**1. Introduction**

362 Prostate Cancer – Diagnostic and Therapeutic Advances

[21] Merrick GS, Butler WM, Galbreath RW, et al. Erectile function after permanent prostate

[22] Asterling A and Greene DR. Prospective evaluation of sexual function in patients

receiving cryotherapy as a primary radical treatment for localized prostate cancer.

brachy therapy. *Int J Radiat Oncol Biol Phys* 2002; 52: 893-902.

*BJU int* 2008; 103: 788-92.

Prostate cancer is among the most common types of malignancies and causes of cancerrelated deaths in men worldwide (Fitzpatrick, *et al*., 2009). Primary tumor involvement outside the prostatic capsule or relapse following radical prostatectomy results generally in incurability (Lassi & Dawson, 2009). Androgen deprivation therapy has progressed since attempts in 1941, when surgical castration had been shown to improve outcomes for the first time. Palliative treatment consists of hormonal manipulation to deprive the cancer cells of androgenic stimulation by orchidectomy or use of LHRH analogs and steroidal or nonsteroidal antiandrogens (Kollmeier & Zelefsky, 2008). Although continuous androgen suppression therapy (CAS) has been a cornerstone of the management of prostate cancer for more than 50 years, controversy remains regarding its optimum application. Generally, androgen suppression (AS) is performed as continuous treatment, resulting in apoptotic regression of the tumor cells in a high percentage of cases. However, surgical or medical castration results in median progression-free survival of only 2–3 years, with no other effective treatment left (Mellado, *et al*., 2009). Responses to cytotoxic therapy are low and only recently several studies revealed a possible benefit of incorporating chemotherapeutic agents in treatment regimen for prostate cancer (Chang & Kibel, 2009). Taxanes like docetaxel and cabazitaxel, therapeutic cancer vaccines and newly developed agents targeting androgen receptor signaling are expected to improve therapy (Madan *et al*., 2011).

Following experimental research using animal models, intermittent androgen suppression (IAS) was introduced as new clinical concept, assuming that during limited regrowth in the treatment cessation periods tumorigenic cells are residing in an androgen-responsive state (Goldenberg, *et al*., 1995). Since induction of androgen independence may occur early after treatment initiation, cessation of antiandrogen therapy prior to this switch is expected to maintain the apoptotic potential of the tumor cells and keep them sensitive to retreatment. Providing an easy method for selection of the type of treatment and early assessment of tumor growth during the off-periods serial serum PSA determinations made IAS feasible. In detail, IAS consists of an initial androgen suppression period of up to nine months combining LHRH antagonists and antiandrogens, which is followed by treatment cessation

Intermittent Androgen Suppression Therapy

PC

heterogeneous hormone-insensitive cells.

refractory tumor occurs.

androgen-independent during the fifth cycle (figure 2).

Androgen suppression

Treatment cessation

Fig. 2. Experimental animal model of IAS using androgen-dependent Shionogi tumor cells. Tumor cells are androgen-depleted by castration and, following tumor shrinkage to a defined degree, treatment cessation re-establishes a hormone-sensitive tumor. This cycle can

The average duration of one cycle was 30 days and progression to androgen-insensitivity was observed after 150 days. After castration, the concentration of testosterone in the tumor was demonstrated to decline more rapidly than the dihydrotestosterone levels and spontaneous recurrent growth was not accompanied by significant elevation of the wholetissue concentration of either androgen. These results suggested that a recurrent tumor may contain hormone-sensitive cells, which are capable of resuming growth in an androgendepleted environment. The data also imply that progression from the androgen-dependent to the androgen-autonomous condition involves the selection and outgrowth of

The effects of castration on gene expression were measured in the androgen-dependent Shionogi mouse tumor model (Rennie, *et al.,* 1988). During the first 48-72 h after castration, the tumor continued to increase its mass, but began to regress at 72-144 h. In the surviving cells there were no major decreases in RNA synthesis. Under these conditions, selected genes become overexpressed and, in particular, the concentration of the transcripts encoding testosterone-repressed prostate message-2 (TRPM-2/clusterin) was enhanced only when tumor regression was most evident, i.e. 72-144 h after castration. The TRPM-2 (clusterin) gene also became expressed constitutively in non-regressing tumors after the first and subsequent cycles of androgen withdrawal. TRPM-2 is a membrane-stabilizing protein that appears to be involved in limiting the autophagic lysis of epithelial cells during

be repeated four times until androgen-insensitive cells constitute a population of approximately 50% of all tumor cells in the fifth cycle and progression to a hormone-

