to be more closely related to the non-inhibitory clade B serpins. As maspin's RCL is shorter than those of inhibitory serpins and unlike multiple other serpins, maspin does not undergo a stressed-relaxed conformational change to inhibit protease activity. Despite the reported activity as a serine protease inhibitor, several studies argue that the tumor suppres‐ sor activity of maspin is due to its ability to inhibit proteolysis. Some studies demonstrate the efficacy of maspin in the inhibition of activity of tissue-type plasminogen activator [4]. Further‐ more,maspinhasbeenshowntomediatetheinhibitionofurokinase-typeplasminogenactivator (uPA)onthe surfaceofprostate cancer cells [5].Althoughmaspinmightlacktheabilitytoinhibit serine proteases, its biological function can be attributed to RCL, which can be derived from its crystal structure [6]. Recent studies of maspin have provided evidence for its ability to regu‐ late cell adhesion, motility, apoptosis, and angiogenesis, which has been of utmost interest in medical field attempting to use maspin as a method of therapeutic intervention for prostate

cancer and other forms of malignancies [3–7].

198 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

**2. Expression of maspin in normal prostate and cancer**

Maspin has been localized to the cell surface, nucleus, cytoplasm, and extracellular matrix of epithelial cells of different tissues [8]. In normal breast and prostate epithelial cells, maspin is highly expressed and found to be localized mostly in the cytoplasm but has also been detected in the nucleus, secretory vesicles, and occasionally at the cell surface [6–8]. Maspin expression is almost completely suppressed in the human prostate cancer LNCaP, DU145, and PC-3 cell lines [9]. At the tissue level, maspin's function seems to be directly correlated with its locali‐ zation. In benign prostate epithelium, maspin expression is uniformly noted in basal cells at high levels. In contrast, maspin expression is predominantly absent in benign secretory cells and elevated in secretory cells at the transition site between benign prostatic hyperplasia and high-grade PIN lesions [9, 10]. Pierson et al. noted higher expression of maspin in HGPIN lesions, particularly within secretory cells [11]. Elevated maspin immunoreactivity in the secretory cells appeared at the transition area between benign prostate tissue and HGPIN, whereas less intense maspin staining was observed in neoplastic cells adjacent to HGPIN. No change in maspin expression was observed in the basal cells near HGPIN, compared to normal basal cells [12]. Moreover, an inverse relationship between maspin expression and tumor progression was noted in clinical specimens with gradual disappearance in primary prostate cancer. Due to the loss of basal layer during prostate cancer progression, strong immunohis‐ tochemical staining of maspin was lost in the basal cells [12, 13]. A progressive decrease in maspin expression was noted with increase in Gleason grade, with complete loss of maspin in high grade and metastatic tumors (**Figure 1**). Maspin expression was significantly higher in

Maspin expression is regulated by a promoter with two response elements—Ets and a promiscuous hormone response element that binds glucocorticoid receptor and progesterone receptor [15]. Regulation of maspin by androgen receptor (AR) seems complex because the hormone response element of maspin's promoter appears to function as a negative regulator. Zou et al. have demonstrated that androgen-responsive LNCaP cells cultured in androgendepleted medium exhibit induction of maspin promoter activity in a promoter luciferase reporter assay [16]. Furthermore, castration of nude mice induces maspin expression in LNCaP xenograft tumors. These data indicate that maspin may be transiently upregulated in early stages of prostate tumor development and remains sensitive to AR repression.

Another major regulatory mechanism for maspin is the involvement of p53 signaling pathway [16]. A consensus p53 site was identified in the maspin promoter, which induced its expression upon binding to p53. Maspin expression was increased in adenoviral-mediated expression of wild-type p53 in maspin-null prostate cancer cell lines. During cellular stress, p53-responsive pathways were induced in cells possessing wild-type p53, whereas mutant p53 failed to induce its expression. Purified p53 protein bound to regions within the promoter from −297 bp and p53 antibody supershifted maspin bands. In support of these data, a later study (using tissue microarray analysis) reported an inverse relationship between mutated p53 and maspin in human tumors [17]. The implication of maspin involvement in the p53 pathway demonstrated a potential hierarchy of tumor suppressor pathways. Studies suggest that maspin may act as an effector molecule downstream of the p53 stress-induced pathway. Interestingly, other proteins and signaling pathways related to maspin regulation were reported to be dependent on p53. Transforming growth factor β (TGFβ) was also found to increase maspin expression and required wild-type p53 activity [18]. This work demonstrated two p53 binding sites in the maspin promoter that were either in close proximity or overlapped with a Smad binding element, leading to recruitment of Smad2/3 and p53 to the maspin promoter following TGFβ signaling. In addition, Smad2/3 increased the binding of p53 to the maspin promoter demon‐ strating transcriptional co-regulation. Reports suggest that there are other factors that regulate maspin expression, independent of p53 function. Expression of the antioxidant manganese superoxide dismutase in prostate cells led to an increase in stability of maspin mRNA, and this effect persisted in the presence of wild-type or mutant p53 [19]. Furthermore, the activating transcription factor (ATF-2) was shown to induce maspin expression independently of p53 by binding to a CRE-like sequence downstream of the transcription start site [20]. In addition, other members of the p53 family, specifically the p63 isoform TAp63γ, induce expression of maspin by binding to the same consensus p53 promoter element and can substitute for activation in the absence of p53 [21]. Other transcription factors have also been noted to bind to maspin promoter and regulate its expression as shown in **Figure 2**.

**Figure 2.** Regulation of maspin by various transcription factors. Maspin promoter has several transcription factor bind‐ ing sites that regulate its expression and function. Most of maspin regulation by the transcription factors is unclear, but the regulation by p53 is widely studied and involves the TGF-β signaling.

Recent evidences suggest that maspin can be epigenetically regulated and that its expression in relationship with tissue specificity directly correlates with DNA methylation [2, 22]. Treatment of maspin-null cancer cell lines with 5-aza-2′-deoxycytidine resulted in the induc‐ tion of maspin expression [22]. Furthermore, promoter methylation was found to serve as a mechanism for tissue and cell-specific expression of maspin [2, 22]. Epigenetic changes regulating maspin expression have been demonstrated to occur at the 5′ regulatory region of the maspin gene that involves methylation of cytosine, histone deacetylation, and the acces‐ sibility of chromatin. Two defining epigenetic events are DNA methylation and histone lysine methylation or deacetylation, which are categorized as chromatin modification events. In support of this, we and others have shown that treatment of prostate cancer cell lines with histone deacetylase (HDAC) inhibitors, namely sodium butyrate and trichostatin A, led to induction of maspin at mRNA and protein level [23]. Re-expression of maspin using deme‐ thylating agents and HDAC inhibitors in combination has been confirmed in additional studies. Furthermore, studies using prostate cancer cells and clinical specimens, we have further demonstrated that maspin expression was only induced by inhibition of class I histone deacetylases regardless of promoter methylation status, highlighting that chromatin conden‐ sation alone may determine its transcriptional activity [23].

Maspin is a non-glycosylated protein; however, phosphorylated forms have been identified and detected in various tumors [24]. Early studies demonstrate abundance of tyrosine phosphorylation in both endogenously expressed maspin in normal epithelial cells and after induction of maspin in transfected tumor cells [18]. Although the kinases responsible for this phosphorylation have yet to be identified, incubation of rMaspin with the TKD38 EGFR kinase domain led to tyrosine phosphorylation in a cell-free system. In addition, serine and threonine phosphorylation sites have been recently identified on maspin secreted from cornea cells using a mass spectrometry approach [25]. Whether this phenomenon is cell type specific requires further studies. In addition to phosphorylation studies, maspin also contains eight cysteine residues; however, intramolecular disulfide bonding had not previously been observed nor predicted from maspin crystallography. Under oxidative stress, maspin adopted an oxidized, disulfide-bonded structure, which was analyzed under non-reducing conditions in epithelial cells [26]. In this state, maspin was no longer able to bind glutathione *S*-transferase (GST), a binding partner for maspin, which suggested a potential difference in protein functionality.

#### **4. Protein binding partners of maspin**

microarray analysis) reported an inverse relationship between mutated p53 and maspin in human tumors [17]. The implication of maspin involvement in the p53 pathway demonstrated a potential hierarchy of tumor suppressor pathways. Studies suggest that maspin may act as an effector molecule downstream of the p53 stress-induced pathway. Interestingly, other proteins and signaling pathways related to maspin regulation were reported to be dependent on p53. Transforming growth factor β (TGFβ) was also found to increase maspin expression and required wild-type p53 activity [18]. This work demonstrated two p53 binding sites in the maspin promoter that were either in close proximity or overlapped with a Smad binding element, leading to recruitment of Smad2/3 and p53 to the maspin promoter following TGFβ signaling. In addition, Smad2/3 increased the binding of p53 to the maspin promoter demon‐ strating transcriptional co-regulation. Reports suggest that there are other factors that regulate maspin expression, independent of p53 function. Expression of the antioxidant manganese superoxide dismutase in prostate cells led to an increase in stability of maspin mRNA, and this effect persisted in the presence of wild-type or mutant p53 [19]. Furthermore, the activating transcription factor (ATF-2) was shown to induce maspin expression independently of p53 by binding to a CRE-like sequence downstream of the transcription start site [20]. In addition, other members of the p53 family, specifically the p63 isoform TAp63γ, induce expression of maspin by binding to the same consensus p53 promoter element and can substitute for activation in the absence of p53 [21]. Other transcription factors have also been noted to bind

to maspin promoter and regulate its expression as shown in **Figure 2**.

the regulation by p53 is widely studied and involves the TGF-β signaling.

200 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

**Figure 2.** Regulation of maspin by various transcription factors. Maspin promoter has several transcription factor bind‐ ing sites that regulate its expression and function. Most of maspin regulation by the transcription factors is unclear, but

Recent evidences suggest that maspin can be epigenetically regulated and that its expression in relationship with tissue specificity directly correlates with DNA methylation [2, 22]. Treatment of maspin-null cancer cell lines with 5-aza-2′-deoxycytidine resulted in the induc‐ tion of maspin expression [22]. Furthermore, promoter methylation was found to serve as a mechanism for tissue and cell-specific expression of maspin [2, 22]. Epigenetic changes regulating maspin expression have been demonstrated to occur at the 5′ regulatory region of the maspin gene that involves methylation of cytosine, histone deacetylation, and the acces‐ sibility of chromatin. Two defining epigenetic events are DNA methylation and histone lysine methylation or deacetylation, which are categorized as chromatin modification events. In support of this, we and others have shown that treatment of prostate cancer cell lines with histone deacetylase (HDAC) inhibitors, namely sodium butyrate and trichostatin A, led to induction of maspin at mRNA and protein level [23]. Re-expression of maspin using deme‐ thylating agents and HDAC inhibitors in combination has been confirmed in additional studies. Furthermore, studies using prostate cancer cells and clinical specimens, we have Through the yeast two-hybrid assays, screening studies identified possible protein-protein interactions with maspin [27]. A short list of candidate intracellular partners of maspin include heat shock proteins (Hsp90 and Hsp70), glutathione *S*-transferase (GST), interferon regulatory factor 6 (IRF6), histone deacetylase 1 (HDAC1), early growth response protein 1 (Egr-1), and GC-binding factor 2 (GCF2). These studies provide new dimensions in understanding the role of maspin from that of a serine protease inhibitory serpin to that of a stress-responsive chaperone and role of maspin in tumor suppression.

Hsp90 is one of the most abundant stress-responsive chaperones that shuttle between the cytoplasm and nucleus to protect its client proteins from degradation [28]. Hsp90 binds and protects the native conformation of AR. AR attains activation and becomes functional as a consequence of agonists binding to its receptor, or when phosphorylated by Akt or due to mutation is ensued by its nuclear translocation. In the nucleus, AR employs co-activators that facilitate its binding to the promoter sequence of responsive genes to activate their expression [29]. Inhibition of Hsp90 destabilizes both wild type and mutant AR, presumably by releasing AR from the chaperone complex, to be subjected to degradation by the proteasome. Once in the nucleus, AR is acetylated, which may be mediated by its co-activators such as p300. Acetylated AR has been shown to specifically interact with and be deacetylated by HDAC1 [30]. Reports suggest that molecular interactions of maspin with HDAC1 and/or Hsp90 may underlie the positive correlation between nuclear maspin and better prognosis of cancer [31]. Inhibition of HDAC specifically upregulates genes that promote cell differentiation, cell cycle arrest, or cell death and downregulates genes that promote tumor survival and epithelialmesenchymal transition, which correlate with higher levels of maspin, whereas both Hsp90 and HDAC have been implicated as key regulators of AR activity and stability. Lockett et al. have proposed a model where maspin may negatively regulate AR-dependent survival/ proliferation of both hormone-sensitive and hormone-refractory prostate epithelial cells [32]. This model proposes that genetic engineering approaches to induce maspin expression may prove to be effective in blocking Hsp90-mediated stability, and/or HDAC1-mediated tran‐ scriptional activation, of AR in prostate cancer cells.

The maspin/GST interaction was initially characterized [27]. Endogenous maspin has been shown to correlate with increased cellular GST activity, even though purified maspin does not affect the activity of GST *in vitro*. Furthermore, maspin transfected tumor cells exhibit markedly lower basal levels of ROS, compared to the control transfected cells. In contrast, siRNA knockdown of maspin in prostate cancer PC-3 cells increased the basal ROS level. Tahmatzo‐ poulos et al. have shown that treatment of human prostate cancer DU145 cells with H2O2 (or PMA) but not with TRAIL further increases the maspin/GST interaction and significantly attenuated H2O2-induced ROS generation and VEGF expression [33]. This study further demonstrates that maspin transfected tumor cells produced less VEGF than the transfection control cells. Interestingly, a single point mutation at the RSL p1 position of maspin (MasR340A) greatly reduced the affinity for GST. Consistently, treatment with purified wildtype maspin, but not MasR340A, significantly increased cellular GST activity.

Studies by Bailey et al. reported specific interaction between maspin and interferon regulatory factor 6 (IRF6) [34, 35]. IRF6 is a member of the IRF family associated with epithelial-tomesenchymal transition through increase in N-cadherin. The interaction between maspin and IRF6 appears to be regulated by IRF6 phosphorylation and may negatively regulate the IRF6 activity. Other maspin-binding proteins identified by yeast two-hybrid approach include transcription factors, such as early growth response protein 1 (Egr-1), GC-binding factor 2 (GCF2), and RNA-binding protein KHDRBS3 and FBX032, which are involved in ubiquitin protein ligase reactions [25]. Additional studies to understand how these proteins interact with maspin and their role during prostate cancer progression are needed.

