Androgen Receptor Splice Variant 7 Protein Research Articles

Preclinical Study Using ABT263 to Increase Enzalutamide Sensitivity to Suppress Prostate Cancer Progression Via Targeting BCL2/ROS/USP26 Axis Through Altering ARv7 Protein Degradation.

Background: The recently developed antiandrogen, Enzalutamide (Enz), has reformed the standard of care for castration resistant prostate cancer (CRPC) patients. However, Enz-resistance inevitably emerges despite success of Enz in prolonging CRPC patients’ survival. Here we found that Enz-resistant prostate cancer (PCa) cells had higher BCL2 expression. We aimed to test whether targeting BCL2 would influence Enz sensitivity of prostate cancer (PCa) and identify the potential mechanism. Methods: The study was designed to target Enz-induced BCL2 with inhibitor ABT263 and test Enz sensitivity in Enz-resistant PCa cells by MTT assay. Cellular reactive oxygen species (ROS) levels were detected with dihydroethidium staining, and in vitro deubiquitinating enzyme activity assay was used to evaluate ubiquitin specific protease 26 (USP26) activity. Results: ABT263 could increase Enz sensitivity in both Enz-sensitive and Enz-resistant PCa cells via inducing ROS generation. Elevated cellular ROS levels might then inhibit USP26 activity to increase the ubiquitination of androgen receptor (AR) and AR splice variant 7 (ARv7) and their ubiquitin/proteasome-dependent degradation, which contributed to the increase of Enz sensitivity. In vivo mouse model also demonstrates that ABT263 will suppress the PCa progression. Conclusion: This study demonstrated that targeting Enz-induced BCL2 with inhibitor ABT263 could increase Enz sensitivity in both Enz-sensitive and Enz-resistant PCa cells through induction of cellular ROS levels and suppression of USP26 activity with a consequent increase of ubiquitin/proteasome-dependent degradation of AR and ARv7 protein expression.

Targeting GRP78-dependent AR-V7 protein degradation overcomes castration-resistance in prostate cancer therapy.

Rationale: Androgen receptor splice variant 7 (AR-V7) is a leading cause of the development of castration-resistant prostate cancer (CRPC). However, the regulation and function of AR-V7 at levels of post-translational modifications in prostate cancer therapy remain poorly understood. Here, we conducted a library screen of natural products to identify potential small molecules responsible for AR-V7 protein degradation in human prostate cancer cell lines.Methods: A natural product library was used to screen the inhibitor of AR-V7. Co-IP and biomass spectrum assays were used to identify the AR-V7-interacting proteins, whereas western blot, confocal microscopy, RNA interfering, and gene transfection were used to validate these interactions. Cell viability, EDU staining, and colony formation assays were employed to detect cell growth and proliferation. Flowcytometry assays were used to detect the distribution of cell cycle. Mouse xenograft models were used to study the anti-CRPC effects in vivo.Results: This screen identified rutaecarpine, one of the major components of the Chinese medicine Evodia rutaecarpa, as a novel chemical that selectively induces AR-V7 protein degradation via K48-linked ubiquitination. Mechanically, this effect relies on rutaecarpine inducing the formation of a GRP78-AR-V7 protein complex, which further recruits the E3 ligase SIAH2 to directly promote the ubiquitination of AR-V7. Consequently, the genetic and pharmacological activation of the GRP78-dependent AR-V7 protein degradation restores the sensitivity of castration-resistant prostate cancer to anti-androgen therapy in cell culture and animal models.Conclusions: These findings not only provide a new approach for overcoming castration-resistance in prostate cancer therapy, but also increase our understanding about the interplay between molecular chaperones and ubiquitin ligase in shaping protein stability.

Recruitment of miR-8080 by luteolin inhibits androgen receptor splice variant 7 expression in castration-resistant prostate cancer.

