Changxue Lu

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.).

Distinct transcriptional programs mediated by the ligand-dependent full-length androgen receptor and its splice variants in castration-resistant prostate cancer

Continued androgen receptor (AR) signaling is an established mechanism underlying castration-resistant prostate cancer (CRPC), and suppression of androgen receptor signaling remains a therapeutic goal of CRPC therapy. Constitutively active androgen receptor splice variants (AR-Vs) lack the androgen receptor ligand-binding domain (AR-LBD), the intended target of androgen deprivation therapies including CRPC therapies such as abiraterone and MDV3100. While the canonical full-length androgen receptor (AR-FL) and AR-Vs are both increased in CRPCs, their expression regulation, associated transcriptional programs, and functional relationships have not been dissected. In this study, we show that suppression of ligand-mediated AR-FL signaling by targeting AR-LBD leads to increased AR-V expression in two cell line models of CRPCs. Importantly, treatment-induced AR-Vs activated a distinct expression signature enriched for cell-cycle genes without requiring the presence of AR-FL. Conversely, activation of AR-FL signaling suppressed the AR-Vs signature and activated expression programs mainly associated with macromolecular synthesis, metabolism, and differentiation. In prostate cancer cells and CRPC xenografts treated with MDV3100 or abiraterone, increased expression of two constitutively active AR-Vs, AR-V7 and ARV567ES, but not AR-FL, paralleled increased expression of the androgen receptor-driven cell-cycle gene UBE2C. Expression of AR-V7, but not AR-FL, was positively correlated with UBE2C in clinical CRPC specimens. Together, our findings support an adaptive shift toward AR-V-mediated signaling in a subset of CRPC tumors as the AR-LBD is rendered inactive, suggesting an important mechanism contributing to drug resistance to CRPC therapy.

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.

Clinical Significance of Androgen Receptor Splice Variant-7 mRNA Detection in Circulating Tumor Cells of Men With Metastatic Castration-Resistant Prostate Cancer Treated With First- and Second-Line Abiraterone and Enzalutamide

Purpose We reported previously that the detection of androgen receptor splice variant-7 (AR-V7) mRNA in circulating tumor cells (CTCs) correlated with poor outcomes from the use of abiraterone and enzalutamide in patients with castration-resistant prostate cancer (CRPC). Here, we expanded our cohort size to better characterize the prognostic significance of AR-V7 in this setting. Methods We prospectively enrolled 202 patients with CRPC starting abiraterone or enzalutamide and investigated the prognostic value of CTC detection (+ v -) and AR-V7 detection (+ v -) using a CTC-based AR-V7 mRNA assay. We examined ≥ 50% prostate-specific antigen (PSA) responses, PSA progression-free survival, clinical and radiologic progression-free survival, and overall survival. We constructed multivariable models adjusting for PSA, Gleason sum, number of prior hormone therapies, prior abiraterone or enzalutamide use, prior taxane use, presence of visceral metastases, and Eastern Cooperative Oncology Group score. We also separately examined the first-line and second-line novel hormonal therapy (NHT) settings. Results Median follow-up times were 15.0, 21.7, and 14.6 months for CTC-, CTC+/AR-V7- and CTC+/AR-V7+ patients, respectively. CTC+/AR-V7+ patients were more likely to have Gleason scores ≥ 8 ( P = .05), metastatic disease at diagnosis ( P = .01), higher PSA ( P < .01), prior abiraterone or enzalutamide use ( P = .03), prior taxane use ( P = .02), and Eastern Cooperative Oncology Group ≥ 1 ( P = .01). Outcomes for the overall cohort (and separately for the first-line and second-line NHT cohorts) were best for CTC- patients, intermediate for CTC+/AR-V7- patients, and worse for CTC+/AR-V7+ patients. These correlations remained significant in multivariable models. Conclusion This expanded analysis further characterizes the importance of CTC-based AR-V7 mRNA detection in predicting outcomes in patients with CRPC receiving first- and second-line NHT and, to the best of our knowledge, is the first to suggest that this assay be interpreted using three separate prognostic categories: CTC-, CTC+/AR-V7-, and CTC+/AR-V7+.

Decoding the androgen receptor splice variants.

