Mary Nakazawa

AR-V7 and Resistance to Enzalutamide and Abiraterone in Prostate Cancer

BACKGROUND The androgen-receptor isoform encoded by splice variant 7 lacks the ligand-binding domain, which is the target of enzalutamide and abiraterone, but remains constitutively active as a transcription factor. We hypothesized that detection of androgen-receptor splice variant 7 messenger RNA (AR-V7) in circulating tumor cells from men with advanced prostate cancer would be associated with resistance to enzalutamide and abiraterone. METHODS We used a quantitative reverse-transcriptase-polymerase-chain-reaction assay to evaluate AR-V7 in circulating tumor cells from prospectively enrolled patients with metastatic castration-resistant prostate cancer who were initiating treatment with either enzalutamide or abiraterone. We examined associations between AR-V7 status (positive vs. negative) and prostate-specific antigen (PSA) response rates (the primary end point), freedom from PSA progression (PSA progression-free survival), clinical or radiographic progression-free survival, and overall survival. RESULTS A total of 31 enzalutamide-treated patients and 31 abiraterone-treated patients were enrolled, of whom 39% and 19%, respectively, had detectable AR-V7 in circulating tumor cells. Among men receiving enzalutamide, AR-V7-positive patients had lower PSA response rates than AR-V7-negative patients (0% vs. 53%, P=0.004) and shorter PSA progression-free survival (median, 1.4 months vs. 6.0 months; P<0.001), clinical or radiographic progression-free survival (median, 2.1 months vs. 6.1 months; P<0.001), and overall survival (median, 5.5 months vs. not reached; P=0.002). Similarly, among men receiving abiraterone, AR-V7-positive patients had lower PSA response rates than AR-V7-negative patients (0% vs. 68%, P=0.004) and shorter PSA progression-free survival (median, 1.3 months vs. not reached; P<0.001), clinical or radiographic progression-free survival (median, 2.3 months vs. not reached; P<0.001), and overall survival (median, 10.6 months vs. not reached, P=0.006). The association between AR-V7 detection and therapeutic resistance was maintained after adjustment for expression of full-length androgen receptor messenger RNA. CONCLUSIONS Detection of AR-V7 in circulating tumor cells from patients with castration-resistant prostate cancer may be associated with resistance to enzalutamide and abiraterone. These findings require large-scale prospective validation. (Funded by the Prostate Cancer Foundation and others.).

Androgen Receptor Splice Variant 7 and Efficacy of Taxane Chemotherapy in Patients With Metastatic Castration-Resistant Prostate Cancer

IMPORTANCE We previously showed that detection of androgen receptor splice variant 7 (AR-V7) in circulating tumor cells (CTCs) from men with castration-resistant prostate cancer (CRPC) was associated with primary resistance to enzalutamide and abiraterone therapy, but the relevance of AR-V7 status in the context of chemotherapy is unknown. OBJECTIVE To investigate whether AR-V7-positive patients would retain sensitivity to taxane chemotherapy and whether AR-V7 status would have a differential impact on taxane-treated men compared with enzalutamide- or abiraterone-treated men. DESIGN, SETTING, AND PARTICIPANTS We examined CTCs for AR-V7 mRNA using a reverse-transcription polymerase chain reaction assay. From January 2013 to July 2014, we prospectively enrolled patients with metastatic CRPC initiating taxane chemotherapy (docetaxel or cabazitaxel) at a single academic institution (Johns Hopkins). Our prespecified statistical plan required a sample size of 36 taxane-treated men. MAIN OUTCOMES AND MEASURES We evaluated associations between AR-V7 status and prostate-specific antigen (PSA) response rates, PSA progression-free survival (PSA PFS), and clinical and/or radiographic progression-free survival (PFS). After incorporating updated data from our prior study of 62 patients treated with enzalutamide or abiraterone, we also investigated the interaction between AR-V7 status (positive or negative) and treatment type (taxane vs enzalutamide or abiraterone). RESULTS Of 37 taxane-treated patients enrolled, 17 (46%) had detectable AR-V7 in CTCs. Prostate-specific antigen responses were achieved in both AR-V7-positive and AR-V7-negative men (41% vs 65%; P = .19). Similarly, PSA PFS (hazard ratio [HR], 1.7, 95% CI, 0.6-5.0; P = .32) and PFS (HR, 2.7, 95% CI, 0.8-8.8; P = .11) were comparable in AR-V7-positive and AR-V7-negative patients. A significant interaction was observed between AR-V7 status and treatment type (P < .001). Clinical outcomes were superior with taxanes compared with enzalutamide or abiraterone therapy in AR-V7-positive men, whereas outcomes did not differ by treatment type in AR-V7-negative men. In AR-V7-positive patients, PSA responses were higher in taxane-treated vs enzalutamide- or abiraterone-treated men (41% vs 0%; P < .001), and PSA PFS and PFS were significantly longer in taxane-treated men (HR, 0.19 [95% CI, 0.07-0.52] for PSA PFS, P = .001; HR, 0.21 [95% CI, 0.07-0.59] for PFS, P = .003). CONCLUSIONS AND RELEVANCE Detection of AR-V7 in CTCs from men with metastatic CRPC is not associated with primary resistance to taxane chemotherapy. In AR-V7-positive men, taxanes appear to be more efficacious than enzalutamide or abiraterone therapy, whereas in AR-V7-negative men, taxanes and enzalutamide or abiraterone may have comparable efficacy. Circulating tumor cell-based AR-V7 detection may serve as a treatment selection biomarker in CRPC.