#### **5. Biological functions of maspin**

Maspin downregulation correlates with increased tumor growth and metastasis [36]. Several published studies using cell lines and animal models underscore the critical role of maspin in tumor growth and invasion. Treatment with recombinant maspin protein was found to inhibit tumor invasion and motility of prostate cancer cells (LNCaP, DU145, and PC-3) in culture by binding specifically to the cell surface. This surface action was further supported by the ability of maspin to block urokinase-type plasminogen activator on cell surface of prostate cancer DU145 cells [5]. Cher et al. used a SCID human intraosseous tumor model that ectopically overexpresses maspin in prostate cancer DU145 cells to demonstrate maspin's ability to abrogate bone matrix remodeling, repress bone tumor growth, and prevent angiogenesis [37]. In another study, Hall et al. using maspin overexpressed PC-3 tumor cells injected to athymic nude mice demonstrated a decrease in bone metastasis but failed to suppress the ability of tumor cells spread/metastasize to distant sites providing new insight in the underlying inhibitory role of tumor cell homing into the bone [38]. Interestingly, in genetically engineered mouse model of prostate cancer, TRAMP the mechanism of maspin repression occurred through association of receptor activator of NF-kB (RANK) with ligand RANKL that facilitates IkB kinase α (IKKα) nuclear translocation, which in turn suppresses maspin transcription allowing progression of prostate cancer [39].

mesenchymal transition, which correlate with higher levels of maspin, whereas both Hsp90 and HDAC have been implicated as key regulators of AR activity and stability. Lockett et al. have proposed a model where maspin may negatively regulate AR-dependent survival/ proliferation of both hormone-sensitive and hormone-refractory prostate epithelial cells [32]. This model proposes that genetic engineering approaches to induce maspin expression may prove to be effective in blocking Hsp90-mediated stability, and/or HDAC1-mediated tran‐

The maspin/GST interaction was initially characterized [27]. Endogenous maspin has been shown to correlate with increased cellular GST activity, even though purified maspin does not affect the activity of GST *in vitro*. Furthermore, maspin transfected tumor cells exhibit markedly lower basal levels of ROS, compared to the control transfected cells. In contrast, siRNA knockdown of maspin in prostate cancer PC-3 cells increased the basal ROS level. Tahmatzo‐ poulos et al. have shown that treatment of human prostate cancer DU145 cells with H2O2 (or PMA) but not with TRAIL further increases the maspin/GST interaction and significantly attenuated H2O2-induced ROS generation and VEGF expression [33]. This study further demonstrates that maspin transfected tumor cells produced less VEGF than the transfection control cells. Interestingly, a single point mutation at the RSL p1 position of maspin (MasR340A) greatly reduced the affinity for GST. Consistently, treatment with purified wild-

Studies by Bailey et al. reported specific interaction between maspin and interferon regulatory factor 6 (IRF6) [34, 35]. IRF6 is a member of the IRF family associated with epithelial-tomesenchymal transition through increase in N-cadherin. The interaction between maspin and IRF6 appears to be regulated by IRF6 phosphorylation and may negatively regulate the IRF6 activity. Other maspin-binding proteins identified by yeast two-hybrid approach include transcription factors, such as early growth response protein 1 (Egr-1), GC-binding factor 2 (GCF2), and RNA-binding protein KHDRBS3 and FBX032, which are involved in ubiquitin protein ligase reactions [25]. Additional studies to understand how these proteins interact with

Maspin downregulation correlates with increased tumor growth and metastasis [36]. Several published studies using cell lines and animal models underscore the critical role of maspin in tumor growth and invasion. Treatment with recombinant maspin protein was found to inhibit tumor invasion and motility of prostate cancer cells (LNCaP, DU145, and PC-3) in culture by binding specifically to the cell surface. This surface action was further supported by the ability of maspin to block urokinase-type plasminogen activator on cell surface of prostate cancer DU145 cells [5]. Cher et al. used a SCID human intraosseous tumor model that ectopically overexpresses maspin in prostate cancer DU145 cells to demonstrate maspin's ability to abrogate bone matrix remodeling, repress bone tumor growth, and prevent angiogenesis [37]. In another study, Hall et al. using maspin overexpressed PC-3 tumor cells injected to athymic

type maspin, but not MasR340A, significantly increased cellular GST activity.

maspin and their role during prostate cancer progression are needed.

**5. Biological functions of maspin**

scriptional activation, of AR in prostate cancer cells.

202 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

The anti-angiogenic effects of maspin were demonstrated by Zhang et al. using endothelial cells [40]. Increasing concentrations of rMaspin inhibited both the growth and migration of endothelial cells toward vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) *in vitro*. *In vivo* experiments using human prostate cancer LNCaP cells grown in immunodeficient xenograft model, tumor growth and neovascularization were reduced following rMaspin treatment [40]. A chimeric bone cancer model in which human cancer cells were injected into human bone that had been implanted into SCID/SCID mice demonstrated that DU145 human prostate cancer cells transfected with maspin exhibited less tumor neo‐ vascularization from murine endothelial cells compared to controls. This effect was associated with a decrease in tumor growth and bone destruction [36, 37]. Another study demonstrated that conditioned media (CM) from maspin-expressing human keratinocytes inhibited the ability of human endothelial cells to migrate toward angiogenic factors, namely VEGF, bFGF, and interleukin-8 (IL-8), in a dose-dependent manner [40]. Using maspin neutralizing antibody to the CM, the cells resumed their ability to migrate, providing evidence for a paracrine antiangiogenic role of maspin.

Increased cell adhesion and cell-cell contact are negative factors for cell cycle progression. Increased cell adhesion to ECM is shown to cause certain cell types to arrest in G1 phase [41]. Studies suggest that maspin has ability to increase prostate epithelial cell adhesion to different matrix proteins and inhibit prostate tumor progression through increased cell adhesion to matrices. Recent study provides evidence that maspin controls cell adhesion through its interaction with integrin β1 [42]. Interestingly, loss of one copy of maspin gene in *Maspin*+/− heterozygous knockout mice leads to the development of prostate hyperplastic lesions, accompanied with a changed pattern of matrix deposition and a loss of epithelial cell polarity [41]. It was also demonstrated that maspin may be able to inhibit surface-bound urokinase plasminogen activator in prostate tumor cells. However, the aforementioned results were not easily observed *in vitro* or *in vivo*. *In vitro*, tumor suppression was not observed with the use of maspin, while *in vivo*, only 50% of tumors that expressed maspin showed a significant reduction [36, 37].

Maspin has been observed to be involved in the regulation of apoptosis. Pro-apoptotic effects from maspin have been demonstrated in prostate cancer cells. Studies by Mckenzie et al. underline maspin as a pertinent therapeutic target to overcome hypoxia in prostate cancer. Overexpression of maspin in DU145 cells leads to apoptosis by abrogating AKT activation induced by hypoxia. According to recent studies, it has been shown that maspin expression (endogenously) is able to sensitize prostate cancer LNCaP and DU145 cells to apoptosis [9]. Watanabe et al. used adeno-associated virus (AAV, serotype 2) vector encoding maspin as a means for introducing *in vivo* gene therapy subcutaneously formed human prostate cancer LNCaP or DU145 tumors in nude mice [43]. In this study, intratumoral AAV-mediated maspin expression significantly upregulated the number of apoptotic cells when compared with AAV-LacZ treatment. Moreover, significantly fewer CD31-positive micro-vessels were observed in AAV-maspin-treated tumors when compared with the control tumors, which correlated with persistent maspin expression. Studies on mechanics have demonstrated that maspin could possibly lead to apoptosis of tumor cells through the manipulation of mitochondrial permea‐ bility and the initiation of degradation through apoptosis [44]. Maspin has also been shown to induce prostate tumor cell dedifferentiation and to increase tumor cell sensitivity to druginduced apoptosis. Suppression of maspin may partly be attributed to the involvement of AR in prostate tumorigenesis and cancer progression. Maspin's role as a tumor suppressor in prostate cancer cells irrespective of their AR status makes it an ideal candidate with therapeutic capabilities against hormone-refractory prostate cancer. Other biological functions of maspin are listed in **Figure 3**.

**Figure 3.** Biological functions of maspin. Post-translational modification of maspin and its nuclear localization sup‐ presses angiogenesis, tumor metastasis, cell invasion, and migration and inhibits bone tumor growth and proliferation, while inducing apoptosis.

#### **6. Therapeutic application of maspin**

Recent studies have investigated the anticancer effects of maspin expression and the use of maspin as a therapeutic agent against cancer. Tumors with LNCaP cells expressing adenoviral maspin (AAV-GFP) resulted in a higher percentage of apoptotic cell death and a decrease in the number of CD31 positive vessels when compared with tumors with empty vector (GFP), thereby substantiating the role of maspin in gene therapy [44]. These results confirm maspin as a therapeutic agent/target in inhibiting prostate cancer progression. Furthermore, animal studies using targeted delivery of maspin by liposome/DNA and/or adenoviral constructs to tumor and/or tumor vasculature have supported a viable approach in cancer treatment [45].

Several reports suggest that exogenously added rMaspin, or maspin-derived peptide frag‐ ments, can act at the surface of numerous migratory cell types to suppress cell motility and movement by enhancing cellular adhesion to the extracellular matrix components laminin, fibronectin, and collagen [43, 45]. McGoven et al. have shown that purified recombinant maspin produced in baculovirus-infected *Spodoptera frugiperda* Sf9 insect cells [rMaspin] binds specifically to the surface of human prostate cancer DU145 cells, inhibits the DU145 cell surface-bound uPA, and forms a stable complex. Similar results with rMaspin were observed in *in vivo* studies transplanted with human prostate cells [5]. In fact, biological assays of invasion by prostate tumor cells through Matrigel membranes and of motility have shown that rMaspin inhibits invasion and migration of these cells [46].

means for introducing *in vivo* gene therapy subcutaneously formed human prostate cancer LNCaP or DU145 tumors in nude mice [43]. In this study, intratumoral AAV-mediated maspin expression significantly upregulated the number of apoptotic cells when compared with AAV-LacZ treatment. Moreover, significantly fewer CD31-positive micro-vessels were observed in AAV-maspin-treated tumors when compared with the control tumors, which correlated with persistent maspin expression. Studies on mechanics have demonstrated that maspin could possibly lead to apoptosis of tumor cells through the manipulation of mitochondrial permea‐ bility and the initiation of degradation through apoptosis [44]. Maspin has also been shown to induce prostate tumor cell dedifferentiation and to increase tumor cell sensitivity to druginduced apoptosis. Suppression of maspin may partly be attributed to the involvement of AR in prostate tumorigenesis and cancer progression. Maspin's role as a tumor suppressor in prostate cancer cells irrespective of their AR status makes it an ideal candidate with therapeutic capabilities against hormone-refractory prostate cancer. Other biological functions of maspin

**Figure 3.** Biological functions of maspin. Post-translational modification of maspin and its nuclear localization sup‐ presses angiogenesis, tumor metastasis, cell invasion, and migration and inhibits bone tumor growth and proliferation,

Recent studies have investigated the anticancer effects of maspin expression and the use of maspin as a therapeutic agent against cancer. Tumors with LNCaP cells expressing adenoviral maspin (AAV-GFP) resulted in a higher percentage of apoptotic cell death and a decrease in the number of CD31 positive vessels when compared with tumors with empty vector (GFP), thereby substantiating the role of maspin in gene therapy [44]. These results confirm maspin as a therapeutic agent/target in inhibiting prostate cancer progression. Furthermore, animal studies using targeted delivery of maspin by liposome/DNA and/or adenoviral constructs to tumor and/or tumor vasculature have supported a viable approach in cancer treatment [45]. Several reports suggest that exogenously added rMaspin, or maspin-derived peptide frag‐ ments, can act at the surface of numerous migratory cell types to suppress cell motility and

are listed in **Figure 3**.

while inducing apoptosis.

**6. Therapeutic application of maspin**

204 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

Maspin expression in tumor cells can also be increased by the use of various phytochemicals. He et al. have demonstrated that gum mastic, a natural resin, can upregulate maspin expression in prostate cancer cells [47]. Gum mastic induced maspin mRNA and protein expression, as well as maspin promoter activity in LNCaP and DU145 cells by suppressing ARE binding activity and enhancing Sp1 binding activity, and the increased activity in the maspin promoter. Sheth et al. have demonstrated that resveratrol (trans-3,49,5-trihydroxystilbene), a polyphe‐ nolic antioxidant found in peanuts, grapes, and red wine inhibits cell proliferation, induces apoptosis of human prostate cancer cells mediated by increase in maspin levels through Akt/ miR-21 pathway [48]. Natural compound curcumin, a hydrophobic polyphenol derived from turmeric (the rhizome of the herb *Curcuma longa*), has been shown to inhibit the AR gene activity in androgen-responsive human prostate cancer LNCaP cells suppressing AR transac‐ tion, thereby affecting AR-regulated genes including maspin [49]. As AR negatively regulates maspin expression, curcumin-mediated inhibition of AR may induce the expression of maspin through a mechanism, which is presently unclear. Nevertheless, increased expression of maspin occurs only in prostate cancer cells, which harbor wild-type p53. As p53 activates maspin promoter by binding directly to its p53 consensus-binding site [16], we studied the effect of apigenin, a natural plant flavone in potentially restoring p53-mediated maspin levels. Exposure of LNCaP and 22Rv1 cells, harboring wild-type p53, with apigenin resulted in dose dependent increase in maspin expression and p53 activation through acetylation at the Lys305 residue by inhibiting class I HDACs. Apigenin withdrawal in LNCaP cells caused loss of maspin and p53 acetylation. Furthermore, proteasome inhibitor MG132 inhibited apigeninmediated proteasomal degradation of class I HDACs in these cells. The increased apigeninmediated p53 acetylation enhanced its binding on maspin promoter, which was associated with decrease in tumor cell invasion and migration. Apigenin treatment also caused accumu‐ lation of acetylated histone H3 in total cellular chromatin, increasing accessibility to bind with the promoter sequences of maspin, consistent with the effects elicited by HDAC inhibitor, trichostatin A. Similar observations were noted after feeding apigenin to 22Rv1 tumor xenograft implanted in nude mice [50]. Other natural compounds that have been reported to enhance prostate cancer suppression and inhibit invasion and migration through upregulation of maspin are tanshinone IIA and apple peel extract [51, 52].

A study by Jiang et al. revealed differential expression patterns of maspin mRNA and protein expression following treatment of cancer cells with essential fatty acids (EFAs) [53]. Addition of omega-6, EFAs arachidonic acid and α-linolenic acid had no effect on maspin expression, while treatment with γ-linolenic acid led to rapid increase in maspin mRNA [53]. Consumption

of γ-linolenic acid has been linked to many beneficial effects in humans. Interestingly in the same study, another omega-6 EFA, linoleic acid, resulted in decreased maspin expression. This study highlighted specific effects of EFAs on maspin that may have significant implications in cancer biology as well as a role for maspin in lipid signaling and processing. Not surprisingly, *in vitro* promoter activity study showed that, in addition to p53 [16], a list of stress-related signals including DNA-damaging agents, cytotoxic drugs [16], peroxisome proliferatoractivated receptor-gamma [54], nitric oxide [55], and manganese superoxide dismutase (MnSOD) [56] activates maspin expression. Consistently, several stress signals that induce maspin expression also induced more differentiated phenotypes [16, 54–56]. Furthermore, epigenetic treatment of prostate cancer cell lines with trichostatin A, a histone deacetylase (HDAC) inhibitor, led to induction of maspin mRNA [20, 50]. Re-expression of maspin using demethylating agents and HDAC inhibitors has been confirmed in additional studies [50, 57].

#### **7. Conclusions and future directions**

Much remains to be understood about the molecular mechanism(s) of action of maspin in the normal prostate and during cancer progression. Studies in our laboratory are ongoing to determine the post-translational modifications of maspin, which may help elucidate the role of maspin in signaling pathways relevant to cell adhesion and angiogenesis. The ability of maspin and its post-translationally modified forms to act as effective therapeutic agents in prostate cancer is also being pursued. Additional studies are required to unravel the multi‐ faceted interrogation of how maspin expression can alter the malignant phenotype of some cell types and not others. Nevertheless, recent findings and ongoing studies should encourage researchers to continue to explore the molecular mechanisms underlying maspin's biological effects in prostate cancer.