In the past five years, multiple structurally and functionally distinct androgen receptor (AR) splice variants have been decoded and characterized. The mature transcripts for the majority of the fully decoded AR splice variants contain a transcribed “intronic” sequence, capable of encoding a short variant-specific peptide to replace the AR ligand-binding domain (LBD). Functionally, AR splice variants represent a diverse group of molecules often demonstrating cell context-specific genomic functions that may or may not be coupled with the functions of the canonical full-length AR (AR-FL). However, the full spectrum of their functional diversity and the underlying mechanistic basis remains very poorly characterized. In clinical specimens derived from men treated with a variety of hormone therapy regimens, AR splice variants are almost always expressed at detectable, yet lower levels when compared to that of AR-FL. In spite of the collective in vitro data supporting the putative role of AR splice variants in therapeutic resistance to hormone therapies, the extent to which AR splice variants mediate resistance to each individual regimen is not known and awaits thorough investigations in a clinically relevant setting using specimens from men undergoing treatments. Among the AR splice variants, AR-V7 is more abundantly and frequently expressed in castration-resistant prostate cancer (CRPC) and remains the most important variant identified so far. The relative importance of different AR molecules, including AR-FL, should be functionally dissected in the setting of castration-resistant prostate cancer, particularly in tumors resistant to more potent inhibitors of AR-FL recently approved by the FDA. In this review, we will focus on the discovery and characterization of AR splice variants, their putative functions and roles in mediating constitutively active AR signaling, and key areas of investigation that are necessary to establish their clinical relevance.

Analytic Validation of RNA In Situ Hybridization (RISH) for AR and AR-V7 Expression in Human Prostate Cancer

Purpose: RNA expression of androgen receptor splice variants may be a biomarker of resistance to novel androgen deprivation therapies in castrate-resistant prostate cancer (CRPC). We analytically validated an RNA in situ hybridization (RISH) assay for total AR and AR-V7 for use in formalin-fixed paraffin-embedded (FFPE) prostate tumors. Experimental Design: We used prostate cell lines and xenografts to validate chromogenic RISH to detect RNA containing AR exon 1 (AR-E1, surrogate for total AR RNA species) and cryptic exon 3 (AR-CE3, surrogate for AR-V7 expression). RISH signals were quantified in FFPE primary tumors and CRPC specimens, comparing to known AR and AR-V7 status by IHC and RT-PCR. Results: The quantified RISH results correlated significantly with total AR and AR-V7 levels by RT-PCR in cell lines, xenografts, and autopsy metastases. Both AR-E1 and AR-CE3 RISH signals were localized in nuclear punctae in addition to the expected cytoplasmic speckles. Compared with admixed benign glands, AR-E1 expression was significantly higher in primary tumor cells with a median fold increase of 3.0 and 1.4 in two independent cohorts (P < 0.0001 and P = 0.04, respectively). While AR-CE3 expression was detectable in primary prostatic tumors, levels were substantially higher in a subset of CRPC metastases and cell lines, and were correlated with AR-E1 expression. Conclusions: RISH for AR-E1 and AR-CE3 is an analytically valid method to examine total AR and AR-V7 RNA levels in FFPE tissues. Future clinical validation studies are required to determine whether AR RISH is a prognostic or predictive biomarker in specific clinical contexts. Clin Cancer Res; 22(18); 4651–63. ©2016 AACR.

Novel Junction-specific and Quantifiable In Situ Detection of AR-V7 and its Clinical Correlates in Metastatic Castration-resistant Prostate Cancer

