Association of AR-V7 on Circulating Tumor Cells as a Treatment-Specific Biomarker With Outcomes and Survival in Castration-Resistant Prostate Cancer

Abstract

Importance: A critical decision in the management of metastatic castration-resistant prostate cancer (mCRPC) is when to administer an androgen receptor signaling (ARS) inhibitor or a taxane.

Objective: To determine if pretherapy nuclear androgen-receptor splice variant 7 (AR-V7) protein expression and localization on circulating tumor cells (CTCs) is a treatment-specific marker for response and outcomes between ARS inhibitors and taxanes.

Design, setting, and participants: For this cross-sectional cohort study at Memorial Sloan Kettering Cancer Center, 265 men with progressive mCRPC undergoing a change in treatment were considered; 86 were excluded because they were not initiating ARS or taxane therapy; and 18 were excluded for processing time constraints, leaving 161 patients for analysis. Between December 2012 and March 2015, blood was collected and processed from patients with progressive mCRPC immediately prior to new line of systemic therapy. Patients were followed up to 3 years.

Main outcomes and measures: Prostate-specific antigen (PSA) response, time receiving therapy, radiographic progression-free survival (rPFS), and overall survival (OS).

Results: Overall, of 193 prospectively collected blood samples from 161 men with mCRPC, 191 were evaluable (128 pre-ARS inhibitor and 63 pretaxane). AR-V7-positive CTCs were found in 34 samples (18%), including 3% of first-line, 18% of second-line, and 31% of third- or greater line samples. Patients whose samples had AR-V7-positive CTCs before ARS inhibition had resistant posttherapy PSA changes (PTPC), shorter rPFS, shorter time on therapy, and shorter OS than those without AR-V7-positive CTCs. Overall, resistant PTPC were seen in 65 of 112 samples (58%) without detectable AR-V7-positive CTCs prior to ARS inhibition. There were statistically significant differences in OS but not in PTPC, time on therapy, or rPFS for patients with or without pretherapy AR-V7-positive CTCs treated with a taxane. A multivariable model adjusting for baseline factors associated with survival showed superior OS with taxanes relative to ARS inhibitors when AR-V7-positive CTCs were detected pretherapy (hazard ratio, 0.24; 95% CI, 0.10-0.57; P = .035).

Conclusions and relevance: The results validate CTC nuclear expression of AR-V7 protein in men with mCRPC as a treatment-specific biomarker that is associated with superior survival on taxane therapy over ARS-directed therapy in a clinical practice setting. Continued examination of this biomarker in prospective studies will further aid clinical utility.

Figure 1. Immunofluorescence Staining for AR-V7 Positivity and Nuclear Localization

Panels include DNA (blue), CD45 (leukocyte common antigen) (green), pan-cytokeratin (red), and AR-V7 (white); CTCs with AR-V7 protein signal greater than 3.2-fold background intensity with clear nuclear localization are scored as AR-V7–positive and can be seen in (A) single CTCs, (B) CTC clusters, and (C) CK-negative CTCs. Cells without the requisite AR-V7 protein signal intensity or localization are classified as AR-V7–negative and are shown in contrast in (D) single CTCs. Samples with at least one AR-V7–positive CTC per 2 slides assayed is scored as AR-V7 positive in this study. AR-V7 indicates nuclear androgen-receptor splice variant 7; CKs, cytokeratins; Composite, all immunofluorescent channels together; CTCs, circulating tumor cells; DAPI, (4′,6-diamidino-2-phenylindole).

Figure 2. Prevalence and Frequency of AR-V7 CTC Positivity by Line of Therapy

The burden of intact, nonapoptotic CTCs (CTCs, CTC clusters, CK-negative CTCs) per 2 slides is displayed volumetrically normalized to CTC/mL. The subpopulation of AR-V7–positive CTCs detected per sample is shown in orange and the AR-V7–negative CTCs are represented in blue. Samples are separated by those collected prior to (A) first line, (B) second line, and (C) third or greater line of therapy in the castration-resistant metastatic setting. (D) A summary chart of AR-V7–positive CTC incidence and subclonal contribution is shown by line of therapy. AR-V7 indicates androgen-receptor splice variant 7 protein; CKs, cytokeratins; CTCs, circulating tumor cells.

Figure 3. Presence of AR-V7–Positive CTCs and Response to AR Signaling Inhibitors

Waterfall plots, colored by presence of AR-V7–positive CTCs, show the percent PSA change from baseline at (A) 12 weeks on ARS inhibitors and (B) 12 weeks (or best decline if after 12 weeks) on taxanes. The dotted horizontal line indicates the threshold for binary PSA response criteria. Overall survival is shown, separated by AR-V7 status, for samples from patients receiving (C) ARS inhibitors or (D) taxanes. AR indicates androgen receptor; AR-V7, nuclear androgen-receptor splice variant 7; CTCs, circulating tumor cells; PSA, prostate-specific antigen.

Figure 4. Patients With Pretherapy AR-V7–Positive CTCs and Overall Survival on Taxanes and/or AR Signaling Inhibitors

Individual covariates were tested for additive predictive power to predict outcome using a Cox proportional hazards model. The P values are the result of compensating for the other factors listed. The interaction of therapy and AR-V7 status was further investigated using a multivariable Cox proportional hazards model. The forest plot shows the hazard ratios and 95%confidence intervals. AR indicates androgen receptor; AR-V7, nuclear androgen-receptor splice variant 7; CTCs, circulating tumor cells.