A need exists for seeking effective treatments for castration-resistant prostate cancer (CRPC) in response to its emergence following androgen deprivation therapy as a major clinical problem. In the present study, we investigated the chemopreventive and chemotherapeutic potential of luteolin, a flavonoid with antioxidative properties, on prostate cancer, including CRPC. Luteolin inhibited the progression of rat prostate carcinogenesis by induction of apoptosis in a transgenic rat for adenocarcinoma of prostate (TRAP) model. Luteolin decreased cell proliferation in a dose-dependent manner and induced apoptosis with the activation of caspases 3 and 7 in both rat (PCai1, established from a TRAP prostate tumor) and human (22Rv1) CRPC cells. Dietary luteolin also suppressed tumor growth via an increase in apoptosis and inhibition of angiogenesis in PCai1 and 22Rv1 xenografts implanted in castrated nude mice. We also focused on androgen receptor splice variant 7 (AR-V7), which contributes to cell proliferation and therapeutic resistance in CRPC. Luteolin dramatically suppressed AR-V7 protein expression in 22Rv1 cells in vitro and ex vivo. Microarray analysis identified MiR-8080, which contains a possible target sequence for AR-V7 3′-UTR, as a gene upregulated by luteolin. MiR-8080 transfection decreased the AR-V7 expression level and the induction of apoptosis in 22Rv1 cells. Furthermore, miR-8080 knockdown canceled luteolin decreasing AR-V7 and the cell growth of 22Rv1. MiR-8080 induced by luteolin intake enhanced the therapeutic effect of enzalutamide on 22Rv1 xenografts under castration conditions. These results indicate luteolin inhibits CRPC by AR-V7 suppression through miR-8080, highlighting luteolin and miR-8080 as promising therapeutic agents for this disease.

Clinical Utility of the Nuclear-localized AR-V7 Biomarker in Circulating Tumor Cells in Improving Physician Treatment Choice in Castration-resistant Prostate Cancer

BackgroundProof of the clinical utility of a biomarker is when its use informs a management decision and improves patient outcomes relative to when it is not used. ObjectiveTo model the clinical benefit of the nuclear-localized androgen receptor splice variant 7 (AR-V7) test for men with progressing metastatic castration-resistant prostate cancer (mCRPC) at the second line of therapy or greater to inform the choice of an androgen receptor signaling inhibitor (ARSI) or a taxane. Design, setting, and participantsThe study population was a cross-sectional cohort of 193 unique patients with progressing mCRPC from whom 255 samples were drawn at the time of the second line or later treatment decision who then received an ARSI or taxane, with up to 3 yr of additional follow-up Circulating tumor cells (CTCs) were identified from blood samples and tested for AR-V7. Physicians were blinded to AR-V7 status and the testing laboratory was blinded to outcomes. Outcome measurements and statistical analysesWe measured physician propensity for choosing an ARSI or taxane based on patient prognosis. We also measured overall survival (OS) adjusted for physician propensity by drug class; OS data were analyzed both without and with knowledge of nuclear-localized AR-V7 status. Results and limitationsTreating physicians had a propensity for choosing a taxane over an ARSI for patients with more advanced disease or who received an ARSI as the immediate prior therapy. After adjusting for physician propensity, discernible OS differences were not observed between taxane- and ARSI-treated patients (median 15.6 vs 14.4 mo; p =0.11). Patients with detectable nuclear-localized AR-V7 in CTCs had superior survival with taxanes over ARSIs (median 9.8 vs 5.7 mo; p = 0.041). AR-V7–negative patients had superior survival on ARSIs over taxanes (p = 0.033) but overlapping curves limit the interpretation. Mutivariable models showed a robust interaction between AR-V7 status and drug, and a lower risk of death on taxanes for AR-V7–positive men. ConclusionsUse of the nuclear-localized AR-V7 CTC test to inform treatment choice can improve patient outcomes relative to decisions based solely on standard-of-care measures. Patient summaryMen with metastatic prostate cancer who test positive for AR-V7 protein in circulating tumor cells are likely to live longer if taxane chemotherapy is used.