BACKGROUND Androgen receptor splice variant 7 (AR-V7) has been implicated in resistance to abiraterone and enzalutamide treatment in men with metastatic castration-resistant prostate cancer (mCRPC). Tissue- or cell-based in situ detection of AR-V7, however, has been limited by lack of specificity. OBJECTIVE To address current limitations in precision measurement of AR-V7 by developing a novel junction-specific AR-V7 RNA in situ hybridization (RISH) assay compatible with automated quantification. DESIGN, SETTING, AND PARTICIPANTS We designed a RISH method to visualize single splice junctions in cells and tissue. Using the validated assay for junction-specific detection of the full-length AR (AR-FL) and AR-V7, we generated quantitative data, blinded to clinical data, for 63 prostate tumor biopsies. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS We evaluated clinical correlates of AR-FL/AR-V7 measurements, including association with prostate-specific antigen progression-free survival (PSA-PFS) and clinical and radiographic progression-free survival (PFS), in a subset of patients starting treatment with abiraterone or enzalutamide following biopsy. RESULTS AND LIMITATIONS Quantitative AR-FL/AR-V7 data were generated from 56 of the 63 (88.9%) biopsy specimens examined, of which 44 were mCRPC biopsies. Positive AR-V7 signals were detected in 34.1% (15/44) mCRPC specimens, all of which also co-expressed AR-FL. The median AR-V7/AR-FL ratio was 11.9% (range 2.7-30.3%). Positive detection of AR-V7 was correlated with indicators of high disease burden at baseline. Among the 25 CRPC biopsies collected before treatment with abiraterone or enzalutamide, positive AR-V7 detection, but not higher AR-FL, was significantly associated with shorter PSA-PFS (hazard ratio 2.789, 95% confidence interval 1.12-6.95; p=0.0081). CONCLUSIONS We report for the first time a RISH method for highly specific and quantifiable detection of splice junctions, allowing further characterization of AR-V7 and its clinical significance. PATIENT SUMMARY Higher AR-V7 levels detected and quantified using a novel method were associated with poorer response to abiraterone or enzalutamide in prostate cancer.

Bipolar androgen therapy in men with metastatic castration-resistant prostate cancer after progression on enzalutamide: an open-label, phase 2, multicohort study

BACKGROUND Prostate cancer that progresses after enzalutamide treatment is poorly responsive to further antiandrogen therapy, and paradoxically, rapid cycling between high and low serum testosterone concentrations (bipolar androgen therapy [BAT]) in this setting might induce tumour responses. We aimed to evaluate BAT in patients with metastatic castration-resistant prostate cancer that progressed after enzalutamide. METHODS We did this single-centre, open-label, phase 2, multicohort study in the USA. We included patients aged 18 years or older who had histologically confirmed and radiographically documented metastatic castration-resistant prostate cancer, with no more than two previous second-line hormonal therapies, and a castrate concentration of testosterone. Patients were asymptomatic, with Eastern Cooperative Oncology Group performance status of 0-2, and did not have high-risk lesions for tumour flare (eg, >5 sites of visceral disease or bone lesions with impending fracture). For the cohort reported here, we required patients to have had progression on enzalutamide with a continued prostate-specific antigen (PSA) rise after enzalutamide treatment discontinuation. Patients received BAT, which consisted of intramuscular testosterone cipionate 400 mg every 28 days until progression and continued luteinising hormone-releasing hormone agonist therapy. Upon progression after BAT, men were rechallenged with oral enzalutamide 160 mg daily. The co-primary endpoints were investigator-assessed 50% decline in PSA concentration from baseline (PSA50) for BAT (for all patients who received at least one dose) and for enzalutamide rechallenge (based on intention-to-treat analysis). These data represent the final analysis for the post-enzalutamide cohort, while two additional cohorts (post-abiraterone and newly castration-resistant prostate cancer) are ongoing. The trial is registered with ClinicalTrials.gov, number NCT02090114. FINDINGS Between Aug 28, 2014, and May 18, 2016, we accrued 30 eligible patients and treated them with BAT. Nine (30%; 95% CI 15-49; p<0·0001) of 30 patients achieved a PSA50 to BAT. 29 patients completed BAT and 21 proceeded to enzalutamide rechallenge, of whom 15 (52%; 95% CI 33-71; p<0·0001) achieved a PSA50 response. During BAT, the only grade 3-4 adverse event occurring in more than one patient was hypertension (three [10%] patients). Other grade 3 or worse adverse events occurring during BAT in one [3%] patient each were pulmonary embolism, myocardial infarction, urinary obstruction, gallstone, and sepsis. During enzalutamide retreatment, no grade 3-4 toxicities occurred in more than one patient. No treatment-related deaths were reported during either BAT or enzalutamide retreatment. INTERPRETATION BAT is a safe therapy that resulted in responses in asymptomatic men with metastatic castration-resistant prostate cancer and also resensitisation to enzalutamide in most patients undergoing rechallenge. Further studies with BAT are needed to define the potential clinical role for BAT in the management of metastatic castration-resistant prostate cancer and the optimal strategy for sequencing between androgen and antiandrogen therapies in metastatic castration-resistant prostate cancer to maximise therapeutic benefit to patients. FUNDING National Institutes of Health and National Cancer Institute.