Androgen Receptor Splice Variants in the Era of Enzalutamide and Abiraterone

The FDA approvals of enzalutamide and abiraterone have rapidly changed the clinical landscape of prostate cancer treatment. Both drugs were designed to further suppress androgen receptor (AR) signaling, which is restored following first-line androgen deprivation therapies. Resistance to enzalutamide and abiraterone, however, is again marked by a return of AR signaling, indicating a remarkable “addiction” of prostate cancer cells to the AR pathway. Several mechanisms of castration resistance have been uncovered in the past decades, featuring a wide spectrum of molecular alterations that may explain sustained AR signaling in castration-resistant prostate cancers (CRPC). Among these, the androgen receptor splice variants (AR-Vs), particularly variant 7 (AR-V7), have been implicated in resistance to enzalutamide and abiraterone in preclinical studies, and they cannot be targeted by currently available AR-directed drugs. Drug development for AR-V-associated CRPC may therefore be necessary to augment the preexisting treatment repertoire. In this mini-review, we will discuss general mechanisms of resistance to AR-directed therapies, with a focus on the role of androgen receptor splice variants in the new era of treating advanced prostate cancer with enzalutamide and abiraterone.

Resistance to androgen-pathway drugs in prostate cancer.

To the Editor: Antonarakis and colleagues (Sept. 11 issue) 1 reported a significant association between the presence of androgen-receptor splice variant 7 messenger RNA (AR-V7) in circulating tumor cells and shorter prostate-specific antigen (PSA) progression–free survival, clinical or radiographic progression–free survival, and overall survival in men with metastatic castration-resistant prostate cancer who were treated with enzalutamide or abiraterone. Several treatment options (abiraterone, enzalutamide, sipuleucel-T, and radium-223) have recently been shown to prolong survival in men with metastatic castration-resistant prostate cancer who have not previously been treated with chemotherapy; the identification of reliable biomarkers that can predict response should optimize treatment with these approaches. A study by Loriot and colleagues 2 suggests that the duration of sensitivity to previous androgen-deprivation therapy (ADT) is predictive of the efficacy of subsequent endocrine manipulations in patients with castrationresistant prostate cancer. Loriot et al. found that 58% of patients with a duration of sensitivity to previous therapy of at least 16 months had a PSA response to subsequent treatment, as compared with 18% of patients with a duration of sensitivity of less than 16 months (P = 0.01). Antonarakis et al. observed no PSA response to abiraterone or enzalutamide in men who tested positive for AR-V7. We wonder about the relationship between AR-V7 positivity and the duration of response to previous ADT.To the Editor: Antonarakis and colleagues (Sept. 11 issue)1 reported a significant association between the presence of androgen-receptor splice variant 7 messenger RNA (AR-V7) in circulating tumor cells and shorter prostate-specific antigen (PSA) progression–free survival, clinical or radiographic progression–free survival, and overall survival in men with metastatic castration-resistant prostate cancer who were treated with enzalutamide or abiraterone. Several treatment options (abiraterone, enzalutamide, sipuleucel-T, and radium-223) have recently been shown to prolong survival in men with metastatic castration-resistant prostate cancer who have not previously been treated with chemotherapy; the identification of reliable biomarkers that can predict response should optimize treatment with these approaches. A study by Loriot and colleagues2 suggests that the duration of sensitivity to previous androgen-deprivation therapy (ADT) is predictive of the efficacy of subsequent endocrine manipulations in patients with castrationresistant prostate cancer. Loriot et al. found that 58% of patients with a duration of sensitivity to previous therapy of at least 16 months had a PSA response to subsequent treatment, as compared with 18% of patients with a duration of sensitivity of less than 16 months (P = 0.01). Antonarakis et al. observed no PSA response to abiraterone or enzalutamide in men who tested positive for AR-V7. We wonder about the relationship between AR-V7 positivity and the duration of response to previous ADT.