IAS cycle

for Prostate Cancer Patients: A Choice for Improved Quality of Life? 365

potential. To determine the effect of intermittent application of androgens on the androgendependent Shionogi carcinoma, the tumor was transplanted into a succession of male mice, each of which was castrated when the estimated tumor weight became about 3g. After the tumor had regressed to 30% of the original weight, it was transplanted into the next noncastrated male (Akakura, *et al*., 1993). This cycle of transplantation and castrationinduced regression was successfully repeated four times before tumor growth became

Androgen-independent

Androgen-dependent

Tumor progression

until a certain PSA threshold is reached; then, androgen suppression is reinitiated until a maximal effect is observed again. In initial pilot trials regrowing tumors of patients undergoing IAS were consistently reported to be sensitive over several cycles of androgen withdrawal (figure 1).

Fig. 1. Schematic model of CAS and IAS. CAS treatment of prostate cancer rapidly results in appearance of androgen-insensitive cells and tumor progression within 2–3 years in advanced stages. On the contrary, IAS preserves the hormone sensitivity of the cells through cycling between androgen suppression and treatment cessation phases in order to prolong the time to hormone refractoriness and possibly survival.

Therefore, the primary goal of IAS has been the prolongation of the hormone-sensitivity of the tumors, which in turn has been expected to result in increased survival eventually. Based on the available evidence, IAS nowadays represents a valid treatment option for patients with nonmetastatic prostate cancer, including those with locally advanced disease, either with or without lymph node involvement, and those who relapsed following apparently curative treatment. IAS has been researched since the mid-1980s in a number of clinical phase II and III trials in an effort to prolong hormone-dependency and reduce adverse effects and costs of CAS (Goldenberg, et al., 1995; Bales, et al., 1996; Buchan & Goldenberg, 2010, da Silva, 2011). With preclinical evidence suggesting a potential benefit of IAS in terms of time to androgen independence, with phase II and phase III studies producing optimistic results and with the potential for decreased costs and complications, IAS has now become a popular modality of therapy worldwide.

#### **2. Experimental development of intermittent androgen suppression**

The concept of IAS was experimentally developed using the androgen-dependent Shionogi mouse mammary tumor, investigating regular phases of growth, regression and recurrence of xenograft tumors during serial transplantation (Bruchovsky, *et al*., 1985). Since postcastrational progression of tumors towards an androgen-independent state appears to be linked to the cessation of androgen-induced differentiation of tumorigenic stem cells, it was hypothesized that the replacement of androgens at the end of apoptotic regression might result in the reappearance of differentiated tumor cells that maintain their apoptotic

until a certain PSA threshold is reached; then, androgen suppression is reinitiated until a maximal effect is observed again. In initial pilot trials regrowing tumors of patients undergoing IAS were consistently reported to be sensitive over several cycles of androgen

> **IAS CAS**

**024**

appearance of androgen-insensitive cells and tumor progression within 2–3 years in

Fig. 1. Schematic model of CAS and IAS. CAS treatment of prostate cancer rapidly results in

advanced stages. On the contrary, IAS preserves the hormone sensitivity of the cells through cycling between androgen suppression and treatment cessation phases in order to prolong

Therefore, the primary goal of IAS has been the prolongation of the hormone-sensitivity of the tumors, which in turn has been expected to result in increased survival eventually. Based on the available evidence, IAS nowadays represents a valid treatment option for patients with nonmetastatic prostate cancer, including those with locally advanced disease, either with or without lymph node involvement, and those who relapsed following apparently curative treatment. IAS has been researched since the mid-1980s in a number of clinical phase II and III trials in an effort to prolong hormone-dependency and reduce adverse effects and costs of CAS (Goldenberg, et al., 1995; Bales, et al., 1996; Buchan & Goldenberg, 2010, da Silva, 2011). With preclinical evidence suggesting a potential benefit of IAS in terms of time to androgen independence, with phase II and phase III studies producing optimistic results and with the potential for decreased costs and complications,

**Years**

withdrawal (figure 1).

**0**

the time to hormone refractoriness and possibly survival.

IAS has now become a popular modality of therapy worldwide.