Clinical studies performed this far in prostate cancer demonstrate the significance of maspin expression as a useful prognostic and possibly predictive marker for patients undergoing definitive therapy. Focusing on the malignancies in which maspin exhibited a positive prognostic value, therapeutic approaches studied so far aimed to re-activate this dormant tumor suppressor gene by transcription factors that regulate its expression and/or to identify natural substances that can determine the activation and the expression of maspin or possibly deliver this molecule in tumor cell through gene therapy capable of upregulating maspin in an attempt to reduce invasiveness and the risk of metastasis. Maspin packaged as an adenoviral construct or a liposomal DNA has been utilized to reduce tumors and tumor vasculatures in *in vivo* studies corroborates as a feasible approach toward cancer therapy. Nevertheless, the approach using viral vectors or liposomal DNA complexes is accompanied with several safety and efficacy issues that modify their association upon interaction with serum components, which is a subject of rigorous studies, and further evaluation is required through clinical trials. The usefulness of rMaspin in targeted therapy is debatable because proteins undergo rapid clearance from the body through proteolysis, liver clearance and filtered through the kidneys. These issues can be resolved by the use of nanotechnology, which has provided a valuable option for targeted delivery of genes, drugs, and proteins.

Recently performed studies highlight reversible nature of epigenetic silencing of maspin in prostate cancer, which offers a unique opportunity for therapeutic intervention by several epigenetic modifiers. Considering the anti-angiogenic and pro-apoptotic properties of nuclear maspin shown by recent studies, re-activated nuclear maspin in association with antiangiogenic or chemotherapeutic drugs may be effective in the treatment of advance-stage prostate cancer. Undoubtedly, the challenges are numerous, but the prospects for improved therapeutic approaches through maspin application for this debilitating disease could be immense.

#### **Acknowledgements**

of γ-linolenic acid has been linked to many beneficial effects in humans. Interestingly in the same study, another omega-6 EFA, linoleic acid, resulted in decreased maspin expression. This study highlighted specific effects of EFAs on maspin that may have significant implications in cancer biology as well as a role for maspin in lipid signaling and processing. Not surprisingly, *in vitro* promoter activity study showed that, in addition to p53 [16], a list of stress-related signals including DNA-damaging agents, cytotoxic drugs [16], peroxisome proliferatoractivated receptor-gamma [54], nitric oxide [55], and manganese superoxide dismutase (MnSOD) [56] activates maspin expression. Consistently, several stress signals that induce maspin expression also induced more differentiated phenotypes [16, 54–56]. Furthermore, epigenetic treatment of prostate cancer cell lines with trichostatin A, a histone deacetylase (HDAC) inhibitor, led to induction of maspin mRNA [20, 50]. Re-expression of maspin using demethylating agents and HDAC inhibitors has been confirmed in additional studies [50, 57].

Much remains to be understood about the molecular mechanism(s) of action of maspin in the normal prostate and during cancer progression. Studies in our laboratory are ongoing to determine the post-translational modifications of maspin, which may help elucidate the role of maspin in signaling pathways relevant to cell adhesion and angiogenesis. The ability of maspin and its post-translationally modified forms to act as effective therapeutic agents in prostate cancer is also being pursued. Additional studies are required to unravel the multi‐ faceted interrogation of how maspin expression can alter the malignant phenotype of some cell types and not others. Nevertheless, recent findings and ongoing studies should encourage researchers to continue to explore the molecular mechanisms underlying maspin's biological

Clinical studies performed this far in prostate cancer demonstrate the significance of maspin expression as a useful prognostic and possibly predictive marker for patients undergoing definitive therapy. Focusing on the malignancies in which maspin exhibited a positive prognostic value, therapeutic approaches studied so far aimed to re-activate this dormant tumor suppressor gene by transcription factors that regulate its expression and/or to identify natural substances that can determine the activation and the expression of maspin or possibly deliver this molecule in tumor cell through gene therapy capable of upregulating maspin in an attempt to reduce invasiveness and the risk of metastasis. Maspin packaged as an adenoviral construct or a liposomal DNA has been utilized to reduce tumors and tumor vasculatures in *in vivo* studies corroborates as a feasible approach toward cancer therapy. Nevertheless, the approach using viral vectors or liposomal DNA complexes is accompanied with several safety and efficacy issues that modify their association upon interaction with serum components, which is a subject of rigorous studies, and further evaluation is required through clinical trials. The usefulness of rMaspin in targeted therapy is debatable because proteins undergo rapid clearance from the body through proteolysis, liver clearance and filtered through the kidneys. These issues can be resolved by the use of nanotechnology, which has provided a valuable

**7. Conclusions and future directions**

206 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

option for targeted delivery of genes, drugs, and proteins.

effects in prostate cancer.

The original work from author's laboratory outlined in this chapter was supported by the United States Public Health Service grants R01CA108512, R21CA193080, R03CA186179, Department of Defense grant W81XWH-15-1-0558, and Department of Veteran Affairs grant 1I01BX002494 to SG.

#### **Conflict of interest**

The authors have no competing interest.

#### **Author details**

Eswar Shankar1 , Mario Candamo2 , Gregory T. MacLennan3 and Sanjay Gupta1,4,5,6\*

\*Address all correspondence to: sanjay.gupta@case.edu

1 Department of Urology, Case Western Reserve University, University Hospitals Case Medical Center, Cleveland, Ohio, United States

2 Department of Biology, School of Undergraduate Studies, Case Western Reserve Universi‐ ty, Cleveland, Ohio, United States

3 Department of Pathology, Case Western Reserve University, University Hospitals Case Medical Center, Cleveland, Ohio,United States

4 Department of Nutrition, Case Western Reserve University, Cleveland, Ohio, United States

5 Division of General Medical Sciences, Case Comprehensive Cancer Center, Cleveland, Ohio, United States

6 Department of Urology, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleve‐ land, Ohio, United States

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Meeting of the American Association for Cancer Research; 2015 Apr 18–22; Philadel‐ phia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4649. doi: 10.1158/1538-7445.AM2015-4649


## **The Emerging Role of PARP Inhibitors in the Treatment of Prostate Cancer**

Jue Wang, Brent B. Freeman and Paul Mathew

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63283

#### **Abstract**

Meeting of the American Association for Cancer Research; 2015 Apr 18–22; Philadel‐ phia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4649.

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Poly (ADPribose) polymerase (PARP) is a critical DNA repair enzyme involved in DNA single-strand break repair through the base excision repair pathway. PARP inhibitors have been shown to sensitize tumors to DNA-damaging agents and selectively kill homologous recombination repair-defective cancers, such as those arising in BRCA1 and BRCA2 mutation carriers. In addition to its well-documented role in DNA damage repair (DDR), emerging evidence has indicated that PARP1 plays an important role in mediating the transcriptional activities of androgen receptor (AR) and ETS gene rearrangement in prostate cancer. Preclinical and clinical research suggested that the activity of PARP inhibitors is not limited to those with BRCA mutations. PARP inhibitors may have activity in cancers deficient in other DNA repair genes or signaling pathways that mitigate DNA repair.

Based on results of the TOPARP-A Phase 2 trial, the US Food and Drug Administra‐ tion (FDA) has granted Breakthrough Therapy designation to olaparib (Lynparza) for monotherapy treatment of BRCA1/2 or ataxia telangiectasia mutated (ATM) gene mutated in patients with metastatic castration-resistant prostate cancer who received a prior taxane-based chemotherapy and at least one newer hormonal agent. Future research is needed to address the optimal timing, combination, and to identify predictive biomarker for PARP inhibition.

**Keywords:** prostate cancer, BRCA1, BRCA2, poly(ADP-ribose) polymerases, PARP in‐ hibitors

© 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**

Prostate cancer is the most commonly diagnosed cancer in men and is the second leading cause of cancer-related deaths in men each year [1]. Androgen-deprivation therapy has been the gold standard of care for metastatic prostate cancer for decades. While this treatment strategy initially shows benefit, the disease inevitably progresses to metastatic castration-resistant prostate cancer (mCRPC) for which there is limited treatment options [2]. Although chemo‐ therapies, immunotherapy, and novel androgen signaling pathway inhibitors therapies [3–7] have shown some benefit, mCRPC remain incurable with overall survival well under 5 years. Further development of novel agents is needed for the treatment of prostate cancer.

#### **2. Poly(ADPribose) polymerase and DNA repair**

Prostate cancer, like most other cancers, is a genetic disease resulting from the accumulation of genetic alterations that enable cancer cells to survive, proliferate, and metastasis [8, 9]. Such enrichment of genomic instability could be attributed to diminished DNA repair in mCRPC [8–11]. To maintain genomic integrity, there exists conserved checkpoint signaling pathways to facilitate cell cycle delay, DNA repair, and/or apoptosis in response to DNA damage [11].

BRCA1 and BRCA2 are the best characterized DNA repair genes associated with cancer development [12]. Germline-mutated prostate cancer is particularly frequent in young patients (<65 years), with BRCA2 more prevalent than BRCA1 (1.4 and 0.44% of all prostate cancer, respectively). Germline mutation carriers have higher Gleason scores, lower overall survival (OS) and cancer-specific survival, higher advanced stages, and globally a worse prognosis compared with noncarrier patients [13]. BRCA proteins have a crucial role in the regulation of homologous recombination (HR) repair, an accurate DNA double-strand break (DSB) repair process. In the absence of BRCAs (and other HR proteins), DNA DSBs increase which induce the accumulation of DNA mutations and thereby promotes tumorigenesis. Although BRCA dysfunction promotes an oncogenic advantage, it also renders cancer cells reliant on alterna‐ tive DNA repair pathways, such as base excision repair (BER).

Poly(ADPribose) polymerase (PARP) are a family of enzymes that catalyze Nicotinamide adenine dinucleotide (NAD) NAD+-dependent ADP ribosylation of DNA. PARP1 has been implicated in several DNA repair mechanisms, including DNA single-strand breaks (SSB) which repair through the BER pathway. PARP1 recognizes DNA SSB and orchestrates the recruitment and assembly of a DNA repair complex [14, 15]. As a result, PARP inhibition induces the accumulation of unrepaired SSB, which are subsequently converted into DSBs at fork replication. Cells with deficient HR repair systems (i.e., for BRCA1/2 mutations) treated with PARP inhibitors are overcome by DNA DSBs, which lead to further chromosomal instability, cell cycle arrest, and apoptosis [16]. Therefore, PARP-1 is an important therapeutic target in cancer therapy including prostate cancer, especially in patients harboring the BRCA mutations.

#### **3. PARP-1 and prostate cancer**

**1. Introduction**

mutations.

Prostate cancer is the most commonly diagnosed cancer in men and is the second leading cause of cancer-related deaths in men each year [1]. Androgen-deprivation therapy has been the gold standard of care for metastatic prostate cancer for decades. While this treatment strategy initially shows benefit, the disease inevitably progresses to metastatic castration-resistant prostate cancer (mCRPC) for which there is limited treatment options [2]. Although chemo‐ therapies, immunotherapy, and novel androgen signaling pathway inhibitors therapies [3–7] have shown some benefit, mCRPC remain incurable with overall survival well under 5 years.

Prostate cancer, like most other cancers, is a genetic disease resulting from the accumulation of genetic alterations that enable cancer cells to survive, proliferate, and metastasis [8, 9]. Such enrichment of genomic instability could be attributed to diminished DNA repair in mCRPC [8–11]. To maintain genomic integrity, there exists conserved checkpoint signaling pathways to facilitate cell cycle delay, DNA repair, and/or apoptosis in response to DNA damage [11].

BRCA1 and BRCA2 are the best characterized DNA repair genes associated with cancer development [12]. Germline-mutated prostate cancer is particularly frequent in young patients (<65 years), with BRCA2 more prevalent than BRCA1 (1.4 and 0.44% of all prostate cancer, respectively). Germline mutation carriers have higher Gleason scores, lower overall survival (OS) and cancer-specific survival, higher advanced stages, and globally a worse prognosis compared with noncarrier patients [13]. BRCA proteins have a crucial role in the regulation of homologous recombination (HR) repair, an accurate DNA double-strand break (DSB) repair process. In the absence of BRCAs (and other HR proteins), DNA DSBs increase which induce the accumulation of DNA mutations and thereby promotes tumorigenesis. Although BRCA dysfunction promotes an oncogenic advantage, it also renders cancer cells reliant on alterna‐

Poly(ADPribose) polymerase (PARP) are a family of enzymes that catalyze Nicotinamide adenine dinucleotide (NAD) NAD+-dependent ADP ribosylation of DNA. PARP1 has been implicated in several DNA repair mechanisms, including DNA single-strand breaks (SSB) which repair through the BER pathway. PARP1 recognizes DNA SSB and orchestrates the recruitment and assembly of a DNA repair complex [14, 15]. As a result, PARP inhibition induces the accumulation of unrepaired SSB, which are subsequently converted into DSBs at fork replication. Cells with deficient HR repair systems (i.e., for BRCA1/2 mutations) treated with PARP inhibitors are overcome by DNA DSBs, which lead to further chromosomal instability, cell cycle arrest, and apoptosis [16]. Therefore, PARP-1 is an important therapeutic target in cancer therapy including prostate cancer, especially in patients harboring the BRCA

Further development of novel agents is needed for the treatment of prostate cancer.

**2. Poly(ADPribose) polymerase and DNA repair**

214 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

tive DNA repair pathways, such as base excision repair (BER).

Several lines of evidence point to a potential role of PARP-1 in prostate cancer progression. PARP-1 expression is markedly elevated in prostate cancer relative to that in benign prostatic hyperplasia (BPH) tissues, which may imply the involvement of PARP-1 and PAR in the development of prostate cancer [17]. Augmented immunodetection of PARP-1 was associated with prostate cancer progression and biochemical recurrence [18].

In addition to its well-documented role in BER, emerging evidence has indicated that PARP1 plays an important role in mediating the transcriptional activities of androgen receptor (AR) and ETS gene rearrangement [19]. PARP inhibition results in antitumor activity in TMPRSS2- ERG rearranged cancer models and suppresses AR target gene expression and tumor prolif‐ eration [20]. PARP-1 regulates Smad-dependent responses to Transforming growth factor beta (TGF-β) signaling and potential AR activity, directing both toward epithelial-mesenchymal transition (EMT) in prostate cancer progression [21]. PARP-1 has also been implicated at the chromatin level in AR-mediated cell proliferation in the early and late-stage prostate cancer models [22], with suppression of PARP-1 resulting in reduced cell proliferation. In androgenindependent PC3 cells, PARP inhibition significantly decreased cell viability, migration, invasion, chromatin loop dimensions, and histone acetylation. Thus PARP could play a key role in the compartmentalization of chromatin and in the development of the more aggressive phenotype [23].

#### **4. BRCAness and prostate cancer**

McCabe et al. [24] demonstrated that deficiencies of several proteins in the HR DNA repair pathway, such as the DNA damage sensors ataxia telangiectasia mutated (ATM) and ataxia telangiectasia (ATR and RAD3-related protein), lead to HR deficiency and subsequent PARP inhibition sensitivity. This concept known as 'BRCAness' has been used to describe the phenotype arising in sporadic cancers that have intact BRCA1/2 genes but share features with the BRCA1/2 mutation-related tumors, such as profound platinum sensitivity [25]. For example, based on its high proliferation rate, sensitivity to platinum-based therapy and the rapid development of chemotherapy resistance [26], small cell/anaplastic prostate cancer clearly fits into the clinical phenotype of BRCAness. Studies of these tumor samples at the DNA level will likely reveal genetic alterations in DNA repair genes. Theoretically, PARP inhibitors may enhance the chemotherapy-induced DNA damage in anaplastic/small cell prostate cancer.

