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BackgroundFor patients with advanced non-small cell lung carcinoma (NSCLC), immune checkpoint inhibitor (ICPI) and chemotherapy (chemo) ICPI represent two distinct first-line standard-of-care regimens without clear and established biomarkers to inform the optimal choice for individual patients. Here, we examined the complementary roles of tumor mutational burden (TMB) and programmed death ligand-1 (PD-L1) immunohistochemistry (IHC) to inform first-line therapy using a large real-world (rw) data set.Materials and methodsThe study included patients with NSCLC from an rw de-identified clinico-genomic database. All patients underwent genomic testing using Foundation Medicine’s tissue comprehensive genomic profiling assay and PD-L1 IHC assay scored for tumor cell staining (TS).ResultsOf 2165 patients included in the analysis, 150 exhibited durable benefit from first-line ICPI regimens (these patients were enriched for PD-L1 TS ≥50, non-squamous histology, and TMB ≥20 mutations/megabase (muts/Mb)). Comparing low TMB (ConclusionThis study provides evidence that higher TMB cut-offs, such as 20 muts/Mb, can identify patients with prolonged benefit from ICPI. TMB ≥20 muts/Mb is a potential biomarker that may identify patients in whom an ICPI without chemo could be considered, even in the setting of lower PD-L1 levels. Prospective validation of these findings could increase access to chemo-sparing regimens for the first-line treatment of advanced NSCLC.

AR-V7-expressing metastatic prostate cancer is an aggressive phenotype with poor progression-free survival (PFS) and overall survival (OS). Preliminary evidence suggests that AR-V7-positive tumors may be enriched for DNA-repair defects, perhaps rendering them more sensitive to immune-checkpoint blockade. We enrolled 15 metastatic prostate cancer patients with AR-V7-expressing circulating tumor cells into a prospective phase-2 trial. Patients received nivolumab 3 mg/kg plus ipilimumab 1 mg/kg every 3 weeks for four doses, then maintenance nivolumab 3 mg/kg every 2 weeks. Targeted next-generation sequencing was performed to determine DNA-repair deficiency (DRD) status. Outcomes included PSA response rates, objective response rates (ORR), PSA progression-free survival (PSA-PFS), clinical/radiographic PFS and OS. Median age of participants was 65, median PSA was 115 ng/mL, 67% had visceral metastases, and 60% had ≥4 prior systemic therapies. Six of 15 men (40%) had DRD mutations (three in BRCA2, two in ATM, one in ERCC4; none had microsatellite instability). Overall, the PSA response rate was 2/15 (13%), ORR was 2/8 (25%) in those with measurable disease, median PSA-PFS was 3.0 (95%CI 2.1–NR) months, PFS was 3.7 (95%CI 2.8–7.5) months, and OS was 8.2 (95%CI 5.5–10.4) months. Outcomes appeared generally better in DRD+ vs. DRD– tumors with respect to PSA responses (33% vs. 0%; P=0.14, nonsignificant), ORR (40% vs. 0%; P=0.46, nonsignificant), PSA-PFS (HR 0.19; P

Tailoring therapeutic regimens to individual patients with ovarian cancer is informed by severity of disease using a variety of clinicopathologic indicators. Although DNA repair variations are increasingly used for therapy selection in ovarian cancer, molecular features are not widely used for general assessment of patient prognosis and disease severity.To distill a highly dynamic characteristic, signature of copy number variations (CNV), into a risk score that could be easily validated analytically or repurposed for use given existing US Food and Drug Administration (FDA)-approved multigene assays.This genetic association study used the Cancer Genome Atlas Ovarian Cancer database to assess for genome-wide survival associations agnostic to gene function. Regions enriched for significant associations were compared to associations from scrambled data. CNV associations were condensed into a risk score, which was internally validated using bootstrapping. The participants were patients with serous ovarian cancer (stages I-IV) diagnosed from 1992 to 2013. Statistical analysis was performed from April to July 2020.Overall survival (OS).Among 564 patients with serous ovarian cancer, the mean (SD) age was 59.7 (11.5) years; 34 (6%) identified as Black or African American. A total of 13 genome regions, comprising 14 alterations, were identified as significantly risk associated. Composite risk score was independent of total CNV burden, total mutational burden, BRCA status, and open-source genome-wide DNA repair deficiency signatures. Binned terciles yielded high-, standard-, and low-risk groups with respective median OS estimates of 2.9 (95% CI, 2.3-3.2) years, 4.1 (95% CI, 3.7-4.8) years, and 5.7 (95% CI, 4.7-7.4) years, respectively (P .001). Associated 5-year survival estimates in each tercile were 15% (95% CI, 10%-22%), 36% (95% CI, 29%-46%), and 53% (95% CI, 45%-62%). The risk score had more discriminatory ability to prognosticate OS than age, clinical stage, grade, and race combined, and was strongly additive to significant clinical features (P .001). Simulated adaptation of FDA-approved assays showed similar performance. Gene ontology analyses of identified regions showed an enrichment for regulatory miRNAs and protein kinase regulators.This study found that a CNV-based risk score is independent to and stronger than current or near-future ovarian cancer genomic biomarkers to prognosticate OS. CNV regions identified were not strongly associated with canonical ovarian cancer biological pathways, identifying candidates for future mechanistic investigations. External validation of the CNV risk score, especially in concert with more extensive clinical features, could be pursued via existing FDA-approved assays.