Clinical Utility of Circulating Tumour Cell Androgen Receptor Splice Variant-7 Status in Metastatic Castration-resistant Prostate Cancer

BackgroundDetection of androgen receptor splice variant-7 (AR-V7) mRNA in circulating tumour cells (CTCs) is associated with worse outcome in metastatic castration-resistant prostate cancer (mCRPC). However, studies rarely report comparisons with CTC counts and biopsy AR-V7 protein expression. ObjectiveTo determine the reproducibility of AdnaTest CTC AR-V7 testing, and associations with clinical characteristics, CellSearch CTC counts, tumour biopsy AR-V7 protein expression and overall survival (OS). Design, setting, and participantsCTC AR-V7 status was determined for 227 peripheral blood samples, from 181 mCRPC patients with CTC counts (202 samples; 136 patients) and matched mCRPC biopsies (65 samples; 58 patients). Outcome measurements and statistical analysisCTC AR-V7 status was associated with clinical characteristics, CTC counts, and tissue biopsy AR-V7 protein expression. The association of CTC AR-V7 status and other baseline variables with OS was determined. Results and limitationsOf the samples, 35% were CTC+/AR-V7+. CTC+/AR-V7+ samples had higher CellSearch CTC counts (median CTC; interquartile range [IQR]: 60, 19–184 vs 9, 2–64; Mann-Whitney test p<0.001) and biopsy AR-V7 protein expression (median H-score, IQR: 100, 63–148 vs 15, 0–113; Mann-Whitney test p=0.004) than CTC+/AR-V7− samples. However, both CTC− (63%) and CTC+/AR-V7− (62%) patients had detectable AR-V7 protein in contemporaneous biopsies. After accounting for baseline characteristics, there was shorter OS in CTC+/AR-V7+ patients than in CTC− patients (hazard ratio [HR] 2.13; 95% confidence interval [CI] 1.23–3.71; p=0.02); surprisingly, there was no evidence that CTC+/AR-V7+ patients had worse OS than CTC+/AR-V7− patients (HR 1.26; 95% CI 0.73–2.17; p=0.4). A limitation of this study was the heterogeneity of treatment received. ConclusionsStudies reporting the prognostic relevance of CTC AR-V7 status must account for CTC counts. Discordant CTC AR-V7 results and AR-V7 protein expression in matched, same-patient biopsies are reported. Patient summaryLiquid biopsies that determine circulating tumour cell androgen receptor splice variant-7 status have the potential to impact treatment decisions in metastatic castration-resistant prostate cancer patients. Robust clinical qualification of these assays is required before their routine use.

Measurement science of the androgen receptor splice variant-7 protein in primary and castration-resistant prostate cancer tissue.

151 Background: Liquid biopsies demonstrate the constitutively active androgen receptor splice variant-7 (AR-V7) associates with reduced benefit from endocrine therapies in castration resistant prostate cancer (CRPC). These studies provide little information pertaining to AR-V7 expression in PC tissue. Methods: AR-V7 protein expression was determined for 358 primary PC samples and 293 metastatic CRPC biopsies by immunohistochemistry. Associations with disease progression, full length AR (AR-FL) expression, response to therapy, gene expression, and circulating tumor cell (CTC) AR-V7 status were investigated. Results: AR-V7 protein is rarely expressed ( &lt; 1% of 358 cases) in primary PC but is frequently detected (75% of 40 cases) following primary androgen deprivation therapy (ADT) alone (H-score 40; interquartile range 1.25-92.5), with a further significant (p = 0.020) increase following abiraterone or enzalutamide therapy (90; 20-150). In CRPC, AR-V7 expression is mainly nuclear (94% of 144 cases), correlates with AR-FL expression (p = &lt; 0.001), and is homogeneous within single metastases (p = 0.997) but heterogeneous in different metastases from the same patient (p &lt; 0.001). In addition, AR-V7 expression correlates with a 59-gene signature, including HOXB13, a co-regulator of AR-V7 function. Moreover, AR-V7 negative disease associates with better PSA response (p = 0.03) and overall survival (p = 0.02) from endocrine therapies. Finally, CTC+/AR-V7+ blood samples had significantly (p = 0.004) higher AR-V7 protein expression (100; 62.5-147.5) in paired tissue biopsy compared to CTC+/AR-V7- blood samples (15; 0.0-112.5), and AR-V7 protein expression is frequently detected (63% of 16 samples) in tissue of patients with CTC- blood samples. Conclusions: AR-V7 protein is not expressed until castration resistance and occurs after primary ADT alone. Levels of AR-V7 protein vary between metastases, and although AR-V7 associates with response to endocrine therapies, this suggests multiple resistance mechanisms exist in the same patient. If developed, agents targeting AR-V7 may be best explored earlier in the course of disease and in combination with other therapies.