While only a minority of prostate cancer patients carry germline mutations, emerging data suggest that HR defects are common in prostate cancer, potentially conferring a BRCAness phenotype [26–29]. Recent genetic studies have shown somatic mutations of the DNA HR repair system in more than 20% of the patients with CRPC. The genes identified are involved in different steps and mechanisms of HR machinery [29]. In an extensive genome analysis, Robinson et al. compared genetic sequencing data of castration sensitive and CRPC. BRCA2 was the most frequent mutations occurring in 12.7% of cases. Interestingly, the analysis of other DNA repair genes showed overall DNA repair gene aberrations in 22.7% of patients, with ATM and BRCA1 alterations occurring most frequently (in 19.3% of patients). In addition, 3.4% of patients have CDK12, FANCA, RAD51B, and RAD51C mutations [29]. These patients repre‐ sent a distinct subtype with unique clinical characteristics that have important implications for management.

#### **5. Active clinical trials investigating PARP inhibitors in prostate cancer**

The evidence correlating increased PARP-1 activity with tumor progression has opened a new avenue for the utilization of PARP inhibitors, which may impair the DNA repair machine. The clinical experiences with PARP inhibitors initially focused on patients carrying mutations of the BRCA1 or BRCA2 genes, which have been linked to increased sensitivity to PARP-1 inhibitors. Additional evidence has shown that tumors with other mechanisms of impaired DNA repair might benefit from treatment with PARP inhibitors. In addition to the use of singleagent, the PARP inhibitors have been studied in combination with a number of different agents in prostate cancer (**Table 1**) and other cancers (**Table 2**). Other areas of active investigation include the development of biomarkers that may predict clinical benefit from PARP inhibition, as well as the identification of resistance mechanisms to PARP inhibitor therapy.


**Table 1.** Ongoing clinical trials (Phase I/II) with PARP-1 inhibitors combination for prostate cancers (www.clinicaltrials.gov).

#### **5.1. PARP inhibitors as single-agent therapy**

In a Phase I clinical trial with olaparib, 60 patients with various refractory caners were enrolled and treated. Objective antitumor activity was reported only in BRCA mutation carriers, all of whom had ovarian, breast, or prostate cancer and had received multiple treatment regimens. Three patients with mCRPC were recruited, and one of them had a BRCA2 mutation [30]. The patient with BRCA2 mutation had >50% decrease in serum prostate-specific antigen (PSA) levels and a complete response of bone lesions.

Niraparib was tested in a Phase 1 dose-escalation study of 21 mCRPC patients. The investi‐ gators reported 43% of the patients with stable disease, and a median duration of response of 254 days. In total, 30% of the patients had a decrease of circulating tumor cells (CTC), and one of the 21 patients enrolled had >50% PSA reduction. Importantly, the authors did not observe the hypothesized correlation between ERG rearrangements or loss of PTEN expression and treatment response [31].

was the most frequent mutations occurring in 12.7% of cases. Interestingly, the analysis of other DNA repair genes showed overall DNA repair gene aberrations in 22.7% of patients, with ATM and BRCA1 alterations occurring most frequently (in 19.3% of patients). In addition, 3.4% of patients have CDK12, FANCA, RAD51B, and RAD51C mutations [29]. These patients repre‐ sent a distinct subtype with unique clinical characteristics that have important implications

**5. Active clinical trials investigating PARP inhibitors in prostate cancer**

as well as the identification of resistance mechanisms to PARP inhibitor therapy.

**Table 1.** Ongoing clinical trials (Phase I/II) with PARP-1 inhibitors combination for prostate cancers

**Agent(s) [Phase] Cancer Identifier** Olaparib + Arbiraterone [II] Metastatic castration-resistant prostate cancer NCT01972217 Veliparib + Arbiraterone [II] Metastatic castration-resistant prostate cancer NCT01576172 Enzalutamide + Niraparib [I] Metastatic castration-resistant prostate cancer NCT02500901 Olaparib + Radical prostatectomy [I] Intermediate/high-risk prostate cancer NCT02324998

In a Phase I clinical trial with olaparib, 60 patients with various refractory caners were enrolled and treated. Objective antitumor activity was reported only in BRCA mutation carriers, all of whom had ovarian, breast, or prostate cancer and had received multiple treatment regimens. Three patients with mCRPC were recruited, and one of them had a BRCA2 mutation [30]. The patient with BRCA2 mutation had >50% decrease in serum prostate-specific antigen (PSA)

Niraparib was tested in a Phase 1 dose-escalation study of 21 mCRPC patients. The investi‐ gators reported 43% of the patients with stable disease, and a median duration of response of 254 days. In total, 30% of the patients had a decrease of circulating tumor cells (CTC), and one of the 21 patients enrolled had >50% PSA reduction. Importantly, the authors did not observe

The evidence correlating increased PARP-1 activity with tumor progression has opened a new avenue for the utilization of PARP inhibitors, which may impair the DNA repair machine. The clinical experiences with PARP inhibitors initially focused on patients carrying mutations of the BRCA1 or BRCA2 genes, which have been linked to increased sensitivity to PARP-1 inhibitors. Additional evidence has shown that tumors with other mechanisms of impaired DNA repair might benefit from treatment with PARP inhibitors. In addition to the use of singleagent, the PARP inhibitors have been studied in combination with a number of different agents in prostate cancer (**Table 1**) and other cancers (**Table 2**). Other areas of active investigation include the development of biomarkers that may predict clinical benefit from PARP inhibition,

for management.

216 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

(www.clinicaltrials.gov).

**5.1. PARP inhibitors as single-agent therapy**

levels and a complete response of bone lesions.


**Table 2.** Ongoing clinical trials (Phase I/II/III) with PARP-1 inhibitors for other cancers (www.clinicaltrials.gov).

In a Phase II clinical trial evaluating the efficacy and safety of olaparib in a spectrum of BRCA1/2-associated cancers, the authors reported 50% response rate, 25% stable disease, and an overall median duration response of 327 days in eight previously heavily treated mCRPC patients with germline BRCA1/2 mutations. Median progression-free survival was 7.2 months with 62.5% of the patients still progression-free at 6 months. Moreover, the Overall Survival (OS) was 18.4 months, with 50% of the patients was still alive at 12 months [32].

The publication of the Phase II clinical trial, TOPARP-A, which tests the efficacy of olaparib in mCRPC patients [33] generated a lot of excitement in prostate cancer field. Fifty mCRPC patients previously treated with docetaxel, most of whom had also been previously treated with abiraterone (Zytiga®) or enzalutamide (Xtandi®), received oral olaparib 400 mg twice daily in 28 day-cycles until disease progression. All patients had received docetaxel; 98% had received abiraterone or enzalutamide; and 58% had received cabazitaxel. Patients underwent biopsies at baseline and during therapy for whole-exome sequencing and transcriptome studies. The primary endpoint was response rate, PSA response, or conversion of the baseline circulating tumor cell count. Sixteen of the 49 patients who could be evaluated had a response rate of 33%, with 12 patients receiving the study treatment for more than 6 months. Median overall survival was 10.1 months. Next-generation sequencing identified homozygous deletions, deleterious mutations, or both in DNA-repair genes--including BRCA1/2, ATM, Fanconi's anemia genes, and CHEK2—in 16 of the 49 patients who could be evaluated (33%). Of these 16 patients, 14 (88%) had a response to olaparib, including all 7 patients with BRCA2 loss (four with biallelic somatic loss and three with germline mutations) and 4 of 5 with ATM aberrations. Anemia and fatigue were the major treatment-related adverse effects. The mutational status of the ERG oncogene and that of the PTEN tumor suppressor gene was not associated with olaparib responses. TOPARP-B, the second stage of this trial, is ongoing and aiming to validate findings of TOPARP-A. Part B of TOPARP (TOPARP-B) is open to the recruitment and aims to recruit a total of 88 patients. Potential participants will have their tumor tissue analyzed and only those with biomarkers predictive of olaparib response will go on to enter TOPARP-B. Forty-four patients will receive 300 mg twice daily, and 44 will receive 400 mg of olaparib twice daily. Part C (TOPARP-C) will be the subsequent Phase II random‐ ized, double-blind, placebo-controlled evaluation of olaparib and is currently in development.

#### **5.2. PARP inhibitors in combination therapy**

#### *5.2.1. Combining the PARP inhibitor with cytotoxic chemotherapy*

Preclinical research has provided a strong rationale for employing PARP inhibitors as chemosensitizers in combination with cytotoxic agents. The PARP inhibitor veliparib has been shown to enhance the antitumor activity of Temozolomide (TMZ) in prostate cancer xenografts [34]. This formed the rationale for testing the safety and efficacy of veliparib and TMZ in 26 patients with mCRPC pretreated with docetaxel [35]. Despite the promising preclinical activity, this combination showed modest activity over TMZ monotherapy. Two patients had a confirmed PSA response and four patients had stable disease (SD) for at least 4 months. The median progression-free survival (PFS) and OS were 2.1 and 9.1 months, respectively. One patient had the TMPRSS2:ERG gene fusion, and this patient achieved stable disease with a progression-free survival of 70 days and overall survival of 277 days. Grade III/IV thrombo‐ cytopenia was noted in 15% of patients.

#### *5.2.2. Combining the PARP inhibitor with androgen deprivation therapy*

The preclinical findings of PARP1 in mediating transcriptional regulation by AR and the ETS fusion protein [20–23] and the potential preclinical synergy of targeting of the PARP and AR pathways provide a strong rationale for the clinical evaluation of combining of these two classes of anticancer drugs.

The primary objective of a multicenter randomized Phase II trial (NCT01576172) is to evaluate whether adding veliparib to abiraterone acetate and prednisone would improve the PSA response rate of the standard abiraterone acetate and prednisone regimen in patients with mCRPC. Secondary and exploratory objectives include PSA decline rate, objective response rate, progression-free survival and toxicity. The investigators will evaluate ETS fusion status in metastatic tumor tissue, and a logistic model will be used to determine the association of TMPRSS2-ERG fusion status with the PSA response in the veliparib plus abiraterone and prednisone arm. This study will provide validation on the value of ETS gene rearrangement as a predicative biomarker for PARP inhibitor-based therapy for mCRPC.

Another Phase I trial evaluating the combination of PARP inhibitor niraparib and enzaluta‐ mide (NCT02500901) was recently activated. The primary goal of this study is to assess whether patients with AR-regulated CRPC can be assessed for synergistic clinical benefit from dual AR blockade and PARP-1 inhibition, with the combination of enzalutamide and niraparib. Secondary and exploratory objectives include PSA kinetics, progression-free survival, and objective response. Correlative studies using quantitative and qualitative measures of CTCs to identify a predictive biomarker of response to the combination. These include dynamic studies of AR and AR splice variant nuclear localization and feasibility studies aim to assess homologous repair deficiency in CTCs.

#### *5.2.3. Combining the PARP inhibitors with radiotherapy*

biopsies at baseline and during therapy for whole-exome sequencing and transcriptome studies. The primary endpoint was response rate, PSA response, or conversion of the baseline circulating tumor cell count. Sixteen of the 49 patients who could be evaluated had a response rate of 33%, with 12 patients receiving the study treatment for more than 6 months. Median overall survival was 10.1 months. Next-generation sequencing identified homozygous deletions, deleterious mutations, or both in DNA-repair genes--including BRCA1/2, ATM, Fanconi's anemia genes, and CHEK2—in 16 of the 49 patients who could be evaluated (33%). Of these 16 patients, 14 (88%) had a response to olaparib, including all 7 patients with BRCA2 loss (four with biallelic somatic loss and three with germline mutations) and 4 of 5 with ATM aberrations. Anemia and fatigue were the major treatment-related adverse effects. The mutational status of the ERG oncogene and that of the PTEN tumor suppressor gene was not associated with olaparib responses. TOPARP-B, the second stage of this trial, is ongoing and aiming to validate findings of TOPARP-A. Part B of TOPARP (TOPARP-B) is open to the recruitment and aims to recruit a total of 88 patients. Potential participants will have their tumor tissue analyzed and only those with biomarkers predictive of olaparib response will go on to enter TOPARP-B. Forty-four patients will receive 300 mg twice daily, and 44 will receive 400 mg of olaparib twice daily. Part C (TOPARP-C) will be the subsequent Phase II random‐ ized, double-blind, placebo-controlled evaluation of olaparib and is currently in development.

Preclinical research has provided a strong rationale for employing PARP inhibitors as chemosensitizers in combination with cytotoxic agents. The PARP inhibitor veliparib has been shown to enhance the antitumor activity of Temozolomide (TMZ) in prostate cancer xenografts [34]. This formed the rationale for testing the safety and efficacy of veliparib and TMZ in 26 patients with mCRPC pretreated with docetaxel [35]. Despite the promising preclinical activity, this combination showed modest activity over TMZ monotherapy. Two patients had a confirmed PSA response and four patients had stable disease (SD) for at least 4 months. The median progression-free survival (PFS) and OS were 2.1 and 9.1 months, respectively. One patient had the TMPRSS2:ERG gene fusion, and this patient achieved stable disease with a progression-free survival of 70 days and overall survival of 277 days. Grade III/IV thrombo‐

The preclinical findings of PARP1 in mediating transcriptional regulation by AR and the ETS fusion protein [20–23] and the potential preclinical synergy of targeting of the PARP and AR pathways provide a strong rationale for the clinical evaluation of combining of these two

The primary objective of a multicenter randomized Phase II trial (NCT01576172) is to evaluate whether adding veliparib to abiraterone acetate and prednisone would improve the PSA response rate of the standard abiraterone acetate and prednisone regimen in patients with

**5.2. PARP inhibitors in combination therapy**

218 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

cytopenia was noted in 15% of patients.

classes of anticancer drugs.

*5.2.1. Combining the PARP inhibitor with cytotoxic chemotherapy*

*5.2.2. Combining the PARP inhibitor with androgen deprivation therapy*

Preclinical study with prostate cancer cell lines reported that combining the PARP inhibitor rucaparib with radiation enhanced the DNA damage and antitumor effects compared with radiation alone. The strongest synergistic activities were observed in LNCaP and VCaP cells, which contain the TMPRSS2-ERG fusion gene [36–38]. However, no association was noted between the loss of PTEN expression and ETS rearrangements, with radiologic assessment of the antitumor activity of niraparib in a Phase I trial [31]. At the present time, there are no active clinical trials investigating the combination of radiation therapy with PARP inhibition in mCRPC.

The combination of a PARP inhibitor with radiation would be an attractive strategy for newly diagnosed ETS fusion-positive, locally advanced, high-risk prostate cancer in adjuvant or neoadjuvant setting, or nonmetastatic CRPC at recurrent setting. However, the risk of overtreatment and long-term safety are the main concerns of testing this strategy.

Combining PARP inhibitors with radiation or radiopharmaceuticals such like Xofigo®(radium 223) could be another reasonable combination for patients with mCRPC to the bone. Currently, there is no active trial testing the combination.

