Glenn J. Bubley

Mutation of the Androgen-Receptor Gene in Metastatic Androgen-Independent Prostate Cancer

BACKGROUND Metastatic prostate cancer is a leading cause of cancer-related death in men. The rate of response to androgen ablation is high, but most patients relapse as a result of the outgrowth of androgen-independent tumor cells. The androgen receptor, which binds testosterone and stimulates the transcription of androgen-responsive genes, regulates the growth of prostate cells. We analyzed the androgen-receptor genes from samples of metastatic androgen-independent prostate cancers to determine whether mutations in the gene have a role in androgen independence. METHODS Complementary DNA was synthesized from metastatic prostate cancers in 10 patients with androgen-independent prostate cancer, and the expression of the androgen-receptor gene was estimated by amplification with the polymerase chain reaction. Exons B through H of the gene were cloned, and mutations were identified by DNA sequencing. The functional effects of the mutations were assessed in cells transfected with mutant genes. RESULTS All androgen-independent tumors expressed high levels of androgen-receptor gene transcripts, relative to the levels expressed by an androgen-independent prostate-cancer cell line (LNCaP). Point mutations in the androgen-receptor gene were identified in metastatic cells from 5 of the 10 patients examined. One mutation was in the same codon as the mutation found previously in the androgen-independent prostate-cancer cell line. The mutations were not detected in the primary tumors from of the two patients. Functional studies of two of the mutant androgen receptors demonstrated that they could be activated by progesterone and estrogen. CONCLUSIONS Most metastatic androgen-independent prostate cancers express high levels of androgen-receptor gene transcripts. Mutations in androgen-receptor genes are not uncommon and may provide a selective growth advantage after androgen ablation.

Eligibility and Response Guidelines for Phase II Clinical Trials in Androgen-Independent Prostate Cancer: Recommendations From the Prostate-Specific Antigen Working Group

PURPOSE Prostate-specific antigen (PSA) is a glycoprotein that is found almost exclusively in normal and neoplastic prostate cells. For patients with metastatic disease, changes in PSA will often antedate changes in bone scan. Furthermore, many but not all investigators have observed an association between a decline in PSA levels of 50% or greater and survival. Since the majority of phase II clinical trials for patients with androgen-independent prostate cancer (AIPC) have used PSA as a marker, we believed it was important for investigators to agree on definitions and values for a minimum set of parameters for eligibility and PSA declines and to develop a common approach to outcome analysis and reporting. We held a consensus conference with 26 leading investigators in the field of AIPC to define these parameters. RESULT We defined four patient groups: (1) progressive measurable disease, (2) progressive bone metastasis, (3) stable metastases and a rising PSA, and (4) rising PSA and no other evidence of metastatic disease. The purpose of determining the number of patients whose PSA level drops in a phase II trial of AIPC is to guide the selection of agents for further testing and phase III trials. We propose that investigators report at a minimum a PSA decline of at least 50% and this must be confirmed by a second PSA value 4 or more weeks later. Patients may not demonstrate clinical or radiographic evidence of disease progression during this time period. Some investigators may want to report additional measures of PSA changes (ie, 75% decline, 90% decline). Response duration and the time to PSA progression may also be important clinical end point. CONCLUSION Through this consensus conference, we believe we have developed practical guidelines for using PSA as a measurement of outcome. Furthermore, the use of common standards is important as we determine which agents should progress to randomized trials which will use survival as an end point.

Increased Expression of Genes Converting Adrenal Androgens to Testosterone in Androgen-Independent Prostate Cancer