Expression pattern of androgen receptor and AR-V7 in androgen-deprivation therapy–naïve salivary duct carcinomas

Androgen-deprivation therapy has been used to treat salivary duct carcinoma (SDC). The androgen receptor splice variant-7 (AR-V7) has been detected in castration-resistant prostate cancer and implicated in resistance to androgen receptor (AR)-targeted therapies. Given the potential role of AR/AR-V7 in SDC treatment, this study focuses on AR/AR-V7 expression in SDC specimens collected before androgen-deprivation therapy. RNA in situ hybridization (ISH) and immunohistochemistry (IHC) to detect total AR and AR-V7 were performed on formalin-fixed, paraffin-embedded SDC specimens from 23 patients. Full-length AR and AR-V7 transcripts were quantified in a subset of tumors by reverse-transcription polymerase chain reaction. Twenty SDCs were positive for total AR by ISH and IHC. Among AR-positive SDCs, 70% (14/20) were positive for AR-V7 messenger RNA by ISH, whereas 15% (3/20) were positive for AR-V7 protein by IHC. The 3 SDCs that expressed the highest levels of AR-V7 were all from female patients; one of them expressed a significant amount of AR-V7 and barely detectable full-length AR transcripts by reverse-transcription polymerase chain reaction. IHC expression of Forkhead box protein A1, prostate-specific antigen, prostatic acid phosphatase, and NKX3.1 was observed in some SDCs regardless of patient sex. Five SDCs demonstrated strong human epidermal growth factor receptor 2 expression. We conclude that treatment-naïve SDCs may express AR-V7 at levels comparable to or even exceeding the levels detected in castration-resistant prostate cancer. Our data support the feasibility to incorporate AR-V7 assessment via ISH and/or IHC in the ongoing clinical trials evaluating the therapeutic benefit of AR-targeted therapies in SDC patients.

Abstract 2640: Detection of androgen receptor (AR) and AR-V7 in small cell prostate carcinoma by immunohistochemistry