#### *5.2.4. Combining the PARP inhibitors with molecularly targeted drugs*

In a Phase II clinical trial (NCT02576444), the investigators evaluated the safety of combining the AKT inhibitor AZD5363 with olaparib. The authors reported that the novel combination of olaparib and AZD5363 was safe and yielded responses in patients with a variety of cancer types, including breast, ovarian, and prostate cancers, regardless of BRCA1/2 mutation status. One patient with BRCA1/2-mutant advanced prostate cancer had a sustained response both by MRI and PSA Working Group 2 response criteria at 11 months. The most commonly observed side effects were nausea, vomiting, fatigue, diarrhea, and anemia.

Several other trails combining the PARP inhibitor with other molecularly targeted drugs are ongoing (**Tables 1** and **2**).

#### *5.2.5. preoperative (neoadjuvant) studies of PARP1 inhibitors*

The testing of novel agents in the preoperative (neoadjuvant) setting approach offers a potentially rapid and efficient strategy for drug development. Neoadjuvant studies allow the assessment of drug effects on the target (pharmacodynamic response) and the development of predictive biomarkers of response.

A Phase I study investigating the feasibility and tolerability of a short course of neoadjuvant treatment with olaparib given prior to radical prostatectomy in men with localized intermedi‐ ate-/high-risk prostate cancer is ongoing (NCT02324998). The primary objective is to determine the pharmacodynamic biomarker effects of olaparib in this patient population. Participants will receive either single-agent olaparib, or olaparib in combination with degarelix (androgen deprivation), for one week prior to routine radical prostatectomy. The degree of PARP inhibition will be measured by the change in immunohistochemistry (IHC) levels of biomark‐ ers such as PAR, gamma H2AX, pH2A (s129), Rad51 foci, FancD2 foci, and ATM/ATR/CHK1/2 in tumor samples taken at baseline and following treatment with olaparib (either alone or in combination with degarelix). Secondary outcome measures include the incidence and severity of adverse events caused by treatment for 7 days with olaparib (either alone or in combination with degarelix) prior to radical prostatectomy [41].

#### **5.3. Safety of PARP inhibitors**

The toxicity profile of PARP inhibitor monotherapy appears to be similar to cytotoxic chemo‐ therapeutic agents [30–33, 39]. The most frequently reported adverse events in published studies are grade 1–2 nausea, vomiting, diarrhea, fatigue, headache, and anemia. Grade 3 or 4 toxicities are rare in early phase clinical trials in patients with prostate cancer being treated with a single-agent. The most common grade 3–4 toxicities were nausea, vomiting, and hematological toxicity, with anemia, lymphopenia, and thrombocytopenia being the most common dose-limiting toxicities in dose-finding studies [31, 32]. In a Phase II trial, in which patients with mCRPC were treated with olaparib tablets at a dose of 400 mg twice a day, anemia (20%) and fatigue (12%) were the most common grade 3 or 4 adverse events. These findings are consistent with previous studies of olaparib [33].

Conversely, dose-limiting toxicities observed in trials of PARP inhibitors in combination with cytotoxic agents include primarily hematologic toxicities. For example, olaparib in combina‐ tion with cisplatin and gemcitabine is associated with myelosuppression even at relatively low doses in a Phase I study of patients with advanced solid tumors [40]. An intermittent schedule of PARP inhibition instead of continuous dosing was investigated [41].

At this time, the long-term safety data on the PAPR inhibitors is still lacking. Enhanced DNA damage with a PARP inhibitor and radiation may lead to genomic instability and more aggressive prostate cancer and secondary malignancy. Few cases of myelodysplastic syn‐ drome and acute myeloid leukemia have been reported in PARP inhibitor trials; most of the patients had been treated with DNA-damaging classic chemotherapeutic agents. Nonetheless, the potential increased risk of developing secondary cancer from DNA damage warrants close attention in future development of PARP inhibitors, especially in the neoadjuvant and adjuvant settings [42].

#### **5.4. Biomarkers for PARP-directed therapy**

Several other trails combining the PARP inhibitor with other molecularly targeted drugs are

The testing of novel agents in the preoperative (neoadjuvant) setting approach offers a potentially rapid and efficient strategy for drug development. Neoadjuvant studies allow the assessment of drug effects on the target (pharmacodynamic response) and the development

A Phase I study investigating the feasibility and tolerability of a short course of neoadjuvant treatment with olaparib given prior to radical prostatectomy in men with localized intermedi‐ ate-/high-risk prostate cancer is ongoing (NCT02324998). The primary objective is to determine the pharmacodynamic biomarker effects of olaparib in this patient population. Participants will receive either single-agent olaparib, or olaparib in combination with degarelix (androgen deprivation), for one week prior to routine radical prostatectomy. The degree of PARP inhibition will be measured by the change in immunohistochemistry (IHC) levels of biomark‐ ers such as PAR, gamma H2AX, pH2A (s129), Rad51 foci, FancD2 foci, and ATM/ATR/CHK1/2 in tumor samples taken at baseline and following treatment with olaparib (either alone or in combination with degarelix). Secondary outcome measures include the incidence and severity of adverse events caused by treatment for 7 days with olaparib (either alone or in combination

The toxicity profile of PARP inhibitor monotherapy appears to be similar to cytotoxic chemo‐ therapeutic agents [30–33, 39]. The most frequently reported adverse events in published studies are grade 1–2 nausea, vomiting, diarrhea, fatigue, headache, and anemia. Grade 3 or 4 toxicities are rare in early phase clinical trials in patients with prostate cancer being treated with a single-agent. The most common grade 3–4 toxicities were nausea, vomiting, and hematological toxicity, with anemia, lymphopenia, and thrombocytopenia being the most common dose-limiting toxicities in dose-finding studies [31, 32]. In a Phase II trial, in which patients with mCRPC were treated with olaparib tablets at a dose of 400 mg twice a day, anemia (20%) and fatigue (12%) were the most common grade 3 or 4 adverse events. These findings

Conversely, dose-limiting toxicities observed in trials of PARP inhibitors in combination with cytotoxic agents include primarily hematologic toxicities. For example, olaparib in combina‐ tion with cisplatin and gemcitabine is associated with myelosuppression even at relatively low doses in a Phase I study of patients with advanced solid tumors [40]. An intermittent schedule

At this time, the long-term safety data on the PAPR inhibitors is still lacking. Enhanced DNA damage with a PARP inhibitor and radiation may lead to genomic instability and more aggressive prostate cancer and secondary malignancy. Few cases of myelodysplastic syn‐ drome and acute myeloid leukemia have been reported in PARP inhibitor trials; most of the

ongoing (**Tables 1** and **2**).

of predictive biomarkers of response.

*5.2.5. preoperative (neoadjuvant) studies of PARP1 inhibitors*

220 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

with degarelix) prior to radical prostatectomy [41].

are consistent with previous studies of olaparib [33].

of PARP inhibition instead of continuous dosing was investigated [41].

**5.3. Safety of PARP inhibitors**

The ultimate clinical need in the development of PARP-directed therapy is to identify bio‐ markers to enrich selection of patients who are most likely to respond to therapy [43]. As monotherapy, findings from clinical trials with olaparib showed that genetic biomarkers could be used to select the patients with mCRPC who will respond to PARP inhibitor therapy. The HR/PARP synthetic lethality model may be more widely applicable in prostate cancer with germline or somatic inactivating mutations in the HR DNA repair genes, CHK2, BRIPI/FANCJ, NBS1, BRCA1, and ATM, collectively estimated to occur in 20–25% of prostate cancer cases. Two of the most common genetic alterations in prostate cancer, TMPRSS2: ERG gene fusion ETS gene rearrangement, and loss of PTEN, have also been linked to increased sensitivity to PARP inhibitor in preclinical models [37, 38]. However, this association has not been confirmed in clinical study [31]. Regardless, these results underscore the complexity and challenge in developing a biomarker for PARP inhibitor activity. Although additional synthetic lethal strategies have been explored in preclinical, or early clinical trial setting [44–47], development of a clinically validated biomarker (companion diagnostic) will depend on the results of welldesigned and conducted Phase III clinical trials.

It is clear that the individual clinical response to PARP inhibition is varied and that currently accepted markers of response (progression-free survival, RECIST, PARP inhibition in periph‐ eral blood mononuclear cells, or hair follicles) are not ideal [48]. The availability of direct imaging tests capable of measuring PARP inhibition locally would thus be of enormous value in such settings [49]. A new radiolabeled compound (18°F) FluorThanatrace ([18 F] FTT), has been generated which can be used to measure PARP1 activity noninvasively and quantitatively using positron emission tomography (PET). Preclinical models show that the uptake of this compound is specific for PARP1 activity and correlates with biochemically determined PARP1 activity. Additional data also suggests that decreased (18 F) FTT uptake predicts tumor response to PARP inhibition with olaparib. A Phase 0 study investigating the feasibility of PET imaging of PARP activity in cancer (NCT02469129) recently opened for enrollment. This technology provides both a biomarker for patient selection as well as a means of monitoring PARP activity during treatment.

#### **6. Conclusion**

Approximately 30% of mCRPC exhibit defective DNA repair via HR, representing a distinct subtype with unique clinical characteristics that have important implications for management. PARP inhibitors are an exciting new class of agents that have already demonstrated promising preclinical and clinical activity in mCRPC. Recent Phase I and II studies have reported singleagent activities with favorable side effect profiles in sporadic and in BRCA‑mutant prostate cancers. Based on results of the TOPARP-A Phase II trial, the US FDA has granted Break‐ through Therapy designation to olaparib (Lynparza) [50]., for monotherapy treatment of BRCA1/2 or ATM gene mutated mCRPC in patients who received a prior taxane-based chemotherapy and at least one newer androgen signaling inhibitor. Currently, there are seven different PARP inhibitors in clinical development for cancer. As we learn more about these agents through ongoing trials, it will be important to identify biomarkers that predict patients who may benefit the most from PARP inhibitor therapy. In addition, it will be important to determine the optimal timing, sequence and clinical setting (neoadjuvant, adjuvant, or maintenance), either as monotherapy or in combination.

#### **Author details**

Jue Wang\* , Brent B. Freeman and Paul Mathew

\*Address all correspondence to: jue.wang@dignityhealth.org

University of Arizona Cancer Center at Dignity Health St. Joseph's, Phoenix, AZ, USA

#### **References**


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agent activities with favorable side effect profiles in sporadic and in BRCA‑mutant prostate cancers. Based on results of the TOPARP-A Phase II trial, the US FDA has granted Break‐ through Therapy designation to olaparib (Lynparza) [50]., for monotherapy treatment of BRCA1/2 or ATM gene mutated mCRPC in patients who received a prior taxane-based chemotherapy and at least one newer androgen signaling inhibitor. Currently, there are seven different PARP inhibitors in clinical development for cancer. As we learn more about these agents through ongoing trials, it will be important to identify biomarkers that predict patients who may benefit the most from PARP inhibitor therapy. In addition, it will be important to determine the optimal timing, sequence and clinical setting (neoadjuvant, adjuvant, or

maintenance), either as monotherapy or in combination.

222 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

, Brent B. Freeman and Paul Mathew

\*Address all correspondence to: jue.wang@dignityhealth.org

University of Arizona Cancer Center at Dignity Health St. Joseph's, Phoenix, AZ, USA

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### **Rehabilitation of Patients with Prostate Cancer**

Meral Huri, Burcu Semin Akel and Sedef Şahin

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63989

#### **Abstract**

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Accessed February 1, 2016.

Cancer rehabilitation involves helping an individual with cancer to regain maximum psychological, physical, cognitive, social, and vocational functioning with the limits up to disease and its treatments in an interdisciplinary team concept. Prostate cancer is one of the most frequent male malignancies in the world. Prostate cancer treatment options have the risk of some side effects including loss of muscle strength, fatigue, pain, urinary incontinence, erectile dysfunction, cognitive problems, decrease in bone density, weight loss, gynecomastia, and hot flushes with stress-related psychosocial problems. Relative to other cancers, the prognosis of men with prostate cancer is much better and the potential treatment-related side effects have important implications which can affect the health-related quality of life (QOL) of this population. Recent studies support the efficiency of multimodal treatment to recognize, prevent, and increase functional recovery with an interdisciplinary rehabilitation team which includes physical and occupational therapists. This chapter describes briefly cancer rehabilitation and rehabilitation approaches at every stage of patients with prostate cancer for minimiz‐ ing the morbidity rate associated with prostate cancer treatment to increase occupa‐ tional participation and improve QOL.

**Keywords:** prostate cancer, rehabilitation, physiotherapy, occupational therapy

#### **1. Introduction**

Cancer rehabilitation involves helping an individual with cancer to regain maximum psycho‐ logical, physical, cognitive, social, and vocational functioning with the limits up to disease and its treatments in an interdisciplinary team concept [1]. Prostate cancer is one of the most frequent male malignancies in the world [2]. The development of serum prostate-specific antigen (PSA) and advanced cancer treatment modalities increased 10-year survival rates from ~60% to >70%. Prostate cancer can occur as a local disease or an advanced metastatic disease. Surgical removal

© 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.

of the prostate gland, hormonal therapy, radiation therapy, cryoablation, and expectant monitoring are some of the treatment options for patients with prostate cancer [3].

These treatment options are associated with the risk of some side effects including fatigue, pain, urinary incontinence, erectile dysfunction, cognitive problems, decrease in bone density, weight loss, gynecomastia, and hot flushes with stress-related psychosocial problems [4]. Relative to other cancers, the prognosis of men with prostate cancer is much better, and the potential treatment-related side effects have important implications which can affect the health-related quality of life (QOL) of the patients; besides, these treatment-related side effects are significant in this population [5].

Increased rates of survival and support required for functional, physical, and psychological status led to a considerable interest in rehabilitation needs and the approaches used to increase the QOL of the patients with prostate cancer [5]. Recent studies support the efficiency of multimodal treatment to recognize, prevent, and increase functional recovery with an interdisciplinary rehabilitation team which includes physical and occupational therapists. These professionals provide inpatient care, outpatient follow-up and education, and services in home care, palliative, and hospice care settings [6].

Physical therapists play a vital role in the rehabilitation of patients with prostate cancer by teaching and implementing weight-bearing and gentle exercise, resistive exercises, and vibration exercises which transmit energy to the body with special techniques that strengthen the posture, balance, and body fitness, maintain or improve bone density, and prevent falls [6, 7]. In addition, pelvic floor training helps alleviate symptoms of urinary incontinence and maintain normal pelvic floor muscle functions [8]. Physical therapy also focuses on restoring the cardiovascular system which helps improve blood flow; this has been shown to improve symptoms associated with cancer-related fatigue and erectile dysfunction. Physical therapists assess the patients and develop individualized intervention programs including exercise programs to increase the endurance, muscle strength, mobility, and balance of patients with prostate cancer [6–8].

Occupational therapists play a vital role in increasing the occupational participation of the patients with prostate cancer [5]. Occupational therapists use training in activities of daily living, assistive technology approaches, education of energy conservation techniques, man‐ agement of treatment-related problems such as pain, fatigue, and nausea. Moreover, occupa‐ tional therapists give occupational balance training for regaining value of engagement in meaningful activities with a holistic view of creative and therapeutic use of activity [5, 9]. Occupational therapists focus on adaptations and offer education assistance for sexual activity for patients where certain sexual positions are limited or impossible due to pain, fatigue, or positioning issues. This complication in prostate cancer treatments is one of the most important limitations of activities of daily living that men face [9]. Occupational therapists offer ways to help patients with prostate cancer to confirm, express, accept, and use problem-solving techniques to present the changes due to prostate cancer and its treatment. Effective stress management must include relaxation and social support in a supportive environment. Such interventions decrease treatment-related symptoms, reduce the physiological accompani‐ ments of stress, and improve mood. Patients who participate in such rehabilitation interven‐ tions are shown to have improved mental health by feeling more controlled and experiencing reduced interpersonal conflicts and distress related to cancer-related intrusive thinking [5]. In addition, cognitive therapy and changing life style with cognitive behavioral therapy are the mostly used occupational therapy interventions for patients with prostate cancer [5]. Futher‐ more, remaining in or returning to work is increasingly important for patients with and survivors of prostate cancer. Occupational therapists support men to remain in or return to work by providing fast-track care, counseling, and monitoring the men in work environment [10].

