Nicole A. Schreiber

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

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.

The Initial Detection and Partial Characterization of Circulating Tumor Cells in Neuroendocrine Prostate Cancer

Purpose: The transition of prostate adenocarcinoma to a predominantly androgen receptor (AR) signaling independent phenotype can occur in the later stages of the disease and is associated with low AR expression +/− the development of small-cell or neuroendocrine tumor characteristics. As metastatic tumor biopsies are not always feasible and are difficult to repeat, we sought to evaluate noninvasive methods to identify patients transitioning toward a neuroendocrine phenotype (NEPC). Experimental Design: We prospectively studied a metastatic tumor biopsy, serum biomarkers, and circulating tumor cells (CTC, Epic Sciences) from patients with castration-resistant prostate cancer (CRPC) including those with pure or mixed NEPC histology present on biopsy. CTCs labeled with the patients clinical status were used to learn features that discriminate NEPC patients, which was then applied to an independent cohort. Results: Twenty-seven patients with CRPC including 12 NEPC and 5 with atypical clinical features suggestive of NEPC transition were studied. CTCs from NEPC patients demonstrated frequent clusters, low or absent AR expression, lower cytokeratin expression, and smaller morphology relative to typical CRPC. A multivariate analysis of protein and morphologic variables enabled distinguishing CTCs of NEPC from CRPC. This CTC classifier was applied to an independent prospective cohort of 159 metastatic CRPC patients and identified in 17/159 (10.7%) of cases, enriched in patients with high CTC burden (P < 0.01) and visceral metastases (P = 0.04). Conclusions: CTCs from patients with NEPC have unique morphologic characteristics, which were also identified in a subset of CRPC patients with aggressive clinical features potentially undergoing NEPC transition. Clin Cancer Res; 22(6); 1510–9. ©2015 AACR.

Phenotypic Heterogeneity of Circulating Tumor Cells Informs Clinical Decisions between AR Signaling Inhibitors and Taxanes in Metastatic Prostate Cancer

The heterogeneity of an individual patients tumor has been linked to treatment resistance, but quantitative biomarkers to rapidly and reproducibly evaluate heterogeneity in a clinical setting are currently lacking. Using established tools available in a College of American Pathologists-accredited and Clinical Laboratory Improvement Amendments-certified clinical laboratory, we quantified digital pathology features on 9,225 individual circulating tumor cells (CTC) from 179 unique metastatic castration-resistant prostate cancer (mCRPC) patients to define phenotypically distinct cell types. Heterogeneity was quantified on the basis of the diversity of cell types in individual patient samples using the Shannon index and associated with overall survival (OS) in the 145 specimens collected prior to initiation of the second or later lines of therapy. Low CTC phenotypic heterogeneity was associated with better OS in patients treated with androgen receptor signaling inhibitors (ARSI), whereas high heterogeneity was associated with better OS in patients treated with taxane chemotherapy. Overall, the results show that quantifying CTC phenotypic heterogeneity can help inform the choice between ARSI and taxanes in mCRPC patients. Cancer Res; 77(20); 5687-98. ©2017 AACR.

Clinical Validity of Detecting Circulating Tumor Cells by AdnaTest Assay Compared With Direct Detection of Tumor mRNA in Stabilized Whole Blood, as a Biomarker Predicting Overall Survival for Metastatic Castration-Resistant Prostate Cancer Patients.