Approximately 15% of men with newly diagnosed prostate cancer have high risk features which increase the risk of recurrence and metastasis. Better predictive biomarkers could allow for earlier detection of biochemical recurrence and change surveillance and adjuvant treatment paradigms. Circulating tumor cells are thought to represent the earliest form of metastases. However, their role as biomarkers in men with high risk, localized prostate cancer is not well defined.Two to 5 months after prostatectomy we obtained blood samples from 37 patients with high risk, localized prostate cancer, defined as stage T3a or higher, Gleason score 8 or greater, or prostate specific antigen 20 ng/ml or greater. Circulating tumor cells were enumerated using a commercial platform. Matched tumor and single circulating tumor cell sequencing was performed.Circulating tumor cells were detected in 30 of 37 samples (81.1%) with a median of 2.4 circulating tumor cells per ml (range 0 to 22.9). Patients with detectable circulating tumor cells showed a trend toward shorter recurrence time (p=0.12). All patients with biochemical recurrence had detectable circulating tumor cells. Androgen receptor over expression was detected in 7 of 37 patients (18.9%). Patients with biochemical recurrence had more circulating tumor cell copy number aberrations (p=0.027). Matched tumor tissue and single circulating tumor cell sequencing revealed heterogeneity.We noted a high incidence of circulating tumor cell detection after radical prostatectomy and shorter time to biochemical recurrence in men with a higher circulating tumor cell burden and more circulating tumor cell copy number aberrations. Genomic alterations consistent with established copy number aberrations in prostate cancer were detectable in circulating tumor cells but often discordant with cells analyzed in bulk from primary lesions. With further testing in appropriately powered cohorts early circulating tumor cell detection could be an informative biomarker to assist with adjuvant treatment decisions.

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.

The heterogeneity of an individual patient's 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.

The caspases are a family of ubiquitously expressed cysteine proteases best known for their roles in programmed cell death. However, caspases play a number of other roles in vertebrates. In the case of caspase-8, loss of expression is an embryonic lethal phenotype, and caspase-8 plays roles in suppressing cellular necrosis, promoting differentiation and immune signaling, regulating autophagy, and promoting cellular migration. Apoptosis and migration require localization of caspase-8 in the periphery of the cells, where caspase-8 acts as part of distinct biosensory complexes that either promote migration in appropriate cellular microenvironments, or cell death in inappropriate settings. In the cellular periphery, caspase-8 interacts with components of the focal adhesion complex in a tyrosine-kinase dependent manner, promoting both cell migration in vitro and metastasis in vivo. Mechanistically, caspase-8 interacts with components of both focal adhesions and early endosomes, enhancing focal adhesion turnover and promoting rapid integrin recycling to the cell surface. Clinically, this suggests that the expression of caspase-8 may not always be a positive prognostic sign, and that the role of caspase-8 in cancer progression is likely context-dependent.

A subset of castration-resistant prostate cancer (CRPC) patients present with atypical clinical features and aggressive disease behavior. These patients have a poor prognosis and may derive increased benefit from platinum-based chemotherapy. Heterogeneity in clinical presentations and insufficient understanding of biological drivers of progression underscore the need for more precise molecular stratification methods. We hypothesized that genomic characterization of circulating tumor cells (CTCs) at the single cell level would identify biomarkers relevant to the classification of aggressive CRPC tumors. Peripheral blood samples were collected before initiation of cabazitaxel +/- carboplatin from a cohort of 62 metastatic CRPC patients with and without clinically defined aggressive variant prostate cancer (AVPC) enrolled in NCT01505868. Samples were processed and analyzed for CTCs using the Epic Sciences detection and characterization platform in a CLIA-like environment. After CTC detection, single cells were picked and individually sequenced to assess gene gains and losses and genomic large-scale segment transitions (LST). Of 62 collected blood samples, 49 were positive for CTCs. Patients with more rapidly progressive disease (as assessed by a clinician) had higher CTC/mL, higher AR+CTC/mL, and higher median LST per CTC (p-values: 0.00669, 0.0358, and 0.00623, respectively). AVPC status, as marked by clinical features, was also associated with a higher number of LSTs in CTCs (median 24 per patient CTC vs. 10.5 in non-AVPC, p = 0.02672), whereas no difference in AR+CTC/mL or CTC/mL enumeration was observed. CTC count and LST were additive to prognosticate death by 12 months (AUC = 0.753 combined). The individual copy number was further assessed across 574 genes to identify alterations more commonly associated with poor prognosis. Myc gain, TP53 loss, and other aberrations in genes affecting DNA repair, resistance to anchorage-independent apoptosis, and cell motility showed significant positive association with LST. The presence of high genomic LST, a marker of genomic instability, in individual CTC was associated with more aggressive CRPC behavior (faster rate of progression, shorter survival) and specific gene copy number aberrations. High LST added independent prognostic significance to CTC enumeration. These data suggest that single CTC genomic analysis may provide clinically relevant insights and point to genomic instability in CTCs as a candidate marker of poor prognosis in advanced prostate cancer. Citation Format: Paymaneh D. Malihi, Ryon P. Graf, Carmen Ruiz Velasco, Anand Kolatkar, Angel E. Rodriguez, Jerry Lee, Ramsay Sutton, Paul G. Corn, Christopher Logothetis, Ana Aparicio, James Hicks, Ryan Dittamore, Amado Zurita, Peter Kuhn. Genomic instability as a marker of poor prognosis in advanced prostate cancer: Subclonal insights from whole-genome sequencing of single circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 448.