This chapter describes briefly cancer rehabilitation and rehabilitation approaches for prostate cancer patients at every stage of the disease for minimizing the morbidity rates associated with prostate cancer treatment to increase occupational participation and improve QOL. The chapter also focuses on physical and occupational therapy approaches for patients with prostate cancer with psychosocial and vocational rehabilitation after prostate cancer treatment.

#### **2. Cancer rehabilitation**

of the prostate gland, hormonal therapy, radiation therapy, cryoablation, and expectant

These treatment options are associated with the risk of some side effects including fatigue, pain, urinary incontinence, erectile dysfunction, cognitive problems, decrease in bone density, weight loss, gynecomastia, and hot flushes with stress-related psychosocial problems [4]. Relative to other cancers, the prognosis of men with prostate cancer is much better, and the potential treatment-related side effects have important implications which can affect the health-related quality of life (QOL) of the patients; besides, these treatment-related side effects

Increased rates of survival and support required for functional, physical, and psychological status led to a considerable interest in rehabilitation needs and the approaches used to increase the QOL of the patients with prostate cancer [5]. Recent studies support the efficiency of multimodal treatment to recognize, prevent, and increase functional recovery with an interdisciplinary rehabilitation team which includes physical and occupational therapists. These professionals provide inpatient care, outpatient follow-up and education, and services

Physical therapists play a vital role in the rehabilitation of patients with prostate cancer by teaching and implementing weight-bearing and gentle exercise, resistive exercises, and vibration exercises which transmit energy to the body with special techniques that strengthen the posture, balance, and body fitness, maintain or improve bone density, and prevent falls [6, 7]. In addition, pelvic floor training helps alleviate symptoms of urinary incontinence and maintain normal pelvic floor muscle functions [8]. Physical therapy also focuses on restoring the cardiovascular system which helps improve blood flow; this has been shown to improve symptoms associated with cancer-related fatigue and erectile dysfunction. Physical therapists assess the patients and develop individualized intervention programs including exercise programs to increase the endurance, muscle strength, mobility, and balance of patients with

Occupational therapists play a vital role in increasing the occupational participation of the patients with prostate cancer [5]. Occupational therapists use training in activities of daily living, assistive technology approaches, education of energy conservation techniques, man‐ agement of treatment-related problems such as pain, fatigue, and nausea. Moreover, occupa‐ tional therapists give occupational balance training for regaining value of engagement in meaningful activities with a holistic view of creative and therapeutic use of activity [5, 9]. Occupational therapists focus on adaptations and offer education assistance for sexual activity for patients where certain sexual positions are limited or impossible due to pain, fatigue, or positioning issues. This complication in prostate cancer treatments is one of the most important limitations of activities of daily living that men face [9]. Occupational therapists offer ways to help patients with prostate cancer to confirm, express, accept, and use problem-solving techniques to present the changes due to prostate cancer and its treatment. Effective stress management must include relaxation and social support in a supportive environment. Such interventions decrease treatment-related symptoms, reduce the physiological accompani‐ ments of stress, and improve mood. Patients who participate in such rehabilitation interven‐

monitoring are some of the treatment options for patients with prostate cancer [3].

are significant in this population [5].

prostate cancer [6–8].

in home care, palliative, and hospice care settings [6].

228 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

Conventionally, function is the most important indicator of activity and is strongly associated with physical performance and interrelated areas such as range of motion, muscular strength, and endurance [11]. The more contemporary function is a perspective that encompasses individual's physical conditions, emotional and psychological states, and the environmental and social circumstances of the individual [12]. The World Health Organization's International Classification of Functioning, Disability and Health (ICF) describes a framework that focuses this multidimensional or biopsychosocial approach for a deeper understanding of function [11, 12]. Within the ICF framework, function is defined as the interactions between an indi‐ vidual, their health conditions, and the social and personal situations in which they thrive [13]. The complex interactions between these variables determine function and disability. In the context of prostate cancer, morbidity associated with the disease and its treatments can lead to functional problems or impairments in physiological, psychological, or behavioral attributes (body functions and structures), potentially leading to limitations in the ability to execute desired tasks (activity) and participation in social demands (participation) [14]. A variety of approaches and a framework for cancer rehabilitation are based on the ICF to diagnosis and treatment of function for prostate cancer survivors [15].

The overall aim in rehabilitation of all cancer types is to overcome all symptoms causing functional difficulties and increase QOL [16]. De Lisa mentioned the importance of maintaining QOL at a high level; therefore, rehabilitation should not only focus on improving function and prognosis [17]. In general, cancer rehabilitation goals are classified as restorative, supportive, palliative, and preventive according to progression and the nature of cancer. *Restorative care* aims at maximal recovery of residual function of the patients. *Supportive* efforts seek to increase ability in daily life and mobility using effective methods such as decrease in functional difficulties and compensate for permanent deficits. In this stage, rehabilitation also aims to prevent disuse for secondary problems such as contractures and loss of muscle strength. *Palliative treatment* aims to reduce or eliminate symptoms such as pain and dyspnea. In the terminal stage of the patient, physical, psychological, and social high QOL as well as wishes of the patient are important, and positioning, heat modalities, low-frequency therapy, breathing– relaxation exercises, or assistive devices can be used. The primary goal of *preventive rehabilitation* is to prevent impairments. Rehabilitation in this stage must include preoperative education, maintenance of strength, and range of motion after treatment. This rehabilitation process starts right after diagnosis [18]. This framework can guide a therapist in all types of cancer.


**Table 1.** Impairment of functional health in prostate cancer.


strength. *Palliative treatment* aims to reduce or eliminate symptoms such as pain and dyspnea. In the terminal stage of the patient, physical, psychological, and social high QOL as well as wishes of the patient are important, and positioning, heat modalities, low-frequency therapy, breathing– relaxation exercises, or assistive devices can be used. The primary goal of *preventive rehabilitation* is to prevent impairments. Rehabilitation in this stage must include preoperative education, maintenance of strength, and range of motion after treatment. This rehabilitation process starts right after diagnosis [18]. This framework can guide a therapist in all types of

Direct operation sequences (wound healing, lymphocele, urinary retention, urinary)

Sequelae cytostatic chemotherapy (polyneuropathy), myelosuppression

Restriction in the field of transportation (incontinence, bone pain, edema)

Limitation of mobility and participation in cultural life (incontinence)

Problems in integrating into the social environment

**Table 1.** Impairment of functional health in prostate cancer.

cancer.

*Somatic*

Urinary incontinence

*Psychosocial*

 Depression Relapse fears Sleep disorders Partnership problems Fatigue syndrome

**Activities**

Social withdrawal

**Participation**

Problems of coping

Post-traumatic stress disorder

Reduction in exercise capacity

Problems with the reintegration

**Body functions and body structures**

Post-therapeutic pain syndromes

Sexual dysfunction (erectile dysfunction)

 Radiation effects (cystitis, proctitis, lymphedema) Treatment-related hormone deficiency symptoms

230 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments


**Table 2.** Rehabilitation goals and the evaluation instruments mostly used for patients with prostate cancer.

In patients with prostate cancer, fatigue, urinary incontinence, sexual dysfunction, impaired physical performance, psychological distress, weight gain, and changes in male body image are stated as the long-term sequelae of disease. Therefore, while considering general rehabilitation framework, special attention has to be given and specific methods must be used while making a treatment plan for patients with prostate cancer.

The evaluation for rehabilitation program of patients with prostate cancer for determining the individual rehabilitation needs can be identified after completion of the primary treatment of prostate cancer to verify the success of rehabilitation intervention [19]. During the follow-up treatment in patients who are taken directly from the acute care settings, the results of the current status of malignancy and PSA levels must be recorded in addition to the results of special rehabilitation evaluation tests, instruments, or interviews on the day of admission to check the rehabilitation capacity of patients with prostate cancer. Patients must be evaluated by functional and goal-oriented evaluation instruments in the somatic, psychosocial, and vocational rehabilitation and participation in daily living activities and public life with contextual factor areas; thus, the therapist can obtain a top-down view of these patients [20]. **Table 1** shows the impairment areas of functional health in prostate cancer which must be analyzed by rehabilitation therapists.

Evaluation tests for assessing body structure and functions, activity, and participation con‐ ducted at the beginning and end of rehabilitation to assess the level of achieving success in terms of the rehabilitation goals. **Table 2** shows the rehabilitation goals and the evaluation instruments which are mostly used by the therapists for patients with prostate cancer.

Cancer rehabilitation must include different therapies from different specialists to improve muscle strength and cardiopulmonary endurance, preserve energy for daily living activities, decrease stress, and especially decrease the effects of prostate cancer and its treatments. Physical therapy and occupational therapy specialists may be involved in the care of prostate cancer patients from the beginning of treatment to the end of a patient's life. They provide evidence-based interventions during inpatient care, outpatient follow-up, education, and services in home care and hospice care settings.

#### **3. Physical therapy**

**General rehabilitation goals Evaluation instruments**

232 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

Reduction of Progression Fear of Progression Questionnaire

Breakdown of family and partnership problems Interview, Couples Climate Scales

Promoting disease management,

emotional stabilization

states and relaxation

dysfunction)

perspectives

improving self-awareness and self-acceptance,

Assistance in transition and when dealing with stroke-related disabilities (incontinence, erectile

Construction of meaning and objective

Treatment goals in the social sphere and Preparation of reintegration, possibly initiating

Obtaining self-sufficiency, financial management and participation in social life and counseling and assistance

for reintegration, placement of self-help groups

Learning to continence training and transfer in

professional promotions

activities of daily living

Reduction of risk behavior

(smoking alcohol abuse, overwork)

meaning of health promotion

Positive influence of eating habits within the

Reduction of insomnia SF, Diary

Coping with stress and anxiety depressive

(LOTCA)

Interview

Loewenstein Occupational Therapy Cognitive Assessment

EORTC, SF-36, Functional Assessment of Cancer Therapy (G:

"Stress thermometer", Hospital Anxiety and Depression Scale (HADS-D), Beck Depression Inventory (BDI), Beck Anxiety

CIO Community Integration Questionnaire, ISSI (Interview

Miktions protocol, Diary, Barthel Index (BI), Instrumental

Diet Protocol, Body Mass Index (BMI), Bioelectrical Impedance

Life Habits Assessment (LIFE-H), Questionnaires

Reintegration to Normal Living Index, Instrumental Activities of

General, F: Fatigue, P: Prostate-FACT),

Inventory (BAI), Visual Analog Scale (VAS)

QLQ-C30, prostate module

Schedule For Social Interaction)

Activities of Daily Living Scale (IADL)

Daily Living Scale (IADL)

Analysis (BIA)

In patients with prostate cancer, fatigue, urinary incontinence, sexual dysfunction, impaired physical performance, psychological distress, weight gain, and changes in male body image are stated as the long-term sequelae of disease. Therefore, while considering general rehabilitation framework, special attention has to be given and specific methods must be used

Vocational rehabilitation Worker Role Interview, Valpar Component Work Samples (VCWS), COPM

**Table 2.** Rehabilitation goals and the evaluation instruments mostly used for patients with prostate cancer.

while making a treatment plan for patients with prostate cancer.

Herein, it is important to remember that rehabilitation programs are driven for men with prostate cancer. Therefore, physical therapists should also be aware of the factors threatening men's health. These factors are stated as obesity, overweight, and bad habits (smoking and drug or alcohol abuse). Epidemics in many diseases are directly related to smoking, poor diet, excess alcohol consumption, and sedentary lifestyles [21]. For a man with prostate cancer and life-threatening habits, the rehabilitation program must also include preparation of a healthy life plan for him as well as his partner. Smoking and other substance abuse should be avoided and such patients can be referred to psychotherapy or cognitive behavioral therapy to redesign their lifestyle. A coordinated plan of rehabilitation team aiming at a healthy diet and lifestyle can lead to good recovery after cancer diagnosis. The main role of physical therapy is to inhibit sedentary behavior and maintain adequate exercise for patients with prostate cancer.

#### **3.1. Muscle strength and loss of bone mineral density**

Prostate cancer and its treatments can cause inactivity and disuse syndrome which must be avoided, while fitness and active lifestyles should be encouraged [16]. It is important to preserve and restore function through exercise as exercise has several, evident positive effects on patients with cancer. Graded exercise has been suggested as a treatment strategy for cancerrelated fatigue that has the strongest evidence [22]. Aerobic exercise has been found to have effects on not only fatigue but also psychological well-being, QOL, physical performance, and weight control [16, 22, 23]. Improvements in the sense of personal worth, self-esteem, selfimage, and confidence have also been stated as good results of exercise. Therefore, exercise improves the positive mood of people and decreases negative moods such as depression and anxiety [16, 22–24]. Some studies also stated the reduced risk of disease recurrence [22–24].

It is stated that aerobic exercise is helpful if it is given in low to moderate intensity (50–70 heart rate%), starting from 15 minutes to 30 minutes duration 3–5 times a week in a progressive way. The current exercise guidelines indicate that cancer survivors should achieve 150-min aerobic exercise per week and resistance (strength) training twice weekly [24, 25]. Most importantly, exercise needs to become a habit. Patients can be encouraged to start exercise with a short duration (15 min a day, several times a week) and then shape the pattern. Electronic monitoring bracelets can be helpful while following this pattern [21]. The patient can also control himself via this bracelet.

Berglund offered a physical training program for men with prostate cancer for an hour lasting for 7 weeks. This training started with light physical training, breathing exercises, and relaxation, and then included exercises of the pelvic floor [26]. The participants stated the benefit of this exercise program. The physical therapist should prepare a patient-specific aerobic exercise plan. Strengthening and endurance exercises should be performed in addition to aerobic training to improve participation in the activities of daily living [16]. Pelvic floor exercises must be added to physical therapy program for prostate cancer patients. These exercises will be mentioned during the management of urinary incontinence.

#### **3.2. Incontinence**

Urinary incontinence is common in patients with prostate cancer who underwent surgery or radiation therapy. Stress incontinence characterized with loss of urine with a cough, sneeze, or laugh is the most common type of urine leakage after prostate surgery, while the need to frequently urinate with episodes of leakage is the most common type seen after radiation therapy [27]. The treatments are as follows:

**a.** *Pelvic floor exercises*: Pelvic floor muscle training was found to be evident in speeding the recovery of continence [27, 28]. Recovery of normal urinary control after surgery normally takes 1 or 2 years. Pelvic-floor reeducation should be used for treating incontinence effectively [16]. First, it is important to train men to control their ability to hold in their urine. For this purpose, men are instructed on the identification and function of the pelvic floor muscles. Training men prior to prostate surgery can help men use their muscles more actively following surgery [27]. Kegel exercises are taught to men to strengthen the pelvic floor muscles. These exercises consist of repeated, high-intensity contractions of the muscles. Similar to providing training, introducing the exercises before surgery is also very beneficial. If possible, the patient is advised to start exercises before the medical treatment. Pelvic floor exercises can be combined with biofeedback programs. In a study, the authors showed positive results on incontinence after a single session of biofeedbackassisted behavioral training. The use of biofeedback may improve a man's ability to isolate the pelvic floor muscle and differentiate between muscle contraction and relaxation [29, 30].