Abstract Background: Small cell prostate carcinoma (SCPC) is a rare and highly malignant subtype of prostate cancer. SCPC only accounts for 0.5%-2% of untreated primary prostate cancer, but can present in up to 25% lethal metastatic prostate cancer autopsies. Small cell prostate carcinoma frequently lacks androgen receptor (AR) and prostate specific antigen (PSA) expression, and often responds poorly to androgen deprivation therapy (ADT). However, the expression pattern of different AR isoforms in SCPC after ADT remains poorly characterized. In this study, we have validated and developed a new immunohistochemical assay for full length AR (AR-FL) and AR splice variant-7 (AR-V7) protein expression in prostate cancer specimens. The expression of AR-FL and AR-V7 protein were determined in post-ADT radical prostatectomy samples from twelve SCPC patients. In this study, we developed and validated a new immunohistochemical assay for the full-length AR (AR-FL) and AR splice variant-7 (AR-V7) proteins. We conducted an IHC study in post-ADT surgical specimens from twelve SCPC patients treated with neoadjuvant ADT. Methods: AR-FL, AR-V7, and total AR proteins were specifically detected using AR C-terminal domain antibody (Sigma-Aldrich), AR-V7 antibody (RevMab), and AR N-terminal domain antibody (AR441, Santa Cruz), respectively. These antibodies were validated using prostate cancer cell lines with known AR profiles, including LNCaP95 cells which express both AR-FL and AR-V7 and the AR-negative PC3 cells. The IHC protocols were optimized for each individual antibody, and applied to the 12 SC/NE tissue specimens. Results: Twelve surgical (RP or TURP) prostate small cell carcinoma specimens were collected from patients after neoadjuvant hormonal therapy at Indiana University School of Medicine. Seventy five percent (9/12) of the specimens showed positive staining for the full-length AR with various intensities. Thirty three percent (4/12) of the specimens showed positive staining for AR-V7. Among the specimens with positive AR-V7 staining, two samples displayed very weak staining, one sample showed weak to moderate staining, and one sample showed strong staining. All positive specimens displayed a heterogeneous pattern of AR/AR-V7 staining. All specimens positive for AR-V7 were also positive for the full length AR. Conclusion and future direction: Our preliminary results indicate that small cell prostate carcinoma may continue to express AR and AR-V7. The functional and clinical relevance of AR/AR-V7 detection in this setting remains unknown. Future investigation will focus on clinical correlation in an expanded patient cohort. Citation Format: Pei Zhao, Yezi Zhu, Liang Cheng, Jun Luo. Detection of androgen receptor (AR) and AR-V7 in small cell prostate carcinoma by immunohistochemistry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2640.

Assessment of the Validity of Nuclear-Localized Androgen Receptor Splice Variant 7 in Circulating Tumor Cells as a Predictive Biomarker for Castration-Resistant Prostate Cancer

ImportanceA blood test to determine whether to treat patients with metastatic castration-resistant prostate cancer (mCRPC) with an androgen receptor signaling (ARS) inhibitor or taxane is an unmet medical need.ObjectiveTo determine whether a validated assay for the nuclear-localized androgen receptor splice variant 7 (AR-V7) protein in circulating tumor cells can determine differential overall survival among patients with mCRPC treated with taxanes vs ARS inhibitors.Design, Setting, and ParticipantsThis blinded correlative study conducted from December 31, 2012, to September 1, 2016, included 142 patients with histologically confirmed mCRPC and who were treated at Memorial Sloan Kettering Cancer Center, The Royal Marsden, or the London Health Sciences Centre. Blood samples were obtained prior to administration of ARS inhibitors or taxanes as a second-line or greater systemic therapy for progressing mCRPC.Main Outcomes and MeasuresOverall survival after treatment with an ARS inhibitor or taxane in relation to pretherapy AR-V7 status.ResultsAmong the 142 patients in the study (mean [SD] age, 69.5 [9.6] years), 70 were designated as high risk by conventional prognostic factors. In this high-risk group, patients positive for AR-V7 who were treated with taxanes had superior overall survival relative to those treated with ARS inhibitors (median overall survival, 14.3 vs 7.3 months; hazard ratio, 0.62; 95% CI, 0.28-1.39; P = .25). Patients negative for AR-V7 who were treated with ARS inhibitors had superior overall survival relative to those treated with taxanes (median overall survival, 19.8 vs 12.8 months; hazard ratio, 1.67; 95% CI, 1.00-2.81; P = .05).Conclusions and RelevanceThis study suggests that nuclear-localized AR-V7 protein in circulating tumor cells can identify patients who may live longer with taxane chemotherapy vs ARS inhibitor treatment.