5075 Background: Prediction of ARSi benefit in mCRPC is an unmet medical need. Recently, the Epic Sciences CTC based nl AR-V7 test validated as a predictive biomarker in two multi-center validation studies and has received Medicare coverage for use in mCRPC. While the nl AR-V7 biomarker is highly specific to resistance and predictive of improved response with taxane Rx, it is a measure of just one mechanism of resistance to ARSis. CTC Het measured by the Shannon Index and CTC chromosomal instability measured by predicted number of Large Scale Transitions (pLST) have both been associated with poor OS to ARSis in previous analysis. Here we investigate the relationship of Het and pLST to nlAR-V7 in order to assess multi-clonal resistance and determine if these biomarkers can provide added sensitivity in the nlAR-V7 negative patient population. Methods: 275 blood samples from 2nd+ line mCRPC patients prior to treatment with ARSi (n=148) or taxanes (n=137) were obtained between 2012 and 2017 from 3 clinical centers. Detectable CTCs in each blood sample were assayed for nlAR-V7, Het, and pLST using the Epic Sciences platform. Biomarkers were analyzed in context of each other and outcomes including clinical co-variates. Results: 94% of samples had detectable CTCs, 84% were evaluable for Het analysis (> 2 CTCs), and 76% were evaluable for pLST (> 3 CTCs). Conclusions: Addition of CTC Het (Shannon Index) and CTC chromosomal instability (pLST) biomarkers to nlAR-V7 identifies an additional 15% of mCRPC pts (38% of total) that are predicted to have poor survival to AR signaling inhibitors. [Table: see text][Table: see text]

Background: Upregulation of HER2 and AR are mechanisms of acquired resistance to endocrine therapy, and are being investigated as treatment-guiding biomarkers. However, measurement of these proteins and their localization requires metastatic biopsies, which are costly, invasive, and prone to under-sampling which limits their utility to guide treatment in late stage metastatic patients. A CTC-based test could expand the clinical utility of these biomarkers. Here we utilized the Epic Sciences CTC platform for CTC detection and characterization. MBC blood samples were characterized for CTC prevalence, HER2 and AR expression at time of disease progression. Material and methods: HER2 and AR expression levels were determined based on model cell lines. A total of 72 blood samples were acquired from ER(+/-)/HER2(-) patients (by standard tissue pathology) at disease progression. 72 samples were analyzed for HER2 and 64 were analyzed for AR using the Epic Platform. Single-cell whole genome sequencing was performed to assess clonality and inter-patient heterogeneity of CTCs detected. Results: 55/72 (76.4%) of patients had CTCs detected across two slides. 13/72 (18.1%) had at least one HER2(+) CTC, 14/64 (21.9%) had at least one AR(+) CTC, and 7/64 (10.9%) had at both AR(+) and HER2(+) CTCs detected on replicate slides. HER2 expression on individual CTCs showed distinctive cytoplasmic membrane staining, and AR expression on individual CTCs showed frequent nuclear localization. Most patient samples showed heterogeneous expression of these markers at disease progression indicating subclonal sensitivity to targeted therapies. Subsequently, these cells will be individually sequenced to better determine the clonality of resistance. Conclusions: CTCs are detected in most MBC pts upon disease progression, with expression of known endocrine therapy resistance markers, HER2 and AR, observed that CTCs could guide subsequent therapy selection. Prospective evaluation of HER2 and AR on MBC pts' CTCs as predictive biomarkers of benefit from inhibitors of these proteins is needed. Citation Format: Ontiveros P, Landaverde C, Graf R, Levin MK, Hippely S, Wang Y, Landers M, Dittamore R, O'Shaughnessy JA. Enabling HER2 and androgen receptor (AR) protein expression and localization in circulating tumor cells (CTCs) of ER(+/-)HER2(-) metastatic breast cancer (MBC) patients (pts) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-07.

Procaspase-8, the zymogen form of the apoptosis-initiator caspase-8, undergoes phosphorylation following integrin-mediated cell attachment to an extracellular matrix substrate. Concordant with cell attachment to fibronectin, a population of procaspase-8 becomes associated with a peripheral insoluble compartment that includes focal complexes and lamellar microfilaments. Phosphorylation of procaspase-8 both impairs its maturation to the proapoptotic form and can promote cell migration. Here we show that the cytoskeletal adaptor protein CrkL promotes caspase-8 recruitment to the peripheral spreading edge of cells, and that the catalytic domain of caspase-8 directly interacts with the SH2 domain of CrkL. We show that the interaction is abolished by shRNA-mediated silencing of Src, in Src-deficient MEFs, and by pharmacologic inhibitors of the kinase. The results provide insight into how tyrosine kinases may act to coordinate the suppression caspase-8 mediated apoptosis, while promoting cell invasion.

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.