Androgen receptor (AR) plays a central role in prostate cancer, and most patients respond to androgen deprivation therapies, but they invariably relapse with a more aggressive prostate cancer that has been termed hormone refractory or androgen independent. To identify proteins that mediate this tumor progression, gene expression in 33 androgen-independent prostate cancer bone marrow metastases versus 22 laser capture-microdissected primary prostate cancers was compared using Affymetrix oligonucleotide microarrays. Multiple genes associated with aggressive behavior were increased in the androgen-independent metastatic tumors (MMP9, CKS2, LRRC15, WNT5A, EZH2, E2F3, SDC1, SKP2, and BIRC5), whereas a candidate tumor suppressor gene (KLF6) was decreased. Consistent with castrate androgen levels, androgen-regulated genes were reduced 2- to 3-fold in the androgen-independent tumors. Nonetheless, they were still major transcripts in these tumors, indicating that there was partial reactivation of AR transcriptional activity. This was associated with increased expression of AR (5.8-fold) and multiple genes mediating androgen metabolism (HSD3B2, AKR1C3, SRD5A1, AKR1C2, AKR1C1, and UGT2B15). The increase in aldo-keto reductase family 1, member C3 (AKR1C3), the prostatic enzyme that reduces adrenal androstenedione to testosterone, was confirmed by real-time reverse transcription-PCR and immunohistochemistry. These results indicate that enhanced intracellular conversion of adrenal androgens to testosterone and dihydrotestosterone is a mechanism by which prostate cancer cells adapt to androgen deprivation and suggest new therapeutic targets.

Biology of Prostate-Specific Antigen

Prostate-specific antigen (PSA) is an androgen-regulated serine protease produced by both prostate epithelial cells and prostate cancer (PCa) and is the most commonly used serum marker for cancer. It is a member of the tissue kallikrein family, some of the members of which are also prostate specific. PSA is a major protein in semen, where its function is to cleave semenogelins in the seminal coagulum. PSA is secreted into prostatic ducts as an inactive 244-amino acid proenzyme (proPSA) that is activated by cleavage of seven N-terminal amino acids. PSA that enters the circulation intact is rapidly bound by protease inhibitors, primarily alpha1-antichymotrypsin, although a fraction is inactivated in the lumen by proteolysis and circulates as free PSA. This proteolytic inactivation, as well as the cleavage of proPSA to PSA, is less efficient in PCa. Serum total PSA levels are increased in PCa, and PSA screening has dramatically altered PCa presentation and management. Unfortunately, although high PSA levels are predictive of advanced PCa, a large fraction of organ-confined cancers present with much lower total PSA values that overlap those levels found in men without PCa. Measurement of free versus total PSA can increase specificity for PCa, and tests under development to measure forms of proPSA may further enhance the ability to detect early-stage PCa. PSA is also widely used to monitor responses to therapy and is under investigation as a therapeutic target. Finally, recent data indicate that there may be additional roles for PSA in the pathogenesis of PCa.

Phase II Multicenter Study of Abiraterone Acetate Plus Prednisone Therapy in Patients With Docetaxel-Treated Castration-Resistant Prostate Cancer

PURPOSE Persistence of ligand-mediated androgen receptor signaling has been documented in castration-resistant prostate cancers (CRPCs). Abiraterone acetate (AA) is a potent and selective inhibitor of CYP17, which is required for androgen biosynthesis in the testes, adrenal glands, and prostate tissue. This trial evaluated the efficacy and safety of AA in combination with prednisone to reduce the symptoms of secondary hyperaldosteronism that can occur with AA monotherapy. PATIENTS AND METHODS Fifty-eight men with progressive metastatic CRPC who experienced treatment failure with docetaxel-based chemotherapy received AA (1,000 mg daily) with prednisone (5 mg twice daily). Twenty-seven (47%) patients had received prior ketoconazole. The primary outcome was > or = 50% prostate-specific antigen (PSA) decline, with objective response by Response Evaluation Criteria in Solid Tumors (RECIST) criteria, and changes in Eastern Cooperative Oncology Group (ECOG) performance status (PS) and circulating tumor cell (CTC) numbers. Safety was also evaluated. RESULTS A > or = 50% decline in PSA was confirmed in 22 (36%) patients, including 14 (45%) of 31 ketoconazole-naïve and seven (26%) of 27 ketoconazole-pretreated patients. Partial responses were seen in four (18%) of 22 patients with RECIST-evaluable target lesions. Improved ECOG PS was seen in 28% of patients. Median time to PSA progression was 169 days (95% CI, 82 to 200 days). CTC conversions with treatment from > or = 5 to < 5 were noted in 10 (34%) of 29 patients. The majority of AA-related adverse events were grade 1 to 2, and no AA-related grade 4 events were seen. CONCLUSION AA plus prednisone was well tolerated, with encouraging antitumor activity in heavily pretreated CRPC patients. The incidence of mineralocorticoid-related toxicities (hypertension or hypokalemia) was reduced by adding low-dose prednisone. The combination of AA plus prednisone is recommended for phase III investigations.