**2. Experimental development of intermittent androgen suppression** 

The concept of IAS was experimentally developed using the androgen-dependent Shionogi mouse mammary tumor, investigating regular phases of growth, regression and recurrence of xenograft tumors during serial transplantation (Bruchovsky, *et al*., 1985). Since postcastrational progression of tumors towards an androgen-independent state appears to be linked to the cessation of androgen-induced differentiation of tumorigenic stem cells, it was hypothesized that the replacement of androgens at the end of apoptotic regression might result in the reappearance of differentiated tumor cells that maintain their apoptotic

**50**

**100**

**Tumor size**

 **(%)** **150**

**200**

potential. To determine the effect of intermittent application of androgens on the androgendependent Shionogi carcinoma, the tumor was transplanted into a succession of male mice, each of which was castrated when the estimated tumor weight became about 3g. After the tumor had regressed to 30% of the original weight, it was transplanted into the next noncastrated male (Akakura, *et al*., 1993). This cycle of transplantation and castrationinduced regression was successfully repeated four times before tumor growth became androgen-independent during the fifth cycle (figure 2).

Fig. 2. Experimental animal model of IAS using androgen-dependent Shionogi tumor cells. Tumor cells are androgen-depleted by castration and, following tumor shrinkage to a defined degree, treatment cessation re-establishes a hormone-sensitive tumor. This cycle can be repeated four times until androgen-insensitive cells constitute a population of approximately 50% of all tumor cells in the fifth cycle and progression to a hormonerefractory tumor occurs.

The average duration of one cycle was 30 days and progression to androgen-insensitivity was observed after 150 days. After castration, the concentration of testosterone in the tumor was demonstrated to decline more rapidly than the dihydrotestosterone levels and spontaneous recurrent growth was not accompanied by significant elevation of the wholetissue concentration of either androgen. These results suggested that a recurrent tumor may contain hormone-sensitive cells, which are capable of resuming growth in an androgendepleted environment. The data also imply that progression from the androgen-dependent to the androgen-autonomous condition involves the selection and outgrowth of heterogeneous hormone-insensitive cells.

The effects of castration on gene expression were measured in the androgen-dependent Shionogi mouse tumor model (Rennie, *et al.,* 1988). During the first 48-72 h after castration, the tumor continued to increase its mass, but began to regress at 72-144 h. In the surviving cells there were no major decreases in RNA synthesis. Under these conditions, selected genes become overexpressed and, in particular, the concentration of the transcripts encoding testosterone-repressed prostate message-2 (TRPM-2/clusterin) was enhanced only when tumor regression was most evident, i.e. 72-144 h after castration. The TRPM-2 (clusterin) gene also became expressed constitutively in non-regressing tumors after the first and subsequent cycles of androgen withdrawal. TRPM-2 is a membrane-stabilizing protein that appears to be involved in limiting the autophagic lysis of epithelial cells during

Intermittent Androgen Suppression Therapy

therapy or clinical trials of new agents.

dependency compared to continuous androgen suppression.

**3. Clinical development of intermittent androgen suppression** 

for Prostate Cancer Patients: A Choice for Improved Quality of Life? 367

The next step included the switch to a human prostate cancer xenograft model using the LNCaP androgen-dependent prostate cell line, where serum PSA levels correlated well with tumor volume and decreased rapidly after castration, followed by appearance of androgenindependency after 3-4 weeks (Gleave, *et al*., 1996). IAS-treated mice were implanted with testosterone pellets two weeks after castration and were subjected to cycles of testosterone replacement for 1 week and withdrawal for 2 weeks until serum PSA levels returned to baseline no longer. IAS therapy prolonged time to androgen-independent PSA production 3-fold, from an average of 26 days in the CAS group to 77 days in the IAS group. It was concluded that IAS in the LNCaP model delayed the onset of androgen-independent PSA gene regulation markedly most likely due to androgen-induced differentiation and/or downregulation of androgen-suppressed gene expression. In summary, the animal experimental data indicated that androgen-dependent tumor xenografts can be subjected to several cycles of androgen withdrawal/replacement and revealed prolonged hormone-

Since the introduction of PSA screening in the late 1980s, more prostate cancers have been detected, and at an earlier stage (Gjertson & Albertsen, 2011). Consequently, the majority of prostate cancers are now detected years before the emergence of clinically evident disease, which usually represents locally advanced or metastatic cancer. PSA screening has remained controversial, because many of the prostate cancers detected are low grade and slow growing and will not need aggressive therapy. Prostate cancer is biologically and clinically a heterogeneous malignancy and its imaging evaluation will need to be tailored to the specific phases of the disease in a patient-specific, risk-adapted manner (Jadvar, 2011). With this long natural history and a median survival without treatment that often approaches at least 15-20 years, many patients will die rather with than of prostate cancer. Approximately onethird of patients who undergo radical prostatectomy will develop a detectable PSA level within 10 years (Tzou, *et al*., 2011). Biochemical relapse is defined as a rising PSA level in the absence of clinical or radiographic evidence of tumor. Management of PSA recurrence is controversial, as prostate cancer may take an indolent course, or it may develop aggressively into metastatic disease. The only potentially curative treatment for biochemical failure after prostatectomy is radiotherapy and the other treatment options include hormone