Overexpression of nuclear AR-V7 protein in primary prostate cancer is an independent negative prognostic marker in men with high-risk disease receiving adjuvant therapy

BackgroundOverexpression of the androgen receptor (AR) splice variant 7 (AR-V7) has recently been reported to be associated with resistance to antihormonal therapy. Herein, we address the question whether tumor cells with AR-V7 expression can be detected at the time of radical prostatectomy, that is, before long-term hormonal manipulation and castration resistance, and what the potential prognostic impact on the biochemical recurrence (BCR)-free survival may be. MethodsAn anti-AR-V7 antibody was first validated in a training set of prostate cancer specimens by a comparison of AR-V7 protein to AR-V7 mRNA expression. We then analyzed nuclear AR-V7 protein expression in the primary tumors and lymph node metastases from 163 predominantly high-risk patients (cohort I) as well as the primary tumors from patients of a second, consecutive patient cohort (n = 238, cohort II) not selected for any clinicopathological features. Staining results were correlated to patient characteristics and BCR-free patient survival. ResultsHigh nuclear AR-V7 protein expression was detected in approximately 30%–40% of patients in cohort I and II at the time of radical prostatectomy. High baseline expression of nuclear AR-V7 protein was associated with an unfavorable BCR-free survival in the high-risk patient cohort I but not in the unselected consecutive cohort II. Remarkably, AR-V7 was an independent negative prognostic factor in high-risk prostate cancer patients of cohort I who were selected to receive adjuvant treatment. ConclusionsProstate cancer cells with high nuclear AR-V7 protein expression can be detected in a substantial proportion of tumors at the time of radical prostatectomy. The presence of AR-V7-positive tumor cells is associated with an unfavorable prognosis for BCR-free survival in a high-risk patient cohort including a subgroup of patients selected to receive adjuvant therapy, in which AR-V7 was an independent negative prognosticator. Overexpression of nuclear AR-V7 protein hence identifies a subset of tumors with remarkably aggressive growth characteristics among clinically and histologically high-risk patients at the time of radical prostatectomy.

PD71-08 ANDROGEN RECEPTOR SPLICE VARIANT 7 (AR-V7) IN PATIENTS WITH LOCAL ADVANCED, METASTATIC AND CRPCM: A NOVEL CAPILLARY NANO-INMUNOASSAY TECHNIQUE