AbstractCirculating tumor cell (CTC) number measured with the CellSearch assay is prognostic for survival in metastatic castration-resistant prostate cancer before and after therapy. Using a standard operating protocol for sample collection, processing, and analysis, we compared detection rates of CellSearch performed using US Food and Drug Administration–cleared methodology with a second positive selection assay, AdnaTest, and a nonselection polymerase chain reaction (PCR)–based (direct detection PCR [DDPCR]) assay in 55 blood samples from 47 men with progressive metastatic castration-resistant prostate cancer. AdnaTest requires processing within 4 hours of the draw and detects KLK3, PSMA, and EGFR transcripts in cells captured on magnetic beads. The DDPCR assay can be processed up to 7 days after a draw and detects KLK2, KLK3, HOXB13, GRHL2, and FOXA1 genes. AdnaTest and DDPCR were considered positive if at least 1 transcript was detected. AdnaTest detected CTCs in 34 samples (62%; 95% confidence interval [CI], 48%–75%), of which 23 (68%) had unfavorable CTC counts by CellSearch. A positive DDPCR result was seen in 38 cases (69%; 95% CI, 55%–81%), including 24 (63%) with unfavorable CellSearch CTC counts. CellSearch found unfavorable CTC counts in 25 samples (45%; 95% CI, 33%–58%). Sensitivities were similar between the AdnaTest and DDPCR assays, and both were more sensitive than CellSearch. Concordance probability estimates (possible values, 0.5–1.0) associating the biomarker result with survival were similar: 0.77 (SE, 0.07) for AdnaTest, 0.72 (SE, 0.08) for DDPCR, and 0.76 (SE, 0.06) for CellSearch. Overall detection rates between the AdnaTest and DDPCR assays were similar, and both were superior to CellSearch. The DDPCR assay required the lowest blood volume, least on-site processing, and longest stability for batch processing.

Abstract 1588: Single cell analysis of AR N terminal, AR C terminal and the ARv7 splice variant in the CTCs of metastatic castration resistant prostate cancer (mCRPC) patients

Background: Androgen signaling directed therapies, including Abiraterone (A) and Enzalutamide (E), prolong survival in patient (pts) with mCRPC and are FDA approved. Resistance to A and E is associated with the presence of the ARv7 splice variant which, along with other AR ligand binding domain (LBD) alterations, may constitutively activate AR. Previous work showed marked heterogeneity in cell types and protein expression in CTCs of mCRPC pts mandating single cell analysis to assess the AR LBD to understand passenger vs. driving clonal subtypes. We have developed and begun clinical validation for AR N terminal (AR N), AR C terminal (AR C), ARv7 protein and ARv7 mRNA enabling single CTC analysis. Methods: Cell lines (VCaP, LnCaP, LnCaP-95, 22RV1, & PC3) expressing varying levels of AR LBD alterations were spiked into healthy donor blood. 47 pt samples were collected for CTC analysis utilizing the Epic Sciences platform which 24 pts (6 treatment naive, 10 post A or E, and 8 post taxane) were selected for AR N/C/v7 analysis due to CTC prevalence across clinical decision point where change in therapy was needed. Epic analysis included ID of traditional CTCs, CK- CTCs, small CTCs, and CTC clusters. Cell lines & pts were analyzed for AR N, AR C and ARv7 IF, and a subset of patients CTC for ARv7 mRNA by RNA FISH. Results: Immunofluorescent protein expression of AR N, AR C and ARv7 in cell lines were consistent with published profiles. AR C & ARv7 were detected only in AR N+ CTCs indicating assay specificity for both targets. ARv7 expression was observed in traditional, CK-, small CTCs and CTC clusters, but no association was seen between ARv7+ and any CTC subtype. AR v7 RNA FISH was specific to AR v7 IF+ pts. Conclusions: AR C loss is more sensitive in detecting AR LBD alterations than ARv7. Variable ARv7/AR N ratios in different cell types suggest intrapatient AR heterogeneity. The low prevalence of ARv7+ CTCs of total and AR N+ CTCs suggests many pts may have other driving resistance mechanisms. Citation Format: James Kelvin, David Lu, Davin Packer, Richard Bambury, Dana Rathkopf, Nicole Schreiber, Zaina Arslan, Natalie Prigozhina, David Brown, Rachel Krupa, Edward Swangren, Mark Landers, Florence Lee, Dena Marrinucci, Ryan Dittamore, Howard I. Scher. Single cell analysis of AR N terminal, AR C terminal and the ARv7 splice variant in the CTCs of metastatic castration resistant prostate cancer (mCRPC) patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1588. doi:10.1158/1538-7445.AM2015-1588

Safety and Efficacy of BIND-014, a Docetaxel Nanoparticle Targeting Prostate-Specific Membrane Antigen for Patients With Metastatic Castration-Resistant Prostate Cancer: A Phase 2 Clinical Trial