Research on hormonal treatment of prostate cancer over the past 20 years has focused on maximizing androgen ablation through combination therapy. This increases treatmentrelated side-effects and expenses and fails to prolong time to progression to androgenindependence (Gleave, *et al*., 1998, Kollmeier & Zelefsky, 2008 ). Preliminary evidence indicates that a low androgen milieu is associated with tumor aggressiveness. Transition to androgen-independence is a complex process and involves both selection and outgrowth of preexisting androgen-resistant clones as well as adaptative upregulation of genes that enable cancer cells to survive and grow after CAS (Corona, et al., 2011). CAS in men with prostate cancer increases the risk of osteoporotic fractures, type 2 diabetes and, possibly, cardiovascular events (Grossmann, *et al*., 2011). The benefits of CAS in treating nonmetastatic prostate cancer need to be carefully weighed against the risks of CAS-induced adverse events. Management of the metabolic sequelae of CAS includes optimal reduction

apoptosis and is possibly preserving the nuclear environment, suppressing the lethal effect of anti-androgenic treatment (Akakura, *et al*., 1993). Therefore, tumor progression, characterized by the loss of the apoptotic potential, appears to be linked in part to the inappropriate activation of the TRPM-2 gene.

Since postcastrational progression of tumors to an androgen-independent state appears to be linked to the cessation of androgen-induced differentiation of tumorigenic stem cells, the replacement of androgens at the end of a period of apoptotic regression might result in the regeneration of differentiated tumor cells with maintained apoptotic potential. (Akakura, *et al*., 1993). The frequency of androgen-dependent and -independent tumorigenic stem cells in parent and recurrent Shionogi tumors was determined with help of an *in vivo* limiting dilution test (Rennie, *et al*., 1990). When assayed in male hosts a marked enrichment of stem cells in the recurrent tumors (1/200 tumor cells) relative to the parent tumors (1/4000 tumor cells) was detectable. By measuring tumor takes in female mice, a 500-fold increase in androgen-independent stem cells was found in the recurrent carcinoma. No enrichment of androgen-independent stem cells was evident in regressing parent tumors. This finding implies that the androgen-independent cells that survived androgen-withdrawal may result from the ability of a small number of initially androgen-independent stem cells to adapt to an altered hormonal environment. These results again indicated that the tumor mass mainly consisted of differentiated cells and that stem cells are initially androgen-dependent, but the apoptosis-inducing effect of androgen withdrawal will be limited to a factor of 100–1000, before compensatory adaptive mechanisms lead to progression of stem cells to an androgen-independent state. In recurrent tumors the amount of dihydrotestosterone was reduced by approximately 85% in comparison to the parent tumor and expression of nuclear androgen receptor was completely abolished within 24 h after castration (Bruchovsky, *et al.*, 1990). However, later on the amount of androgen receptor mRNA in androgen-dependent and -independent cells derived from the Shionogi carcinoma was similar, showing no relationship to progression (Akakura, *et al.,* 1996). The uncoupling of TRPM-2 expression and apoptosis observed in androgen-independent tumor cells implicates that the function of androgen receptor becomes more restricted with tumor progression. There is evidence that the androgen receptor still plays an important role in progression to the castration-resistant incurable state in prostate cancer. While castration proved to be ineffective in castrationresistant prostate tumors in an animal model, knockdown of androgen receptor was demonstrated to decrease serum PSA, inhibit cancer growth and frequently resulted in tumor regression (Snoek, *et al*., 2009). This study provided evidence that elimination of the androgen receptor might constitute a promising therapeutic strategy for treatment of prostate tumors that had progressed to the castration-resistant state.