You have accessJournal of UrologyProstate Cancer: Markers III1 Apr 2017PD71-08 ANDROGEN RECEPTOR SPLICE VARIANT 7 (AR-V7) IN PATIENTS WITH LOCAL ADVANCED, METASTATIC AND CRPCM: A NOVEL CAPILLARY NANO-INMUNOASSAY TECHNIQUE ALVARO RODRIGUEZ, JUAN LUIS GARCIA, JAVIER GARCIA GARCIA, REBECA LOZANO MEJORADA, IRENA MISIEWICZ-KRZEMINSKA, MANUEL HERRERO POLO, JUAN JESUS CRUZ, and FRANCISCO GOMEZ VEIGA ALVARO RODRIGUEZALVARO RODRIGUEZ More articles by this author , JUAN LUIS GARCIAJUAN LUIS GARCIA More articles by this author , JAVIER GARCIA GARCIAJAVIER GARCIA GARCIA More articles by this author , REBECA LOZANO MEJORADAREBECA LOZANO MEJORADA More articles by this author , IRENA MISIEWICZ-KRZEMINSKAIRENA MISIEWICZ-KRZEMINSKA More articles by this author , MANUEL HERRERO POLOMANUEL HERRERO POLO More articles by this author , JUAN JESUS CRUZJUAN JESUS CRUZ More articles by this author , and FRANCISCO GOMEZ VEIGAFRANCISCO GOMEZ VEIGA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.3175AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The androgen receptor (AR) splice variant 7 (AR-V7), has been associated with increased risk of prostate cancer (PC) and with the possibility of progression and responsiveness to the various lines of hormonal treatment and chemotherapy. The aim of this study was to investigate the feasibility of a novel capillary nano-immunoassay in detecting AR-V7 variant in plasma from PC patients METHODS 72 patients with PC were enrolled. Localized, locally advanced, metastasic and CPRC status were allowed due to represent all the PC biological spectrum. Capillary Electrophoresis with immunoassay methodology were performed using the WES (TM) machine according to the manufacturer′s protocol. With this technique, proteins are separated by size, immobilized and probed with specific antibodies. Signal intensity is detected by a horseradish peroxidase-conjugated chemiluminescence system and the data is shown as an electropherogram. The primary antibody used was AR-V7. Quantitative analysis for AR-V7 protein peak was done automatically as area under the peak. Dates from the human prostate cell lines PC3 and VCaP and from 17 healthy donors were used to standardize the assays for AR-V7. The study was carried out in accordance with the ethical standards of the Declaration of Helsinki and was approved by the Institutional Review Board of the University Hospital of Salamanca (Spain). RESULTS AR-V7 signal was detected in 21 (29%) patients: 17 (81%) had a Gleason score ≥ 7, 15 (71%) castration-resistant prostate cancer (CRPC) and 18 (86%) metastatic disease. The complete results are shown in the table 1. CONCLUSIONS This study describes the feasibility of detecting AR-V7 expression from plasma of PC patients by using an automated capillary nano-immunoassay technique WESTM. We provide evidence about this technique, simpler, faster and we have automated platform to make it in comparison with traditional CTCs techniques. AR-V7 has been observed more frequently in aggressive tumors. These findings lay the foundations for liquid biopsy as a means of obtaining biological data in a more convenient way for the patient. These data are needed for personalized treatment decisions, which can prevent patients from receiving inefficient systemic therapy. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1358 Advertisement Copyright & Permissions© 2017MetricsAuthor Information ALVARO RODRIGUEZ More articles by this author JUAN LUIS GARCIA More articles by this author JAVIER GARCIA GARCIA More articles by this author REBECA LOZANO MEJORADA More articles by this author IRENA MISIEWICZ-KRZEMINSKA More articles by this author MANUEL HERRERO POLO More articles by this author JUAN JESUS CRUZ More articles by this author FRANCISCO GOMEZ VEIGA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract 4965: Analytic validation and clinical qualification of a novel immunohistochemical assay for AR-V7 protein expression in metastatic prostate cancer

Abstract BACKGROUND: The androgen-receptor splice variant 7 (AR-V7) has been implicated in the development of castration resistant prostate cancer (CRPC) and resistance to current therapies including enzalutamide and abiraterone. AR-V7 mRNA expression in circulating tumour cells of patients with CRPC correlated with treatment resistance. However, the importance of AR-V7 has been questioned in light of low AR-V7 mRNA levels relative to the full-length androgen receptor in CRPC and it is critically important to develop validated assays that confirm AR-V7 protein levels and its clinical importance in patients with CRPC. METHODS: Following validation of a monoclonal antibody, immunohistochemical analysis of nuclear AR-V7 (alongside a nuclear AR N-terminal domain antibody; AR-NTD) was performed in a patient cohort identified with matched therapy-naive hormone-sensitive primary prostate cancer (HSPC) and CRPC. We determined the levels of nuclear AR-V7 as patients progressed from HSPC to CRPC. We also determined if AR-V7 expression levels associated with overall survival from time of CRPC biopsy. RESULTS: In our patient cohort (n = 39), nuclear AR-V7 (p = &amp;lt;0.0001) and nuclear AR-NTD (p = 0.0006) increased significantly as patients progressed from HSPC to CRPC. Lower nuclear AR-V7 expression was associated with improved overall survival from time of CRPC biopsy in patient groups divided by the 25th (18.7 vs 9.6 months; HR 0.36 [95% CI 0.17-0.62]; p = 0.002), 50th (13.0 vs 9.8 months; HR 0.60 [95% CI 0.29-1.07]; p = 0.09) or 75th (6.0 vs 11.5 months; HR 0.31 [95% CI 0.04-0.34]; p = 0.0004) percentile of AR-V7 expression. Similarly, a lower nuclear AR-V7 to nuclear AR-NTD ratio was associated with improved overall survival from time of CRPC biopsy in patient groups divided by the 25th (17.8 vs 9.1 months; HR 0.42 [95% CI 0.22-0.78], p = 0.01), 50th (14.0 vs 8.8 months; HR 0.43 [95% CI 0.17-0.69], p = 0.005) and 75th (11.5 vs 7.3 months; HR 0.39 [95% CI 0.09-0.69], p = 0.009) percentile. Nuclear AR-NTD did not associate with overall survival from CRPC biopsy. CONCLUSION: We provide first evidence that expression of nuclear AR-V7 protein not only increases with emerging treatment resistance in CRPC but also is associated with overall survival from time of CRPC biopsy. These data support AR-V7 protein being key to CRPC progression and that agents targeting AR splice variants will be important to improve patient outcome in CRPC. Citation Format: Daniel Nava Rodrigues, Jon Welti, Adam Sharp, Shihua Sun, Ruth Riisnaes, Ines Figueiredo, Zafeiris Zafeiriou, Pasquale Rescigno, Johann S. de Bono, Stephen R. Plymate. Analytic validation and clinical qualification of a novel immunohistochemical assay for AR-V7 protein expression in metastatic prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4965.