Loss of IFN-gamma production by invariant NK T cells in advanced cancer.

Invariant NK T cells express certain NK cell receptors and an invariant TCRα chain specific for the MHC class I-like CD1d protein. These invariant NK T cells can regulate diverse immune responses in mice, including antitumor responses, through mechanisms including rapid production of IL-4 and IFN-γ, but their physiological functions remain uncertain. Invariant NK T cells were markedly decreased in peripheral blood from advanced prostate cancer patients, and their ex vivo expansion with a CD1d-presented lipid Ag (α-galactosylceramide) was diminished compared with healthy donors. Invariant NK T cells from healthy donors produced high levels of both IFN-γ and IL-4. In contrast, whereas invariant NK T cells from prostate cancer patients also produced IL-4, they had diminished IFN-γ production and a striking decrease in their IFN-γ:IL-4 ratio. The IFN-γ deficit was specific to the invariant NK T cells, as bulk T cells from prostate cancer patients produced normal levels of IFN-γ and IL-4. These findings support an immunoregulatory function for invariant NK T cells in humans mediated by differential production of Th1 vs Th2 cytokines. They further indicate that antitumor responses may be suppressed by the marked Th2 bias of invariant NK T cells in advanced cancer patients.

Intratumoral De Novo Steroid Synthesis Activates Androgen Receptor in Castration Resistant Prostate Cancer and is Upregulated by Treatment with CYP17A1 Inhibitors

Relapse of castration-resistant prostate cancer (CRPC) that occurs after androgen deprivation therapy of primary prostate cancer can be mediated by reactivation of the androgen receptor (AR). One important mechanism mediating this AR reactivation is intratumoral conversion of the weak adrenal androgens DHEA and androstenedione into the AR ligands testosterone and dihydrotestosterone. DHEA and androstenedione are synthesized by the adrenals through the sequential actions of the cytochrome P450 enzymes CYP11A1 and CYP17A1, so that CYP17A1 inhibitors such as abiraterone are effective therapies for CRPC. However, the significance of intratumoral CYP17A1 and de novo androgen synthesis from cholesterol in CRPC, and the mechanisms contributing to CYP17A1 inhibitor resistance/relapse, remain to be determined. We report that AR activity in castration-resistant VCaP tumor xenografts can be restored through CYP17A1-dependent de novo androgen synthesis, and that abiraterone treatment of these xenografts imposes selective pressure for increased intratumoral expression of CYP17A1, thereby generating a mechanism for development of resistance to CYP17A1 inhibitors. Supporting the clinical relevance of this mechanism, we found that intratumoral expression of CYP17A1 was markedly increased in tumor biopsies from CRPC patients after CYP17A1 inhibitor therapy. We further show that CRPC cells expressing a progesterone responsive T877A mutant AR are not CYP17A1 dependent, but that AR activity in these cells is still steroid dependent and mediated by upstream CYP11A1-dependent intraturmoral pregnenolone/progesterone synthesis. Together, our results indicate that CRPCs resistant to CYP17A1 inhibition may remain steroid dependent and therefore responsive to therapies that can further suppress de novo intratumoral steroid synthesis.