Serial determinations of the proportion of stem cells in the Shionogi tumor revealed a constant part during the first three cycles but a 15-fold increase between the third and fourth cycles (Rennie, *et al*., 1994). In the parent androgen-dependent tumor before androgen ablation they formed 0.8% of the total stem cell compartment. After the fourth cycle the androgen-independent stem cell population increased to 47% and a population of similar size was found in the androgen-independent recurrent of the tumor, which was induced by one-time castration. Therefore, it was concluded that independent of intermittent or continuous androgen withdrawal, conversion to hormone-insensitivity occurs as soon as the tumor has accumulated one-third to one-half of the total stem cell compartment with androgen-independent cells.

apoptosis and is possibly preserving the nuclear environment, suppressing the lethal effect of anti-androgenic treatment (Akakura, *et al*., 1993). Therefore, tumor progression, characterized by the loss of the apoptotic potential, appears to be linked in part to the

Since postcastrational progression of tumors to an androgen-independent state appears to be linked to the cessation of androgen-induced differentiation of tumorigenic stem cells, the replacement of androgens at the end of a period of apoptotic regression might result in the regeneration of differentiated tumor cells with maintained apoptotic potential. (Akakura, *et al*., 1993). The frequency of androgen-dependent and -independent tumorigenic stem cells in parent and recurrent Shionogi tumors was determined with help of an *in vivo* limiting dilution test (Rennie, *et al*., 1990). When assayed in male hosts a marked enrichment of stem cells in the recurrent tumors (1/200 tumor cells) relative to the parent tumors (1/4000 tumor cells) was detectable. By measuring tumor takes in female mice, a 500-fold increase in androgen-independent stem cells was found in the recurrent carcinoma. No enrichment of androgen-independent stem cells was evident in regressing parent tumors. This finding implies that the androgen-independent cells that survived androgen-withdrawal may result from the ability of a small number of initially androgen-independent stem cells to adapt to an altered hormonal environment. These results again indicated that the tumor mass mainly consisted of differentiated cells and that stem cells are initially androgen-dependent, but the apoptosis-inducing effect of androgen withdrawal will be limited to a factor of 100–1000, before compensatory adaptive mechanisms lead to progression of stem cells to an androgen-independent state. In recurrent tumors the amount of dihydrotestosterone was reduced by approximately 85% in comparison to the parent tumor and expression of nuclear androgen receptor was completely abolished within 24 h after castration (Bruchovsky, *et al.*, 1990). However, later on the amount of androgen receptor mRNA in androgen-dependent and -independent cells derived from the Shionogi carcinoma was similar, showing no relationship to progression (Akakura, *et al.,* 1996). The uncoupling of TRPM-2 expression and apoptosis observed in androgen-independent tumor cells implicates that the function of androgen receptor becomes more restricted with tumor progression. There is evidence that the androgen receptor still plays an important role in progression to the castration-resistant incurable state in prostate cancer. While castration proved to be ineffective in castrationresistant prostate tumors in an animal model, knockdown of androgen receptor was demonstrated to decrease serum PSA, inhibit cancer growth and frequently resulted in tumor regression (Snoek, *et al*., 2009). This study provided evidence that elimination of the androgen receptor might constitute a promising therapeutic strategy for treatment of

prostate tumors that had progressed to the castration-resistant state.

androgen-independent cells.

Serial determinations of the proportion of stem cells in the Shionogi tumor revealed a constant part during the first three cycles but a 15-fold increase between the third and fourth cycles (Rennie, *et al*., 1994). In the parent androgen-dependent tumor before androgen ablation they formed 0.8% of the total stem cell compartment. After the fourth cycle the androgen-independent stem cell population increased to 47% and a population of similar size was found in the androgen-independent recurrent of the tumor, which was induced by one-time castration. Therefore, it was concluded that independent of intermittent or continuous androgen withdrawal, conversion to hormone-insensitivity occurs as soon as the tumor has accumulated one-third to one-half of the total stem cell compartment with

inappropriate activation of the TRPM-2 gene.

The next step included the switch to a human prostate cancer xenograft model using the LNCaP androgen-dependent prostate cell line, where serum PSA levels correlated well with tumor volume and decreased rapidly after castration, followed by appearance of androgenindependency after 3-4 weeks (Gleave, *et al*., 1996). IAS-treated mice were implanted with testosterone pellets two weeks after castration and were subjected to cycles of testosterone replacement for 1 week and withdrawal for 2 weeks until serum PSA levels returned to baseline no longer. IAS therapy prolonged time to androgen-independent PSA production 3-fold, from an average of 26 days in the CAS group to 77 days in the IAS group. It was concluded that IAS in the LNCaP model delayed the onset of androgen-independent PSA gene regulation markedly most likely due to androgen-induced differentiation and/or downregulation of androgen-suppressed gene expression. In summary, the animal experimental data indicated that androgen-dependent tumor xenografts can be subjected to several cycles of androgen withdrawal/replacement and revealed prolonged hormonedependency compared to continuous androgen suppression.