Importance Preferential delivery of docetaxel to tumors by prostate-specific membrane antigen (PSMA)–targeted nanoparticles is clinically effective, and the selective reduction of PSMA-positive circulating tumor cells (CTCs) after treatment has implications for patient selection and disease monitoring. Objective To determine the safety and efficacy of BIND-014, a PSMA-directed docetaxel-containing nanoparticle, in patients with metastatic castration-resistant prostate cancer (mCRPC). Design, Setting, and Participants A multicenter open-label, phase 2 clinical trial of 42 chemotherapy-naive patients with progressing mCRPC after treatment with abiraterone acetate and/or enzalutamide was conducted from June 24, 2013, to June 10, 2016. Intervention Treatment with BIND-014 at a dosage of 60 mg/m2 was given intravenously on day 1 of 21-day cycles in combination with prednisone until disease progression or unacceptable toxic effects occurred. Main Outcomes and Measures The primary end point was radiographic progression-free survival according to Prostate Cancer Working Group 2 recommendations and Response Evaluation Criteria in Solid Tumors, version 1.1. Secondary end points included prostate-specific antigen (PSA) response (≥50% reduction from baseline) and changes in CTC number (from ≥5 to <5 cells per 7.5 mL of blood) (CellSearch). Changes in CTC number based on PSMA expression levels on CTCs were also evaluated (Epic Sciences). Results Among the 42 patients (81% white), the median age was 66 (range, 50-85) years, and median number of doses received was 6 (range, 1-21). A PSA response was observed in 12 of 40 patients (30%; 95% CI, 18%-45%), measurable disease response in 6 of 19 (32% [95% CI, 15%-54%]), and CTC conversions in 13 of 26 (50%; 95% CI, 32%-68%). Median radiographic progression-free survival was 9.9 (95% CI, 7.1-12.6) months. With use of the Epic Sciences non-EPCAM-based CTC detection platform, CTCs were detected in 16 of 18 patients (89%); 11 of 18 (61%) had CTCs with PSMA expression above the analytical threshold level (PSMA positive) at baseline (range, 0.4-72.4 CTCs/mL). After treatment, PSMA-positive CTCs were preferentially reduced. Treatment-related adverse events included grade 1 or 2 fatigue (29 of 42 patients [69%]), nausea (23 [55%]), neuropathy (14 [33%]), and neutropenic fever (1 [2%]). Conclusions and Relevance These findings suggest that treatment with BIND-014 is active and well tolerated in patients with chemotherapy-naive mCRPC. Antitumor activity may be related to PSMA expression levels on CTCs, which suggests that patients who are likely to benefit from this treatment can be identified before treatment is initiated. Trial Registration ClinicalTrials.gov Identifier: NCT01812746

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

Importance A 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. Objective To 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 Participants This 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 Measures Overall survival after treatment with an ARS inhibitor or taxane in relation to pretherapy AR-V7 status. Results Among 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 Relevance This 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.

Abstract 4310: Predictive biomarkers of tumor sensitivity to STEAP1 antibody-drug conjugate (ADC) in patients (pts) with metastatic castration-resistant prostate cancer (mCRPC)

ADCs hold promise for enhancing the therapeutic index of cytotoxics. STEAP1 is overexpressed in mCRPC and is the target of an ADC currently under clinical development. To identify pts most likely to benefit from the ADC, we explored STEAP1 expression as a predictive biomarker in tumor tissue using a CLIA-certified IHC assay, and on circulating tumor cells (CTCs) in blood using the CellSearch and EPIC platforms. Sixty pts with progressive mCRPC received doses ranging from 0.3 to 2.8 mg/kg once every three weeks. Response was defined as a ≥50% decline in PSA from baseline, and time on study for >6 months was consistent with continued clinical benefit. At doses of ≥2 mg/kg, the response rate (RR) was 10/45 (22%, 95% CI 9.9-34.1) and was highest in the STEAP1 IHC 3+ group, as was the fraction of patients who remained on study for >6 months. At ≥2 mg/kg, 20/45 pts (44%, 95% CI 29.5-58.5) had unfavorable CTC counts of ≥5/7.5mL at baseline and were considered evaluable. After treatment, conversions from unfavorable to favorable CTC counts of An immuno-fluorescence-based assay developed to measure STEAP1 expression levels on CTCs showed readily detectable signal in 18/42 samples (43%, 95% CI 28-58), with a range from 1-56% of STEAP1-positive CTCs per case. After treatment, a decrease in the fraction of patients with STEAP1-positive and an increase in the STEAP1-negative CTCs was observed compared to baseline (see Table). We are prospectively selecting pts with STEAP1 IHC 2+/3+ tumors and assessing STEAP1 levels on CTCs in an ongoing Ph1 expansion study with the goal of developing a companion diagnostic to enrich for mCRPC pts most likely to benefit from treatment with the STEAP1 ADC. Citation Format: Daniel C. Danila, Howard I. Scher, Edith Szafer-Glusman, Amrita Herkal, Rebecca Suttmann, Martin Fleisher, Nicole A. Schreiber, Kristen Curtis, Houston Gilbert, Daniel Maslyar, Bernard Fine, Ron Firestein, Michael Mamounas, Mark R. Lackner, Omar Kabbarah. Predictive biomarkers of tumor sensitivity to STEAP1 antibody-drug conjugate (ADC) in patients (pts) with metastatic castration-resistant prostate cancer (mCRPC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4310. doi:10.1158/1538-7445.AM2015-4310