Detection fidelity of AR mutations in plasma derived cell-free DNA

Somatic genetic alterations including copy number and point mutations in the androgen receptor (AR) are associated with resistance to therapies targeting the androgen/AR axis in patients with metastatic castration resistant prostate cancer (mCRPC). Due to limitations associated with biopsying metastatic lesions, plasma derived cell-free DNA (cfDNA) is increasingly being used as substrate for genetic testing. AR mutations detected by deep next generation sequencing (NGS) of cfDNA from patients with mCRPC have been reported at allelic fractions ranging from over 25% to below 1%. The lower bound threshold for accurate mutation detection by deep sequencing of cfDNA has not been comprehensively determined and may have locus specific variability. Herein, we used NGS for AR mutation discovery in plasma-derived cfDNA from patients with mCRPC and then used droplet digital polymerase chain reaction (ddPCR) for validation. Our findings show the AR (tTC>cTC) F877L hotspot was prone to false positive mutations during NGS. The rate of error at AR (tTC>cTC) F877L during amplification prior to ddPCR was variable among high fidelity polymerases. These results highlight the importance of validating low-abundant mutations detected by NGS and optimizing and controlling for amplification conditions prior to ddPCR.

Abstract 2910: Androgen receptor splice variant-7 predicts resistance to enzalutamide in patients with castration-resistant prostate cancer

Background: Androgen receptor splice variant-7 (AR-V7) is a truncated form of the androgen receptor (AR) protein which lacks the ligand-binding domain, the target of enzalutamide, but remains constitutively active as a transcription factor. Following from preclinical studies implicating AR-V7 as a mechanism of resistance to novel AR-directed therapies, we hypothesized that the presence of AR-V7 in circulating tumor cells (CTCs) from men with advanced prostate cancer would be associated with primary resistance to enzalutamide. Methods: We used quantitative reverse-transcription polymerase-chain-reaction (qRT-PCR) analysis to interrogate CTCs for the presence or absence of AR-V7 from prospectively enrolled patients with metastatic castration-resistant prostate cancer initiating treatment with enzalutamide. We examined associations between AR-V7 status and PSA response rates (the primary clinical endpoint of the study), PSA-progression-free-survival (PSA-PFS), and clinical/radiographic-progression-free-survival (PFS). Multivariable Cox regressions were performed to determine the independent effect of AR-V7 status on clinical outcomes to enzalutamide treatment. A prespecified sample size of 30 patients would yield 85% power to detect a difference in PSA response rates from 10% (in AR-V7-positive men) to 60% (in AR-V7-negative men), using a two-sided α=0.10. Results: Thirty-one (31) enzalutamide-treated patients were enrolled in the study, of which 38.7% (12/31) had detectable AR-V7 mRNA from CTCs. Compared to AR-V7-negative patients, AR-V7-positive men had worse PSA response rates (0% [0/12] vs 52.6% [10/19], P=0.004); in fact, no patient with detectable AR-V7 achieved a PSA response. Furthermore, AR-V7-positive patients had shorter PSA-PFS (median: 1.4 vs 5.9 months, HR 7.4, 95%CI 2.7-20.6, log-rank P Conclusions: Detection of AR-V7 mRNA in circulating tumor cells from patients with metastatic castration-resistant prostate cancer may be associated with primary resistance to enzalutamide. If confirmed by other investigators in larger-scale prospective studies, this could be used as a biomarker to predict enzalutamide resistance (and to direct AR-V7-positive patients away from further AR-targeting therapies). Citation Format: Emmanuel S. Antonarakis, Changxue Lu, Hao Wang, Brandon Luber, Mary Nakazawa, Jeffrey C. Roeser, Yan Chen, Helen L. Fedor, Tamara L. Lotan, Angelo M. De Marzo, John T. Isaacs, William B. Isaacs, Rosa Nadal, Channing J. Paller, Samuel R. Denmeade, Michael A. Carducci, Mario A. Eisenberger, Jun Luo. Androgen receptor splice variant-7 predicts resistance to enzalutamide in patients with castration-resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2910. doi:10.1158/1538-7445.AM2014-2910