Hormonal Predictors of Prostate Cancer: A Meta-Analysis

PURPOSE Although there is strong circumstantial evidence that androgens are implicated in the etiology of prostate cancer, epidemiologic investigations have failed to demonstrate consistently that one or more steroid hormones are implicated. In contrast, recent epidemiologic studies unequivocally link serum insulin-like growth factor 1 (IGF-1) levels with risk for prostate cancer. METHODS We have performed the first meta-analysis of all previously published studies on hormonal predictors of risk for prostate cancer. RESULTS A meta-analysis restricted to studies that performed mutual adjustment for all measured serum hormones, age, and body mass index indicated that men whose total testosterone is in the highest quartile are 2.34 times more likely to develop prostate cancer (95% confidence interval, 1.30 to 4.20). In contrast, levels of dihydrotestosterone and estradiol do not seem to play a role of equal importance. The only study that provides multivariably adjusted sex hormone-binding globulin data indicates that this binding protein is inversely related to prostate cancer risk (odds ratio, 0.46; 95% confidence interval, 0.24 to 0.89). Finally, all three studies that examined the role of serum IGF-1 have consistently demonstrated a positive and significant association with prostate cancer risk that is similar in magnitude to that of testosterone. CONCLUSION Men with either serum testosterone or IGF-1 levels in upper quartile of the population distribution have an approximately two-fold higher risk for developing prostate cancer.

High dose bicalutamide for androgen independent prostate cancer: Effect of prior hormonal therapy

PURPOSE A pilot study of the antiandrogen bicalutamide at 150 mg. a day for androgen independent prostate cancer was performed. This study was based on the possibility that androgen independent cases might display responses to additional hormonal agents. MATERIALS AND METHODS The study included 31 androgen independent cases with an increasing prostate specific antigen (PSA) and progressive disease. PSA measurements were used as the primary method of assessing response. However, PSA decline was also correlated with clinical status. RESULTS Seven patients demonstrated PSA declines of greater than 50% for 2 months or more, for an overall response rate of 22.5%. Responses were observed almost exclusively in patients treated with long-term flutamide as part of a complete androgen blockade regimen (43% response rate) in contrast to patients treated with androgen deprivation without flutamide (6% response rate). Of the 7 PSA responding patients bicalutamide resulted in a significant improvement in performance status and a decrease in analgesic requirement in 4 and 3 remained asymptomatic. Bicalutamide at 150 mg. a day was well tolerated, with the most frequent side effect being mild exacerbation of hot flashes. CONCLUSIONS Bicalutamide at this dose is modestly effective for some patients with androgen independent prostate cancer, particularly for those previously treated with long-term flutamide. This study indicates that previous antiandrogen therapy alters the response to subsequent hormonal agents.

Phase II Study of Abiraterone Acetate in Chemotherapy-Naive Metastatic Castration-Resistant Prostate Cancer Displaying Bone Flare Discordant with Serologic Response

Purpose: Abiraterone is an oral inhibitor of CYP17, which is essential for androgen biosynthesis. This multicenter study assessed its efficacy in patients with castration-resistant prostate cancer (CRPC), without prior chemotherapy or CYP17-targeted therapy, and frequency of bone scans discordant with prostate-specific antigen (PSA) and clinical response. Experimental Design: Thirty-three patients received abiraterone acetate 1,000 mg daily with prednisone 5 mg twice daily in continuous 28-day cycles. Patients were evaluated monthly for efficacy and safety. Bone scan flare was defined as the combination, after 3 months of therapy, of an interpreting radiologists report indicating “disease progression” in context of a 50% or more decline in PSA level, with scan improvement or stability 3 months later. Results: A 50% or more decline in PSA level at week 12 was confirmed in 22 of 33 (67%) patients. Declines in PSA level of 50% or more were seen in 26 of 33 (79%) patients. Undetectable PSA levels (≤0.1 ng/mL) occurred in 2 patients. Median time on therapy and time to PSA progression were 63 weeks and 16.3 months, respectively. Twenty-three patients were evaluable for bone scan flare. Progression was indicated in radiologists report in 12 of 23 (52%), and 11 of 12 subsequently showed improvement or stability. As prospectively defined, bone scan flare was observed in 11 of 23 (48%) evaluable patients or 11 of 33 (33%) enrolled patients. Adverse events were typically grade 1/2 and consistent with prior published abiraterone reports. Conclusion: Clinical responses to abiraterone plus prednisone were frequent and durable in men with metastatic CRPC. Further investigation is needed to clarify the confounding effect of bone scan flare on patient management and interpretation of results. Clin Cancer Res; 17(14); 4854–61. ©2011 AACR.