#### **3.3. Fatigue management**

**3.1. Muscle strength and loss of bone mineral density**

234 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

via this bracelet.

**3.2. Incontinence**

therapy [27]. The treatments are as follows:

Prostate cancer and its treatments can cause inactivity and disuse syndrome which must be avoided, while fitness and active lifestyles should be encouraged [16]. It is important to preserve and restore function through exercise as exercise has several, evident positive effects on patients with cancer. Graded exercise has been suggested as a treatment strategy for cancerrelated fatigue that has the strongest evidence [22]. Aerobic exercise has been found to have effects on not only fatigue but also psychological well-being, QOL, physical performance, and weight control [16, 22, 23]. Improvements in the sense of personal worth, self-esteem, selfimage, and confidence have also been stated as good results of exercise. Therefore, exercise improves the positive mood of people and decreases negative moods such as depression and anxiety [16, 22–24]. Some studies also stated the reduced risk of disease recurrence [22–24].

It is stated that aerobic exercise is helpful if it is given in low to moderate intensity (50–70 heart rate%), starting from 15 minutes to 30 minutes duration 3–5 times a week in a progressive way. The current exercise guidelines indicate that cancer survivors should achieve 150-min aerobic exercise per week and resistance (strength) training twice weekly [24, 25]. Most importantly, exercise needs to become a habit. Patients can be encouraged to start exercise with a short duration (15 min a day, several times a week) and then shape the pattern. Electronic monitoring bracelets can be helpful while following this pattern [21]. The patient can also control himself

Berglund offered a physical training program for men with prostate cancer for an hour lasting for 7 weeks. This training started with light physical training, breathing exercises, and relaxation, and then included exercises of the pelvic floor [26]. The participants stated the benefit of this exercise program. The physical therapist should prepare a patient-specific aerobic exercise plan. Strengthening and endurance exercises should be performed in addition to aerobic training to improve participation in the activities of daily living [16]. Pelvic floor exercises must be added to physical therapy program for prostate cancer patients. These

Urinary incontinence is common in patients with prostate cancer who underwent surgery or radiation therapy. Stress incontinence characterized with loss of urine with a cough, sneeze, or laugh is the most common type of urine leakage after prostate surgery, while the need to frequently urinate with episodes of leakage is the most common type seen after radiation

**a.** *Pelvic floor exercises*: Pelvic floor muscle training was found to be evident in speeding the recovery of continence [27, 28]. Recovery of normal urinary control after surgery normally takes 1 or 2 years. Pelvic-floor reeducation should be used for treating incontinence effectively [16]. First, it is important to train men to control their ability to hold in their urine. For this purpose, men are instructed on the identification and function of the pelvic floor muscles. Training men prior to prostate surgery can help men use their muscles more actively following surgery [27]. Kegel exercises are taught to men to strengthen the pelvic

exercises will be mentioned during the management of urinary incontinence.

Most cancer patients experience fatigue and loss of energy. This severe and activity-limiting symptom is also common among patients with prostate cancer. Fatigue is mostly related with cancer treatment [16, 31]; however, it may also be present after or before treatment due to cancer [32, 33]. From our experience, in the presence of fatigue, patients, their relatives, and even some professionals suggest to rest and slow down activities. During the day time, many patients sleep a lot and cannot sleep well at night. Prolonged rest and inactivity indu‐ ces muscular catabolism and the time of being fatigue increases [16]. Therefore, cancer pa‐ tients suffering from primary fatigue should not be advised to increase the amount of daily rest. As we have mentioned earlier, exercise has a positive effect on fatigue. Therefore, pa‐ tients should not be advised to rest more but carry out aerobic exercise [34]. It is supported that an 8-week cardiovascular exercise program in patients with localized prostate cancer undergoing radiotherapy improved the overall QOL and helped prevent fatigue [31].

Occupational therapists follow other strategies in terms of physical, psychological, cognitive, and social dimensions of fatigue. Graded activity and diversional should be planned in the manner of giving exercise [33]. Other interventions to reduce the degree of fatigue are stress management, nutritional management, and energy conservation techniques [16]. During energy conservation, patients should be taught to spread out activities through the use of timetables, organize activities to the energy level required, ensure breaks during activities, and use adaptive devices [33]. Providing good rest/sleep patterns, teaching structured sleep is also an important role of a therapist [16]. The patient should be recommended to maintain a schedule of sleeping and waking times, avoid sleeping constantly during day time, open curtains in the morning, and avoid doing things that can affect night sleep [33]. Therapists should remember that fatigue is an important symptom of cancer and help their patients to manage this symptom.

#### **3.4. Lymphedema management**

Lymphedema can be observed in patients with prostate cancer as a result of radiation damage or following the removal of lymph nodes during surgery. It is characterized with the collection of fluids in the lower extremities, and compression therapy helps the fluid to move and reduce swelling which can help the patient move easily and comfortably [34, 35].

Both occupational and physical therapists may decide the kind of compression therapy and the effective manual techniques for patients with prostate cancer. Elevation, exercise, and using custom-fitted compression wear can help increase the lymph flow in the early stages of lymphedema. Compression wears are worn continuously throughout the day and removed at night. They are reapplied as soon as the patient awakens in the early morning. Additionally; to drain the lymph from the extremity pneumatic pump compression which provides sequen‐ tial, active compression can be used in the home [34, 35]. For severe edema, compression bandaging after manual lymphatic drainage using light massage (complete decongestive therapy) can be effective. Manual massage can help collateral lymph vessels to milk the lymphedema. To determine the effectiveness of the treatment, the size of the extremity always must be monitored by the therapist [36–38].

#### **3.5. Peripheral neuropathy**

Peripheral neuropathy is one of the side effects of chemotherapy. It is characterized with tingling, burning, or shooting pain sensation of hands and feet depending on nerve damage. Patients with prostate cancer may also experience loss of sensation which can cause problems on somatosensory perception, finger movements and grasping problems, balance problems, tripping, and/or decreased reflexes. Physical and occupational therapy can help the patients with prostate cancer to improve coordination, balance and gait, fine motor skills, and dexterity. The primary aim of treating peripheral neuropathy is to decrease the risk of falling and injuries [39].

#### **3.6. Scar tissue management**

Radiation therapy can cause scar tissue which may increase pain and decrease the flexibility of the skin. Physical therapists can use manual therapy and tissue techniques for stretching and tissue and nerve mobilization to decrease pain and increase the tissue mobility of the patients with prostate cancer [39].

#### **3.7. Early ambulation**

If patients underwent surgery, early postoperative ambulation and improving physical functions is the main goal of physical therapy [22]. During chemotherapy, physical strength tends to diminish; hence, rehabilitation aims to encourage ambulation consistent with the patient's condition even during chemotherapy and prevent disuse syndrome and maintain physical and muscle strength by performing early ambulation [24, 26].

#### **4. Occupational therapy**

schedule of sleeping and waking times, avoid sleeping constantly during day time, open curtains in the morning, and avoid doing things that can affect night sleep [33]. Therapists should remember that fatigue is an important symptom of cancer and help their patients to

Lymphedema can be observed in patients with prostate cancer as a result of radiation damage or following the removal of lymph nodes during surgery. It is characterized with the collection of fluids in the lower extremities, and compression therapy helps the fluid to move and reduce

Both occupational and physical therapists may decide the kind of compression therapy and the effective manual techniques for patients with prostate cancer. Elevation, exercise, and using custom-fitted compression wear can help increase the lymph flow in the early stages of lymphedema. Compression wears are worn continuously throughout the day and removed at night. They are reapplied as soon as the patient awakens in the early morning. Additionally; to drain the lymph from the extremity pneumatic pump compression which provides sequen‐ tial, active compression can be used in the home [34, 35]. For severe edema, compression bandaging after manual lymphatic drainage using light massage (complete decongestive therapy) can be effective. Manual massage can help collateral lymph vessels to milk the lymphedema. To determine the effectiveness of the treatment, the size of the extremity always

Peripheral neuropathy is one of the side effects of chemotherapy. It is characterized with tingling, burning, or shooting pain sensation of hands and feet depending on nerve damage. Patients with prostate cancer may also experience loss of sensation which can cause problems on somatosensory perception, finger movements and grasping problems, balance problems, tripping, and/or decreased reflexes. Physical and occupational therapy can help the patients with prostate cancer to improve coordination, balance and gait, fine motor skills, and dexterity. The primary aim of treating peripheral neuropathy is to decrease the risk of falling and injuries

Radiation therapy can cause scar tissue which may increase pain and decrease the flexibility of the skin. Physical therapists can use manual therapy and tissue techniques for stretching and tissue and nerve mobilization to decrease pain and increase the tissue mobility of the

If patients underwent surgery, early postoperative ambulation and improving physical functions is the main goal of physical therapy [22]. During chemotherapy, physical strength

swelling which can help the patient move easily and comfortably [34, 35].

manage this symptom.

**3.4. Lymphedema management**

236 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

must be monitored by the therapist [36–38].

**3.5. Peripheral neuropathy**

**3.6. Scar tissue management**

patients with prostate cancer [39].

**3.7. Early ambulation**

[39].

Occupational therapy (OT) offers a client-centered approach to patients with prostate cancer. OT clinical reasoning assessments and interventions focus on functioning and participation by rehabilitating the abilities of the patients with prostate cancer. Therapists guide goaldirected activities that give meaning to the patient's life [40]. According to the ICF, the affected performance areas of prostate cancer on which OT focuses are shown in **Table 3**.


**Table 3.** Affected performance areas and occupational therapy focus in patients with prostate cancer.

As shown in **Table 3**, OT mainly focuses on activity and participation limitations in the rehabilitation phase. Patients with prostate cancer generally require activity education, sensory training breathing and relaxation education, stress management education, sensory stimuli and praxis skills, cognitive therapy, erectile dysfunction and sexual rehabilitation, cognitive therapy, vocational rehabilitation, patient education and counseling, and also rehabilitation during palliative care and supportive care to engage in activities independently.

#### **4.1. Daily living activities education**

Reasons of limitation of activity are both dysmotility and muscle weakness. Activity limita‐ tion interventions are important for improving the activity performance of patients with cancer. The occupational therapist describes and measures activity performance necessitated to be carried out by patients with prostate cancer. After the activity for individual needs is identified, intervention strategies may be determined. The practitioner must determine the appropriate intervention approach for each patient. These strategies are divided into four parts: restoration, compensation, environmental modification, and education of patient [41].

#### *4.1.1. Restoration*

Patients have many different activities related to their roles. It is important to determine the most important activities for their life. The focus of the restorative approach is to develop patient skills and abilities or restore the activity performance of the patient with prostate cancer. A restorative approach is planned specifically to the situation of the patient. In this stage, grading of the activity level can be done. Grading can be done according to the following parameters:


#### *4.1.2. Compensation*

The compensation approach focuses on using the patients' skills to achieve the highest possible stage of functioning in the activities. Therapists may teach the patients with prostate cancer new methods for modifying task performance to compensate for deficient areas of occupation, performance, and individual factors. If the patients still require help for participation in new activities, the occupational therapist should also give some advice regarding the use of adapted techniques or equipment. Patients with prostate cancer may need to use some assisted technology devices such as activity facilitator or computer-aided software to perform the activities. These instruments can help decrease symptoms (i.e., fatigue and pain) of cancer and increase participation in the activities of patients.

#### *4.1.3. Environmental modification*

*4.1.1. Restoration*

parameters:

all of the tasks instead of the patient.

238 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

comfortable during activity education.

familiar to unfamiliar environment.

*4.1.2. Compensation*

Patients have many different activities related to their roles. It is important to determine the most important activities for their life. The focus of the restorative approach is to develop patient skills and abilities or restore the activity performance of the patient with prostate cancer. A restorative approach is planned specifically to the situation of the patient. In this stage, grading of the activity level can be done. Grading can be done according to the following

**•** *Physical assistance*: If the patient with prostate cancer is in need of help, practitioner or caregiver can provide assistance. In this way, patients' skill to complete task may be increased. The presence of some symptoms such as fatigue or pain can cause the patient with prostate cancer to take physical support. This support does not mean that caregiver do

**•** *Supervision and cuing*: This involves a number and types of cues. For example, if the patient forgets some tasks of activity due to cognitive impairment, patient can be supported by verbal, tactile, and written material cues to help the patient with cancer finalize the activity. **•** *Activity demands*: The activity can be changed due to complexity of performance skills. Generally, for patients with prostate cancer, it will be better to select an activity with lowmotor and high-cognitive demands by the reason of symptoms. In activity education, the

**•** *Sequencing of activity*: Activity is divided in order of priority and sequence. The number of steps in tasks and the total number of steps can be purposed to increase. Thus, patient with

**•** *Type of activity*: During activity education, activities can be graded from familiar to unfami‐ liar or from former to new. This method can help the patient with prostate cancer feel more

**•** *Environment*: Activity environment can affect participation in the activities. For instance, patients with prostate cancer may have urinary incontinence. Therefore, patients may need to use toilet frequently. This situation can affect men in a negative way. To avoid negative symptoms such as stress or unhappiness, patients may limit themselves to only familiar environments to find a toilet. In intervention programs, the activity can be graded from a

The compensation approach focuses on using the patients' skills to achieve the highest possible stage of functioning in the activities. Therapists may teach the patients with prostate cancer new methods for modifying task performance to compensate for deficient areas of occupation, performance, and individual factors. If the patients still require help for participation in new activities, the occupational therapist should also give some advice regarding the use of adapted techniques or equipment. Patients with prostate cancer may need to use some assisted technology devices such as activity facilitator or computer-aided software to perform the

motor and cognitive demands of the activity can be increased step by step.

prostate cancer can complete the activity easily without fatigue or pain.

Environmental modifications consist of compensation, modification, and adaptation strategy. The compensation approach directly influences patient functioning. However, environmental modification approach influences patients' functioning indirectly. Patients with prostate cancer will need help for home or work environment. Occupational therapists should give advice to redesign the home or work environment of the patient with prostate cancer where the patients can participate in activity easier than before. Modifications can include low-cost and easily accessible strategies to improve participation in domestic and community activities. Patients can also have problems in a social environment. They might not want to participate in social activities owing to general reluctance to do any activities. Besides, they may be exposed to stigma and pity from other people. For these reasons, occupational therapists must consider both physical and social aspects. Daily living activity education needs to be holistic and must integrate the activity, environment, and the patient with prostate cancer. Using occupation-based activity, education improves participation and supports wellness and QOL of patients with prostate cancer.

#### **4.2. Sensory training**

Treatment modalities such as surgery (e.g., radical prostatectomy), androgen deprivation therapy, radiation therapy, and chemotherapy affect the sensory–neural ways in body. After treatment, some deficiencies can occur in sensory skills. In particular, body composition may be affected if patients receive ADT. The generally observed side effects such as fatigue and pain might negatively affect patient body. Literature evidence demonstrates that sensory training is an important part of intervention program in patients with prostate cancer [42]. The purpose of sensory training is to develop body image by increasing body awareness. The body awareness includes these trainings and sensory stimuli, breathing, and relaxation techniques.