Background Circulating tumor cells (CTCs) expressing AR-V7 protein localized to the nucleus (nuclear-specific) identify metastatic castration-resistant prostate cancer (mCRPC) patients with improved overall survival (OS) on taxane therapy relative to the androgen receptor signaling inhibitors (ARSi) abiraterone acetate, enzalutamide, and apalutamide. Objective To evaluate if expanding the positivity criteria to include both nuclear and cytoplasmic AR-V7 localization ("nuclear-agnostic") identifies more patients who would benefit from a taxane over an ARSi. Design, setting, and participants The study used a cross-sectional cohort. Between December 2012 and March 2015, 193 pretherapy blood samples, 191 of which were evaluable, were collected and processed from 161 unique mCRPC patients before starting a new line of systemic therapy for disease progression at the Memorial Sloan Kettering Cancer Center. The association between two AR-V7 scoring criteria, post-therapy prostate-specific antigen (PSA) change (PTPC) and OS following ARSi or taxane treatment, was explored. One criterion required nuclear-specific AR-V7 localization, and the other required an AR-V7 signal but was agnostic to protein localization in CTCs. Outcome measurements and statistical analyses Correlation of AR-V7 status to PTPC and OS was investigated. Relationships with survival were analyzed using multivariable Cox regression and log-rank analyses. Results and limitations A total of 34 (18%) samples were AR-V7-positive using nuclear-specific criteria, and 56 (29%) were AR-V7-positive using nuclear-agnostic criteria. Following ARSi treatment, none of the 16 nuclear-specific AR-V7-positive samples and six of the 32 (19%) nuclear-agnostic AR-V7-positive samples had ≥50% PTPC at 12 weeks. The strongest baseline factor influencing OS was the interaction between the presence of nuclear-specific AR-V7-positive CTCs and treatment with a taxane (hazard ratio 0.24, 95% confidence interval 0.078-0.79; p=0.019). This interaction was not significant when nuclear-agnostic criteria were used. Conclusions To reliably inform treatment selection using an AR-V7 protein biomarker in CTCs, nuclear-specific localization is required. Patient summary We analyzed outcomes for patients with metastatic castration-resistant prostate cancer on androgen receptor signaling inhibitors and standard chemotherapy. Patients with circulating tumor cells that had AR-V7 protein in the cellular nuclei were very likely to survive longer on taxane-based chemotherapy, and tests unable to distinguish where the protein is located in the cell are not as predictive of benefit.

Background While programmed death 1 (PD-1) and programmed death-ligand 1 (PD-L1) checkpoint inhibitors have activity in a proportion of patients with advanced bladder cancer, strongly predictive and prognostic biomarkers are still lacking. In this study, we evaluated PD-L1 protein expression on circulating tumor cells (CTCs) isolated from patients with muscle invasive (MIBC) and metastatic (mBCa) bladder cancer and explore the prognostic value of CTC PD-L1 expression on clinical outcomes. Methods Blood samples from 25 patients with MIBC or mBCa were collected at UCSF and shipped to Epic Sciences. All nucleated cells were subjected to immunofluorescent (IF) staining and CTC identification by fluorescent scanners using algorithmic analysis. Cytokeratin expressing (CK)+ and (CK)−CTCs (CD45−, intact nuclei, morphologically distinct from WBCs) were enumerated. A subset of patient samples underwent genetic characterization by fluorescence in situ hybridization (FISH) and copy number variation (CNV) analysis. Results CTCs were detected in 20/25 (80 %) patients, inclusive of CK+ CTCs (13/25, 52 %), CK−CTCs (14/25, 56 %), CK+ CTC Clusters (6/25, 24 %), and apoptotic CTCs (13/25, 52 %). Seven of 25 (28 %) patients had PD-L1+ CTCs; 4 of these patients had exclusively CK−/CD45−/PD-L1+ CTCs. A subset of CTCs were secondarily confirmed as bladder cancer via FISH and CNV analysis, which revealed marked genomic instability. Although this study was not powered to evaluate survival, exploratory analyses demonstrated that patients with high PD-L1+/CD45−CTC burden and low burden of apoptotic CTCs had worse overall survival. Conclusions CTCs are detectable in both MIBC and mBCa patients. PD-L1 expression is demonstrated in both CK+ and CK−CTCs in patients with mBCa, and genomic analysis of these cells supports their tumor origin. Here we demonstrate the ability to identify CTCs in patients with advanced bladder cancer through a minimally invasive process. This may have the potential to guide checkpoint inhibitor immune therapies that have been established to have activity, often with durable responses, in a proportion of these patients. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2758-3) contains supplementary material, which is available to authorized users.

conclusively showed catheter position in the centre of SPN (26 cases) NIR spectroscopy probe based forceps were placed at the same place in order to gather tissue information. Mean measurement time was less than one minute after establishing ideal position. Results of spectroscopy measurements from NIR spectroscopy were obtained as differences between spectral characteristics of normal tissue (same side, different lobe) to SPN tissue. Results: The results are expressed as sensitivity of NIR spectroscopy towards EBUS navigated biopsies. Statistical analysis of the results showed very high sensitivity for NIR spectroscopy in confirmation of SPN tissue. From 26 EBUS positive visualisations of SPN there were 26 correct discriminations of SPN tissue, leading to 23 conclusive histological findings. Conclusions: Every confirmatory method brings different information about tissue. EBUS describes volume of the SPN and gives valuable information about the position of catheter in the SPN. NIR spectroscopy brings information about biochemical/ optical characteristics of the tissues. Prototype of intelligent NIR based forceps showed good discriminative ability in the diagnosis of SPN.