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

ImportanceA 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.ObjectiveTo 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 ParticipantsFor 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 MeasuresProstate-specific antigen (PSA) response, time receiving therapy, radiographic progression-free survival (rPFS), and overall survival (OS).ResultsOverall, 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 RelevanceThe 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.

Analytical Validation and Clinical Qualification of a New Immunohistochemical Assay for Androgen Receptor Splice Variant-7 Protein Expression in Metastatic Castration-resistant Prostate Cancer

BackgroundThe androgen receptor splice variant-7 (AR-V7) has been implicated in the development of castration-resistant prostate cancer (CRPC) and resistance to abiraterone and enzalutamide. ObjectiveTo develop a validated assay for detection of AR-V7 protein in tumour tissue and determine its expression and clinical significance as patients progress from hormone-sensitive prostate cancer (HSPC) to CRPC. Design, setting, and participantsFollowing monoclonal antibody generation and validation, we retrospectively identified patients who had HSPC and CRPC tissue available for AR-V7 immunohistochemical (IHC) analysis. Outcome measurements and statistical analysisNuclear AR-V7 expression was determined using IHC H score (HS) data. The change in nuclear AR-V7 expression from HSPC to CRPC and the association between nuclear AR-V7 expression and overall survival (OS) was determined. Results and limitationsNuclear AR-V7 expression was significantly lower in HSPC (median HS 50, interquartile range [IQR] 17.5–90) compared to CRPC (HS 135, IQR 80–157.5; p<0.0001), and in biopsy tissue taken before (HS 80, IQR 30–136.3) compared to after (HS 140, IQR 105–167.5; p=0.007) abiraterone or enzalutamide treatment. Lower nuclear AR-V7 expression at CRPC biopsy was associated with longer OS (hazard ratio 1.012, 95% confidence interval 1.004–1.020; p=0.003). While this monoclonal antibody primarily binds to AR-V7 in PC biopsy tissue, it may also bind to other proteins. ConclusionsWe provide the first evidence that nuclear AR-V7 expression increases with emerging CRPC and is prognostic for OS, unlike antibody staining for the AR N-terminal domain. These data indicate that AR-V7 is important in CRPC disease biology; agents targeting AR splice variants are needed to test this hypothesis and further improve patient outcome from CRPC. Patient summaryIn this study we found that levels of the protein AR-V7 were higher in patients with advanced prostate cancer. A higher level of AR-V7 identifies a group of patients who respond less well to certain prostate cancer treatments and live for a shorter period of time.