#### **4.3. Breathing and relaxation education**

Cancer and its treatment can be stressful for patients with prostate cancer and their partners and caregivers. Relaxation techniques and other body/mind practices can help calm the patient's mind, reduce stress, and sharpen the ability to focus to maintain inner peace. Some patients with prostate cancer use these techniques to help themselves relax while they wait for the results of treatments or tests. Breathing techniques include slow inhalation and exhalation to reduce tension in the shoulders, trunk, and abdomen. The process begins with focusing on normal breathing in a quiet and comfortable place when the patients feel stressful. Patients should perform deep inhalation and slow exhalation. During this phase, the abdominal muscles should be relaxed during inhalation; the abdominal muscles should be contracted during exhalation [41]. Relaxation techniques involve teaching the patient with prostate cancer to cope with stress which results in deficiency of body composition. During relaxation education, the patient with prostate cancer is instructed to contract and relax his major skeletal muscles systematically and then asked to repeat phases silently and finally asked to use purposeful images to achieve the goals [41]. OT practitioners have a core role in providing therapeutic activities that enable the patients with sensory problems to develop body imagi‐ nation in occupational performance.

#### **4.4. Stress management**

Occupational therapists help patients with prostate cancer to acknowledge, express, accept, and use problem-solving techniques to address the changes that result from prostate cancer and its treatments. Effective stress management can include relaxation training, education, a supportive environment, social support, and participation in daily living activities. It is supported that these interventions can help decrease the treatment-related symptoms, the physiological accompaniments of stress, and improve mood of the patient with prostate cancer. Patients who participate in such rehabilitation programs are shown to control and experience reduced interpersonal conflicts and distress related to cancer-related intrusive thinking and have improved mental health [5].

#### **4.5. Sensory stimuli**

The patients involve in many activities which include various sensory stimuli. The basic activities involve tactile and proprioceptive inputs. Occupational therapists impart sensory training to patients with prostate cancer and also suggest somatosensoriel perception activities that involve tactile and proprioceptive inputs especially after chemotherapy or hormonal therapy. Patients use these senses during routines of activity in daily life. In addition, mirror activities and visual perception skills must be added to intervention programs to promote sensorial perception, harmony with the environment, and the body imagination of patients with prostate cancer [43–45].

#### **4.6. Cognitive therapy**

Cognitive therapy approach was generally used in patients with mental health problems. However, patients with prostate cancer may have some deficiencies in cognitive skills owing to cancer and its treatments [16]. OT intervention should focus on cognitive skills and activity function, and it includes orientation, memory, attention, motor planning, and executive functions of the patient with prostate cancer [44, 45].

*Orientation* is the ability to understand the self and the relationship between self and past– present environment time. After receiving the cancer treatments, patients might have orien‐ tation problems about place or time. In the intervention, verbal and external cues are used as reminders and therapy advances by changing numbers and types of cues [46]. *Memory* is described in terms of sensory memory, working or immediate memory, and long-term memory. If there are any problems on these types of memory, patients' tasks may be affected. Occupational therapists evaluate and improve memory abilities. They may also advice to use verbal, visual, and external cues for activity independently [46]. *Attention* is a multidimension that includes five components: sustained attention (concentration), selective attention, divided attention, alternating attention, and shifting of attention. Patients may have problems pertain‐ ing to not only each component of attention but also several components of attention [46]. The occupational therapist provides examples of basic and complex tasks for each component of attention. *Motor planning or praxis* is the ability of individuals to point at how to get their body to do what they want it to do. Motor planning involves the cognitive skills of intending to move, selecting a goal, planning the movement, and anticipating the end result. Impaired motor planning is disabling in cancer patients so that they may not be able to initiate or follow through on tasks [46]. The occupational therapist may give information about the functional properties of an activity. Besides, they might inform about conceptual errors related to creating the idea of the movement, involving object usage information, and sequencing of activity. When evaluating or treating patients with prostate cancer for motor planning abilities, it is important to identify the patients' activities. A series of cues (visual, verbal, or tactile) may be used if the patient experiences difficulty in performing an activity demand in the OT [46]. *Executive functions* consist of organization, problem-solving, and coping skills. It may signifi‐ cantly influence performance of activities of daily living. Occupational therapist using a dynamic interactional approach to the intervention of executive functions and organizational, problem- solving, and coping skills would focus on self-awareness and ability to perform new, unexpected or routine tasks [46]. In conclusion, cognitive impairments can be seen, caused either by cancer or its treatments in patients with prostate cancer. Hence, cognitive skills of prostate cancer patients should not be ignored.

#### **4.7. Erectile dysfunction and sexual rehabilitation**

muscles systematically and then asked to repeat phases silently and finally asked to use purposeful images to achieve the goals [41]. OT practitioners have a core role in providing therapeutic activities that enable the patients with sensory problems to develop body imagi‐

Occupational therapists help patients with prostate cancer to acknowledge, express, accept, and use problem-solving techniques to address the changes that result from prostate cancer and its treatments. Effective stress management can include relaxation training, education, a supportive environment, social support, and participation in daily living activities. It is supported that these interventions can help decrease the treatment-related symptoms, the physiological accompaniments of stress, and improve mood of the patient with prostate cancer. Patients who participate in such rehabilitation programs are shown to control and experience reduced interpersonal conflicts and distress related to cancer-related intrusive thinking and

The patients involve in many activities which include various sensory stimuli. The basic activities involve tactile and proprioceptive inputs. Occupational therapists impart sensory training to patients with prostate cancer and also suggest somatosensoriel perception activities that involve tactile and proprioceptive inputs especially after chemotherapy or hormonal therapy. Patients use these senses during routines of activity in daily life. In addition, mirror activities and visual perception skills must be added to intervention programs to promote sensorial perception, harmony with the environment, and the body imagination of patients

Cognitive therapy approach was generally used in patients with mental health problems. However, patients with prostate cancer may have some deficiencies in cognitive skills owing to cancer and its treatments [16]. OT intervention should focus on cognitive skills and activity function, and it includes orientation, memory, attention, motor planning, and executive

*Orientation* is the ability to understand the self and the relationship between self and past– present environment time. After receiving the cancer treatments, patients might have orien‐ tation problems about place or time. In the intervention, verbal and external cues are used as reminders and therapy advances by changing numbers and types of cues [46]. *Memory* is described in terms of sensory memory, working or immediate memory, and long-term memory. If there are any problems on these types of memory, patients' tasks may be affected. Occupational therapists evaluate and improve memory abilities. They may also advice to use verbal, visual, and external cues for activity independently [46]. *Attention* is a multidimension that includes five components: sustained attention (concentration), selective attention, divided

nation in occupational performance.

240 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

have improved mental health [5].

with prostate cancer [43–45].

functions of the patient with prostate cancer [44, 45].

**4.6. Cognitive therapy**

**4.5. Sensory stimuli**

**4.4. Stress management**

Rehabilitation approach to erectile dysfunction is focused on pelvic floor muscle training and the muscle strength at the base of the penis. After the initial examination and determining an intervention plan, the physical therapist may guide the patient to perform specific pelvic floor muscle exercises and indirectly related muscles such as abdominal and gluteal muscles. These exercises help increase oxygen supply to the tissues. Vacuum therapy can also be used to generate negative pressure that increases the blood flow to the penis [47].

Sexual rehabilitation is one of the most important components of rehabilitation of patients with prostate cancer and significantly related to quality of life. Men with prostate cancer are more stressed about sexual dysfunction if they are younger. Both younger and older men are in need of physical, social, emotional, and psychological treatment assistance for this issue [28].

**Figure 1.** Safe and less fatigue sex positions for patients with prostate cancer.

Sexuality is an intimate issue and occupational therapy practitioners can examine both societal attitudes toward patients with prostate cancer and their own beliefs, values, and attitudes about sexuality. Patients can be emotionally vulnerable and recessive. The occupational therapists may provide information about the sexual rehabilitation. Rehabilitation should consists of an interdisciplinary team including nurses, physiotherapists, occupational thera‐ pists, social workers, sexologists, dieticians, massage therapists and psychologists. Die Perink et al. had conducted a rehabilitation program, with a 4-day course developed based on the experience with rehabilitation of more than 7000 cancer survivors, included physical activity, pelvic floor exercises, couples massage and relaxation, diet, and education of sexuality. They advise to practice sexual rehabilitation about sexual dysfunction [28]. Occupational therapists may give physically advice not only patients, but also partners regarding favorable sex positions (**Figure 1**). These positions can be more comfortable and safe for men. Thus, the occurrence of symptoms such as fatigue and pain will be reduced with occupational therapy intervention. Patients with prostate cancer will thus have normal sexual function. The patients and their partners should be informed about social support. It has many dimensions, including emotional, material assistance, and information. The occupational therapist should give lifestyle advice to patients for applying to their daily life. Thus, the patients and their partners will be improved.

#### **4.8. Vocational Rehabilitation**

Long survival with good quality of life make the patient with prostate cancer to think about returning to work after prostate cancer treatment and also 6 months after the radical prosta‐ tectomy surgery, men can return to their work. This may be a big positive step for men, and men might look forward to re-establishing his usual routine and it is understandable if they feel anxious or worried. But from a view of occupational therapy, having a return-to-work plan can help the patient to make the transition easier. Most of the mentally and physically healthy prostate cancer survivors do not require a job change, while others need some adjustments such as reduced working hours, modified duties, trying to do similar jobs, making self to do lists with time use with fatigue management, or the use of assistive technology. An occupational therapist can help the man determine if he is ready to go back to work, identify accommodations that will help him do his job, and help him get training or seek new employ‐ ment if needed. In addition, improving self-management skills of prostate cancer survival helps him identify his needs and borders which can help him prepare for independent daily life and social reinteraction [48].

#### **4.9. Patient education and counseling**

Patient, partner, family, and caregiver education are an important part of occupational therapy because nearly all of the approaches (restoration, compensation, environmental modification, etc.) involve learning new strategies and combining these strategies into persons' lifestyles. Education contains information about prostate cancer; symptoms create and raise awareness about management skills. At the same time, an occupational therapist can use various materials such as demonstration, written format, pictures, and videotapes to help the patient and family participate in their activities. In addition to general education, it is supported that the main education resource of the patient with prostate cancer is internet but this way may not be helpful for psychosocial healing of the patient with prostate cancer or the survivor. Hence, in recent studies, new education programs were designed such as "Between men," which offers group online therapy sessions and education. The aims of these programs were to give the patients all the available information about prostate cancer, treatment, side effects, and how to deal with the side effects. Program planned once a week for 7 weeks and it included patients' experiences and reactions, patients' communication difficulties especially sexual and emotional effects, prostate cancer disease and treatments, incontinence, sexuality, importance, and problem solving. Online education programs must be improved and generalized for patients and survivors with prostate cancer [5, 49].

#### **5. Rehabilitation in palliative care and hospice care**

In palliative and hospice care, both physical and occupational therapists support men with prostate cancer by minimizing the secondary symptoms related to cancer and its treatments. The role of the occupational therapist and physical therapist in palliative and hospice care is quite similar and important.

At the end of life, physical therapy offers functional training, therapeutic exercise, and soft tissue mobilization. The goals of physical therapy are to improve overall strength, range of motion, and endurance of the patient with prostate cancer. Physical therapists may use heat, cold, and TENS (transcutaneous electrical nerve stimulation) for pain relief and design exercises that improve endurance and positioning regimens that help the patient maintain functional range of motion [50, 51].

In this stage, occupational therapists identify the roles and activities which are meaningful to the patient with prostate cancer and try to present the barriers that limit their performance. Occupational therapists support the patient both for physical and psychosocial/behavioral health requirements and pay close attention to what is most important for the patient. They look at the available activity and environmental resources to increase patient participation. The main goal of occupational therapy is to improve the quality of life according to patients' values and maximize residual functional abilities [50].

#### **6. Conclusion**

about sexuality. Patients can be emotionally vulnerable and recessive. The occupational therapists may provide information about the sexual rehabilitation. Rehabilitation should consists of an interdisciplinary team including nurses, physiotherapists, occupational thera‐ pists, social workers, sexologists, dieticians, massage therapists and psychologists. Die Perink et al. had conducted a rehabilitation program, with a 4-day course developed based on the experience with rehabilitation of more than 7000 cancer survivors, included physical activity, pelvic floor exercises, couples massage and relaxation, diet, and education of sexuality. They advise to practice sexual rehabilitation about sexual dysfunction [28]. Occupational therapists may give physically advice not only patients, but also partners regarding favorable sex positions (**Figure 1**). These positions can be more comfortable and safe for men. Thus, the occurrence of symptoms such as fatigue and pain will be reduced with occupational therapy intervention. Patients with prostate cancer will thus have normal sexual function. The patients and their partners should be informed about social support. It has many dimensions, including emotional, material assistance, and information. The occupational therapist should give lifestyle advice to patients for applying to their daily life. Thus, the patients and their partners

Long survival with good quality of life make the patient with prostate cancer to think about returning to work after prostate cancer treatment and also 6 months after the radical prosta‐ tectomy surgery, men can return to their work. This may be a big positive step for men, and men might look forward to re-establishing his usual routine and it is understandable if they feel anxious or worried. But from a view of occupational therapy, having a return-to-work plan can help the patient to make the transition easier. Most of the mentally and physically healthy prostate cancer survivors do not require a job change, while others need some adjustments such as reduced working hours, modified duties, trying to do similar jobs, making self to do lists with time use with fatigue management, or the use of assistive technology. An occupational therapist can help the man determine if he is ready to go back to work, identify accommodations that will help him do his job, and help him get training or seek new employ‐ ment if needed. In addition, improving self-management skills of prostate cancer survival helps him identify his needs and borders which can help him prepare for independent daily

Patient, partner, family, and caregiver education are an important part of occupational therapy because nearly all of the approaches (restoration, compensation, environmental modification, etc.) involve learning new strategies and combining these strategies into persons' lifestyles. Education contains information about prostate cancer; symptoms create and raise awareness about management skills. At the same time, an occupational therapist can use various materials such as demonstration, written format, pictures, and videotapes to help the patient and family participate in their activities. In addition to general education, it is supported that the main education resource of the patient with prostate cancer is internet but this way may not be

will be improved.

**4.8. Vocational Rehabilitation**

242 Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

life and social reinteraction [48].

**4.9. Patient education and counseling**

Patients with prostate cancer can face problems about body structure and functions, activity, and participation which may limit their participation to life. Patients with prostate cancer require skilled rehabilitation and supportive care from the initial process of diagnosis through clinical reasoning and treatment to posttreatment periods. Qualified interdisciplinary reha‐ bilitation interventions may help men regain their performance and independency and maintain the highest quality of life.

#### **Author details**

Meral Huri\* , Burcu Semin Akel and Sedef Şahin

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

Hacettepe University, Faculty of Health Sciences, Department of Occupational Therapy, Ankara, Turkey

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### *Edited by Ravinder Mohan*

Prostate cancer is diagnosed commonly in the course of general health screening. Very often it is difficult to tell whether the cancer will be aggressive and lethal or will be so slow growing that it will not affect the patient's survival. Treatment can have a major impact on the patient's health-related quality of life. This book presents cutting-edge research in predicting the behavior of localized and metastatic cancer, targeted treatment of cancer that has spread minimally, response to newer methods of treatment, and rehabilitation of patients.

Photo by HeitiPaves / iStock

Prostate Cancer - Leading-edge Diagnostic Procedures and Treatments

Prostate Cancer

Leading-edge Diagnostic Procedures

and Treatments

*Edited by Ravinder Mohan*