Genomic instability is a hallmark of cancer often associated with poor patient outcome and resistance to targeted therapy. Assessment of genomic instability in bulk tumor or biopsy can be complicated due to sample availability, surrounding tissue contamination, or tumor heterogeneity. The Epic Sciences circulating tumor cell (CTC) platform utilizes a non-enrichment based approach for the detection and characterization of rare tumor cells in clinical blood samples. Genomic profiling of individual CTCs could provide a portrait of cancer heterogeneity, identify clonal and sub-clonal drivers, and monitor disease progression. To that end, we developed a single cell Copy Number Variation (CNV) Assay to evaluate genomic instability and CNVs in patient CTCs. For proof of concept, prostate cancer cell lines, LNCaP, PC3 and VCaP, were spiked into healthy donor blood to create mock patient-like samples for downstream single cell genomic analysis. In addition, samples from seven metastatic castration resistant prostate cancer (mCRPC) patients were included to evaluate clinical feasibility. CTCs were enumerated and characterized using the Epic Sciences CTC Platform. Identified single CTCs were recovered, whole genome amplified, and sequenced using an Illumina NextSeq 500. CTCs were then analyzed for genome-wide copy number variations, followed by genomic instability analyses. Large-scale state transitions (LSTs) were measured as surrogates of genomic instability. Genomic instability scores were determined reproducibly for LNCaP, PC3, and VCaP, and were higher than white blood cell (WBC) controls from healthy donors. A wide range of LST scores were observed within and among the seven mCRPC patient samples. On the gene level, loss of the PTEN tumor suppressor was observed in PC3 and 5/7 (71%) patients. Amplification of the androgen receptor (AR) gene was observed in VCaP cells and 5/7 (71%) mCRPC patients. Using an in silico down-sampling approach, we determined that DNA copy number and genomic instability can be detected with as few as 350K sequencing reads. The data shown here demonstrate the feasibility of detecting genomic instabilities at the single cell level using the Epic Sciences CTC Platform. Understanding CTC heterogeneity has great potential for patient stratification prior to treatment with targeted therapies and for monitoring disease evolution during treatment.

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/m 2 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 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

Background: Expression of PD-L1 on tumor and immune markers in tumor tissue is associated with improved response to PD-1 and PD-L1 checkpoint inhibitors. However, each alone has limited predictive utility. Multimodal characterization of both the tumor and host immune system is an unmet medical need for the improved prediction of response to immunotherapy. Metastatic lesions are likely to be undersampled and require a liquid biopsy, given tumor heterogeneity and evolution and temporal changes in the host immune system. We sought to examine expression of PD-L1 on circulating tumor cells (CTCs) as well as characterize rare immune cell populations with a noninvasive liquid biopsy. Examining dynamic biomarker changes in longitudinal samples could enable the development of novel diagnostic tools for response prediction and pharmacodynamics studies related to immunotherapy. Methods: Blood samples from lung cancer patients were collected and shipped to Epic Sciences. Contrived samples were also developed, using cancer cell lines spiked into healthy donor (HD) blood. Red blood cells were lysed and nucleated cells were plated onto glass slides. Slides were stained with DAPI and immune cocktails, then imaged. Targets included pan-CK, CD45, PD-L1, CD4, CD8, Ki-67, and TIM-3. Approximately 3 million nucleated cells per slide were examined through advanced digital pathology pipelines to detect and quantify changes in T-cell populations and to assess circulating tumor burden. Results: Epic Sciences' rare cell detection platform has an analytically validated limit of detection of 2 cells/mL of blood. Immuno-panels were developed to profile leukocyte subpopulations and CTCs from a single blood sample and feasibility was demonstrated in cell lines, HD and lung disease patient blood. Quantitation experiments showed that immunomagnetically purified CD8+ and CD4+ cells spiked in 3:1, 1:1, and 1:3 ratios were detected as 3.1:1, 1:1.1, and 1:3.1. Purified CD4 cells were also spiked into healthy donor WBCs at target ratios of 1-10%. Percentages of detected spike-in CD4 cells were linearly correlated with the target ratios with correlation coefficient (r) = 0.96. 22.8% (18/79) lung cancer patients had PD-L1+ CTCs detected, and the majority (91%) of patients had PD-L1+ leukocyte populations. Conclusions: The low limit of detection of the Epic Sciences CTC platform coupled with the ability to archive patient blood samples allowed for retrospective, precise quantification of leukocyte subpopulations and PD-L1 expression on CTCs. Development of a liquid biopsy-based platform that can simultaneously measure immune biomarkers in CTCs as well as on leukocytes will allow for real-time assessment and monitoring of response to immune checkpoint inhibitors. Citation Format: Rachel Krupa, David Lu, Adam Jendrisak, Angel Rodriguez, Nadia Ebrahim, Robin Richardson, Sean Nisperos, Ryon Graf, Jiyun Byun, Yipeng Wang, Mark Landers, Ryan Dittamore. Simultaneous characterization and quantification of immune cell subpopulations and PD-L1 expressing CTCs in peripheral blood of cancer patients [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 5536.

1084Background: The choice between hormonal therapies and chemotherapy is a frequent decision in the care of mBCa pts. We previously developed quantitative measures of phenotypic CTC heterogeneity ...

5012Background: Response to PARPi in mCRPC patients (pts) particularly those with DNA damage response mutations (DDRm) was observed in the TOPARP trials (utilizing Olaparib) which enrolled heavily ...

5053Background: Biomarkers to predict outcomes for individual patients (pts) on standard of care Rx is an unmet medical need in the treatment of mCRPC. Using baseline (BL) blood draws, we previousl...