Germline DNA-repair Gene Mutations and Outcomes in Men with Metastatic Castration-resistant Prostate Cancer Receiving First-line Abiraterone and Enzalutamide

BACKGROUND Inherited DNA-repair gene mutations are more prevalent in men with advanced prostate cancer than previously thought, but their clinical implications are not fully understood. OBJECTIVE To investigate the clinical significance of germline DNA-repair gene alterations in men with metastatic castration-resistant prostate cancer (mCRPC) receiving next-generation hormonal therapy (NHT), with a particular emphasis on BRCA/ATM mutations. DESIGN, SETTING, AND PARTICIPANTS We interrogated 50 genes for pathogenic or likely pathogenic germline mutations using leukocyte DNA from 172 mCRPC patients beginning treatment with first-line NHT with abiraterone or enzalutamide. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS We assessed the impact of germline DNA-repair gene mutation status on ≥50% and ≥90% PSA responses, PSA progression-free survival (PSA-PFS), clinical/radiologic progression-free survival (PFS), and overall survival (OS). Survival outcomes were adjusted using propensity score-weighted multivariable Cox regression analyses. RESULTS AND LIMITATIONS Among 172 mCRPC patients included, germline mutations (in any DNA-repair gene) were found in 12% (22/172) of men, and germline BRCA/ATM mutations specifically in 5% (9/172) of men. In unadjusted analyses, outcomes to first-line NHT were better in men with germline BRCA/ATM mutations (vs no mutations) with respect to PSA-PFS (hazard ratio [HR] 0.47; p=0.061), PFS (HR 0.50; p=0.090), and OS (HR 0.28; p=0.059). In propensity score-weighted multivariable analyses, outcomes were superior in men with germline BRCA/ATM mutations with respect to PSA-PFS (HR 0.48, 95% confidence interval [CI] 0.25-0.92; p=0.027), PFS (HR 0.52, 95% CI 0.28-0.98; p=0.044), and OS (HR 0.34, 95% CI 0.12-0.99; p=0.048), but not in men with non-BRCA/ATM germline mutations (all p>0.10). These results require prospective validation, and our conclusions are limited by the small number of patients (n=9) with BRCA/ATM mutations. CONCLUSIONS Outcomes to first-line NHT appear better in mCRPC patients harboring germline BRCA/ATM mutations (vs no mutations), but not for patients with other non-BRCA/ATM germline mutations. PATIENT SUMMARY Patients with metastatic castration-resistant prostate cancer and harboring germline mutations in BRCA1/2 and ATM benefit from treatment with abiraterone and enzalutamide.

The role of computed tomography of the chest in the management of small-cell lung cancer.

Despite the wide application of computed tomography (CT) in the diagnosis and management of lung cancer, the role of this diagnostic modality in the management of small-cell lung cancer (SCC) has not yet been defined. We therefore compared information gained from routine chest radiography (CXR) and CT scans performed on 32 patients with SCC who were treated on an intensive chemotherapy-radiotherapy protocol. Seventy-nine pairs of CXRs and CT scans were retrospectively reviewed. We found that although CT delineates a greater extent of intrathoracic disease in each of nine anatomic areas evaluated than does CXR, agreement between CT and CXR was significant for all areas except the pericardium. Pericardial thickening was seen only on CT scan and is more frequent in SCC patients than has previously been appreciated, but both its etiology and prognostic significance are unclear at this time. CT also allowed interpretation of disease status in cases where radiation-induced fibrosis made interpretation of the CXR impossible. We do not recommend routine use of chest CT at time of diagnosis of SCC, but we recommend that its use be reserved for evaluation of new symptoms or suspected relapse, or when radiation fibrosis on CXR is severe.