238PCHARACTERIZATION OF CIRCULATING TUMOR CELLS (CTCS) OF METASTATIC CASTRATION RESISTANT PROSTATE CANCER (MCRPC) PATIENTS IN FIRST, SECOND & THIRD LINE SYSTEMIC THERAPIES

ABSTRACT Aim: New androgen signaling directed therapies (AR Tx), including abiraterone acetate plus prednisone (AA + P) and enzalutamide (E), prolong survival in patients (pts) with mCRPC. Used sequentially, response to E given after AA + P is of shorter duration and less frequent. Assessing the evolution of the disease to a more resistant phenotype following each treatment is necessary to develop more effective treatment(s). We examined changes in CTC subtypes along with AR expression (exp) and AR localization (LOC) in pts receiving the 1st, 2nd and 3rd+ line of systemic therapy for mCRPC. Methods: 110 blood samples from 88 unique pts (26 with no prior tx, 32 with 1 prior tx, and 52 with 2+ prior tx) were analyzed for CTCs utilizing the Epic Sciences platform and CellSearch®. Epic analysis included identification of four specific CTC subtypes: traditional CTCs (CK + , CD45- cells, intact nuclei, morph distinct), CK- CTCs (CK-, CD45-, intact nuclei, morph distinct), small CTCs (CK + , CD45-, intact nuclei, small cell size), and CTC clusters. CTC AR exp and AR subcellular LOC were evaluated. Results: CTCs and CTC subtype frequency increased with lines of systemic therapies. Heterogeneity of CTC subtypes, AR exp and LOC also increased. CTC Events per 7.5mL of Blood 1st line (n = 26) 1st line (n = 26) ≥ 3rd line (n = 52) Mean PSA (range) 116.0 (0.7 - 1479.8) 170.2 (7.9 - 1516.1) 441.4 (8.5 - 2589.9) CellSearch® ≥ 5 CellSearch® CTCs 38% 50% 56% Epic ≥ 5 Traditional CTCs 92% 97% 94% Epic Mean of All Cell Events (range) 384 (0–4133) 238 (23–2985) 684 (8 - 9705) % Traditional CTCs 80% 59% 52% % Non-Traditional CTCs 20% 41% 48% Conclusions: The frequency of traditional CTCs, CTC Clusters, small CTCs, CK- CTCs, and high nuclear & cytoplasmic AR CTCs increased by line of therapy and increasing disease burden. The increased ratio of non-traditional to traditional CTCs with lines of tx may indicate an evolved, more resistant disease due to increased tx exposure. Increased CTC heterogeneity within and between pts suggests multiple mechanisms of resistance including constitutively active AR signaling, AR signaling independence, and epithelial plasticity. A relationship to disease burden cannot be excluded. This heterogeneity may explain the specific response to new therapies in the post AA + P & E mCRPC. Disclosure: H.I. Scher: Research funding from Janssen, Janssen Research, and Medivation Uncompensated advisor for Janssen, Janssen Research, and Medivation; J. Louw, R. Krupa, A. Jendrisak, D. Marrinucci and R. Dittamore: I am an employee and stockholder of Epic Sciences. D.E. Rathkopf: Research funding from Janssen, Medivation Uncompensated adviser for Janssen. All other authors have declared no conflicts of interest.