69 Background: Over 15% of men with newly diagnosed prostate cancer (PCa) have high-risk features that raise the recurrence risk. Better biomarkers could allow for even earlier detection of biochemical recurrence (BCR) and inform adjuvant treatment decisions. Circulating tumor cells (CTCs) may represent the earliest form of metastases, however their role as biomarkers in men with localized PCa is not well defined. Here, we aim to enumerate and molecularly and genomically analyze CTCs using an enrichment-free, unbiased CTC identification technology from men with high-risk, localized PCa after radical prostatectomy (RP) and correlate the analysis with clinical outcomes. Methods: Blood samples from 37 patients with high-risk, localized PCa were obtained 2-5 mos post RP and shipped to Epic. All nucleated cells were subjected to immunofluorescent staining for cytokeratin (CK), CD45, and AR. CTCs were identified using algorithmic analysis. CK+ CTCs were enumerated and subsequently analyzed for AR expression and individually sequenced for copy number alterations (CNA). Patients were followed for BCR, defined as detectable PSA > 0.2ng/dL. Progression free survival (PFS) was calculated using Kaplan-Meier and Cox proportional hazards. Results: CTCs were detected in 81.1%(30/37) of patients with an average of 5.2 CTCs/ml (range: 0 – 22.9) detected per patient. AR expression was detected in 18.9% (7/37) of patients. Ninety nine CTCs from 14 patients were picked and sequenced. CNAs were identified in CTCs in commonly mutated genes in PCa, including MYC amplification and CHD1 deletions. Patients with higher traditional CTC (CK+) burdens exhibited a trend towards shorter PFS (hazard ratio: 1.65; 95% confidence interval: 0.7-3.86; p = 0.13). Conclusions: There was a high incidence of CTC detection after RP in patients with high-risk, localized PCa. A trend toward shorter PFS was seen in those with higher CTC burden. Genomic alterations were detectable in CTCs and consistent with established CNAs in PCa. With further testing in appropriately powered cohorts early CTC detection after primary therapy could represent an informative biomarker to stratify patients with high risk PCa.

273 Background: A previous analysis of 161 patients (pts) tested for nuclear-localized AR-V7(+) CTCs showed a therapy interaction between AR-V7 positivity and improved overall survival (OS) on taxane chemotherapy vs. androgen receptor signaling inhibitors (ARSI). To validate the use of the biomarker result for physician decision making, we prospectively analyzed an independent, multicenter, blinded, cross-sectional cohort (n = 225) to confirm a therapy interaction with AR-V7. We corrected for possible pt selection bias by the treating physician by analyzing the association of therapy to OS in low and high risk groups defined by the test cohort. Methods: Two analyses were performed: (1) the validation of a therapy interaction between AR-V7 positivity and superior OS benefit for pts treated with taxanes in the context of use for 2nd+ line pts; and (2) as the choice between ARSI or taxanes was at the discretion of the attending physician, pt risk was incorporated into the predictive biomarker assessment. A pt-specific risk score was developed from line of therapy and other covariates to stratify pts as low and high risk, and the association of AR-V7 status and OS was performed within each risk group to correct for physician decision making and address possible confounding with treatment assignment. Results: (1) A therapy interaction between AR-V7(+) pts and lower risk of death on taxanes vs. ARSI (HR: 0.23, p = 0.003, 95% CI: 0.09 – 0.61) was validated. (2) In the validation cohort, high risk AR-V7(+) pts had an OS benefit when treated with taxanes (p = 0.02) and the AR-V7(-) pts had an improved OS with ARSI therapy (p = 0.02). AR-V7 incidence was low in the low risk pts, precluding the analysis for this sub-cohort. Conclusions: The results validate that the nuclear-localized AR-V7(+) biomarker has an interaction with therapy and improved survival on taxanes. Further, when adjusting for pt risk, the biomarker is predictive of OS in the high risk group. Nuclear-localized AR-V7 protein in CTCs can aid in the decision between ARSI and taxane chemotherapy in the 2nd or greater line of therapy for mCRPC.

Background: Expression of PD-L1 on tumor and immune markers in tumor tissueis associated with improved response to PD-1 and PD-L1 checkpoint inhibitors. However, the PD-L1 biomarker has limited predictive utility. Multimodal characterization of both tumor and host immune system is an unmet medical need for the improved prediction of immuno-oncology therapy. Given tumor heterogeneity and evolution and temporal changes to the host immune system, metastatic lesions are likely to be undersampled and require a liquid biopsy. We sought to examine the expression of PD-L1 on circulating tumor cells (CTCs) as well as characterize rare immune cell populations with a noninvasive liquid biopsy. Examining dynamic biomarker changes in longitudinal samples could enable the development of novel diagnostic tools for response prediction and pharmacodynamics studies related to immunotherapy. Methods: Blood samples from cancer patients were received and shipped to Epic Sciences. Contrived blood samples were also developed utilizing spike-in of cancer cell line controls into healthy blood samples. RBCs were lysed, and nucleated cells plated onto glass slides utilizing the Epic Sciences process. Slides were stained for nucleus DAPI in addition to IF cocktails and scanned, with targets including CK, CD45, PD-L1, CD4, CD8, Ki-67, LAG-3, and TIM-3. Approximately 3 million nucleated cells were examined through advanced Digital Pathology pipelines to detect and quantify the changes in T-cell populations to infer immune activation, exhaustion, suppression, and circulating tumor burden. Results: Epic’s rare cell detection platform has an analytically validated limit of detection of 1 cell/mL of blood. Three immuno-panels were developed to profile leukocytes subpopulations and CTCs simultaneously. 30 out of 33 lung cancer patients had PD-L1 leukocytes detected, and the incidences of PD-L1+ leukocytes widely ranged from 0 to 0.138% (average 0.0213%, median 0.0118%). PD-L1+ CTCs were observed in the presence of both high and low counts of PD-L1+ leukocyte populations. Conclusions: Epic Sciences CTC platform’s low limit of detection coupled with ability to archive patient blood samples allowed precise quantification of leucocyte subpopulations (CD4 and CD8, etc.) and PD-L1 expression on CTCs retrospectively. Development of a liquid biopsy-based platform that is capable of simultaneously measuring immune biomarkers in CTCs as well as leucocytes will allow real-time assessment and monitoring of response to immune checkpoints inhibitors. Citation Format: Adam Jendrisak, Angel Rodriguez, Nadia Ebrahim, Jiyun Byun, Ryon Graf, Yipeng Wang, Mark Landers, Ryan Dittamore. Simultaneous characterization of rare immune cell subpopulations and PD-L1-expressing CTCs in peripheral blood of cancer patients [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A058.

Retrospective analysis of patient tumour samples is a cornerstone of clinical research. CTC biomarker character‐ ization offers a non-invasive method to analyse patient samples. However, current CTC technologies require prospective blood collection, thereby reducing the ability to utilize archived clinical cohorts with long-term outcome data. We sought to investigate CTC recovery from frozen, archived patient PBMC pellets. Matched samples from both mCRPC patients and mock samples, which were prepared by spiking healthy donor blood with cultured prostate cancer cell line cells, were processed “fresh” via Epic CTC Platform or from “frozen” PBMC pellets. Samples were analysed for CTC enumeration and biomark‐ er characterization via immunofluorescent (IF) biomarkers, fluorescence in-situ hybridization (FISH) and CTC mor‐ phology. In the frozen patient PMBC samples, the median CTC recovery was 18%, compared to the freshly processed blood. However, abundance and localization of cytokeratin (CK) and androgen receptor (AR) protein, as measured by IF, were largely concordant between the fresh and frozen CTCs. Furthermore, a FISH analysis of PTEN loss showed high concordance in fresh vs. frozen. The observed data indicate that CTC biomarker characterization from frozen archival samples is feasible and representative of prospec‐ tively collected samples.

Purpose Androgen receptor (AR) is frequently detected in breast cancers, and AR-targeted therapies are showing activity in AR-positive (AR+) breast cancer. However, the role of AR in breast cancers is still not fully elucidated and the biology of AR in breast cancer remains incompletely understood. Circulating tumor cells (CTCs) can serve as prognostic and diagnostic tools, prompting us to measure AR protein expression and conduct genomic analyses on CTCs in patients with metastatic breast cancer. Methods Blood samples from patients with metastatic breast cancer were deposited on glass slides, subjected to nuclear staining with DAPI, and reacted with fluorescent-labeled antibodies to detect CD45, cytokeratin (CK), and biomarkers of interest (AR, estrogen receptor [ER], and HER2) on all nucleated cells. The stained slides were scanned and enumerated by non-enrichment-based non-biased approach independent of cell surface epithelial cell adhesion molecule (EpCAM) using the Epic Sciences CTC platform. Data were analyzed using established digital pathology algorithms. Results Of 68 patients, 51 (75%) had at least 1 CTC, and 49 of these 51 (96%) had hormone-receptor-positive (HR+)/HER2-negative primary tumors. AR was expressed in CK+ CTCs in 10 patients. Of these 10 patients, 3 also had ER expression in CK+ CTCs. Single cell genomic analysis of 78 CTCs from 1 of these 3 patients identified three distinct copy number patterns. AR+ cells had a lower frequency of chromosomal changes than ER+ and HER2+ cells. Conclusions CTC enumeration and analysis using no enrichment or selection provides a non-biased approach to detect AR expression and chromosomal aberrations in CTCs in patients with metastatic breast cancer. The heterogeneity of intrapatient AR expression in CTCs leads to the new hypothesis that patients with AR+ CTCs have heterogeneous disease with multiple drivers. Further studies are warranted to investigate the clinical applicability of AR+ CTCs and their heterogeneity.

Background: The NOTCH pathway has been identified as a key therapeutic pathway in SCLC. Tarextumab (TRXT, anti-Notch2/3, OMP-59R5) is a fully human monoclonal antibody that targets the Notch2 and Notch3 receptors. PINNACLE is a Phase 1b/2 trial of TRXT in combination with etoposide and platinum therapy (EP) in patients with untreated extensive stage small cell lung cancer (ES-SCLC). Baseline CTCs and post treatment changes in CTCs have previously been shown to predict the response to chemotherapy in SCLC. CTCs may also serve as pharmacodynamic biomarkers. Here we describe a study of baseline and longitudinal CTCs in ES-SCLC patients from the PINNACLE phase 1b trial (clinicaltrials.gov:NCT01859741). Materials and methods: CTCs, CTC clusters, apoptotic CTCs and N-Cadherin+ CTCs were identified and enumerated from patient blood samples using Epic Sciences CTC technology. Baseline CTCs from 26 patients were correlated with clinical outcome: progression-free survival (PFS), overall survival (OS) and best overall response, as well as metastatic status. A mixed effects model was used to investigate the post treatment changes in CTCs among the dose groups. Association of CTCs with PFS/OS and best overall response, including CTCs at each time point, as well as temporal changes of CTC status, were studied. Multivariate analysis was performed to identify CTC numbers in a subset of time points to correlate with response to treatment. Results: CTCs were present in 81% of the patients (21/26). CTC clusters and apoptotic CTCs were detected in 38% and 77% of the patients, respectively. At baseline, CTC counts ≥ 5/mL were significantly associated with poor OS (p=0.04). There was a trend that the presence of CTC clusters was associated with worse OS. With a cut-off of 3.4/mL, apoptotic CTCs showed a trend in association with overall survival. CTC numbers in patients with liver metastasis were significantly higher than in patients without liver metastasis. CTCs were also found to be correlated significantly with the number of metastatic sites. When measuring at Day 7 post dosing, CTC numbers were significantly decreased. Conclusions: Our findings suggest that CTCs are frequently detectable in patients and are a prognostic factor in ES-SCLC. CTCs decrease with TRXT and platinum-based chemotherapy. Updated results will be presented. CTCs will be further evaluated in the phase 2 portion of the PINNACLE trial. Citation Format: Chun Zhang, Ryon Graf, Adam Jendrisak, Amanda K. Anderson, Priscilla Ontiveros, Sarah Orr, Anne Chiang, David Spigel, Charles Rudin, Eric Holmgren, Jakob Dupont, Gretchen Argast, Leonardo Faoro, Lei Zhou, John Lewicki, Ann M. Kapoun. Circulating Tumor Cells (CTCs) in patients with extensive stage small cell lung cancer and their association with clinical outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1727. doi:10.1158/1538-7445.AM2017-1727

Background: Expression of PD-L1 on tumor tissue is associated with improved response to PD-1 and PD-L1 checkpoint inhibitors. Additionally, expression of PD-L1 on infiltrating T cells is associated with immune exhaustion. Currently, PD-L1 analysis as well as measures of infiltrating T cells are examined in surgically removed or biopsied samples, usually taken long before clinical decision points. Additionally, given tumor heterogeneity, metastatic lesions are likely to be under-sampled. We sought to examine expression of PD-L1 on circulating tumor cells (CTCs) and leukocytes cell populations with a non-invasive liquid biopsy. Examining dynamic biomarker changes in longitudinal samples could enable the development of novel diagnostic tools for response prediction and pharmacodynamics studies related to immunotherapy. Methods: Blood samples from cancer patients were received and shipped to Epic Sciences. Contrived blood samples were also developed utilizing spike-in of cancer cell line controls into healthy blood samples. RBCs were lysed, and nucleated cells plated onto glass slides utilizing the Epic Sciences process. Slides are stained with an IF cocktail (CK, DAPI, CD45, PD-L1) and scanned. Approximately 3 million nucleated cells are examined through advanced Digital Pathology pipelines to detect rare CTCs (CK+/-, cancer morphology, CD45-) and leukocytes (CK-, CD45+, leukocyte morphology) and analyze for PD-L1 expression. Results: A limit of detection of 1 cell/mL of blood was analytically validated. Using Epic’s rare cell detection platform, we could detect a wide range of PD-L1+ leukocyte counts in a subset of peripheral blood of cancer patients, observed as low as 0.003% (110 of 3,192,263) of total leukocytes. PD-L1+ CTCs were observed in the presence of both high and low counts of PD-L1+ leukocyte populations. Conclusions: Epic Sciences CTC platform’s low limit of detection coupled with ability to archive patient blood samples allowed precise quantification of PD-L1 expression on CTCs and leucocytes retrospectively. In addition, we have previously demonstrated the platform’s ability to detect leucocyte subtypes such as CD3, CD4 and CD8 cells, which will allow us to further characterize PD-L1 expression in T-lymphocytes and other immune cell types. Development of a liquid biopsy based platform that is capable of simultaneously measuring immune biomarkers in CTCs as well as leucocytes will allow real time assessment of response to immune checkpoints inhibitors. Citation Format: Adam Jendrisak, Jiyun Byun, Mahipal Suraneni, David Lu, Rachel Krupa, Sarah Orr, Ryon Graf, Yipeng Wang, Mark Landers, Ryan Dittamore. Quantification of rare PD-L1 expressing leukocytes and CTCs in peripheral blood of cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 683. doi:10.1158/1538-7445.AM2017-683

Background: Epithelial Plasticity (EP) is a proposed mechanism of immune escape, resistance to programmed cell death, resistance to specific drugs, and promotion of metastasis. EP has been extensively explored in mathematical, cell biological, and animal models, which predict the presence of these tumor cells in circulation. Most studies on CTCs in human subjects are based on enrichment of cells expressing EpCAM prior to enumeration or molecular interrogation, which precludes analysis of cells that might be EP. The non-enrichment Epic Sciences platform was utilized to identify CTCs in metastatic castration-resistant prostate cancer patient samples phenotypically consistent with EP: CTCs that do not express cytokeratins (CK), but do express malignant or EP biomarkers: Androgen Receptor (AR), AR splice variant 7 isoform (AR-V7) or N-cadherin. Phenotypic and genotypic analyses of individual CTCs were performed to assess the malignant origin of CK- CTCs and their prevalence associated with overall patient survival. Methods: 221 pre-therapy mCRPC blood samples were collected at Memorial Sloan-Kettering Cancer Center and shipped to Epic Sciences. Patients represent a diverse number of previous therapies and tumor burdens. All nucleated cells were plated to glass slides and subjected to DAPI and immunofluorescent (IF) staining of cytokeratin (CK), CD45 and another tumor marker (AR, AR-V7, or N-cadherin), followed by CTC identification by fluorescent scanners and malignant morphology. All samples were stained for AR, with a subset of samples stained for AR-V7 or N-Cadherin as well. Additionally, 83 AR(+)/CK(-) and 9 AR-V7(+)/CK(-) CTCs were analyzed for copy number variation (CNV) by low pass whole genome single CTC sequencing. Abnormal genomes were identified by amplifications and deletions of known oncogenes and tumor suppressors. Results: 3931 AR(+) CTCs were detected, of which 2996 (76%) expressed CK and 935 (24%) did not. The AR(+)/CK(-) CTCs had a lower median AR signal, less nucleoli, and more nuclear speckles by digital pathology vs. CK(+) CTCs. AR(+)/CK(-) and AR-V7(+)/CK(-) CTCs had gross genomic alterations consistent with malignant prostate origin (AR gain, PTEN loss, RB1 loss, 8q gain, 8p loss, etc), often sharing the same genomic alterations with CK(+) CTCs in the same patients, with some having unique CNV profiles Presence of AR(+)/CK(-) CTCs was associated with worse overall survival (median 13.7 mo vs. 33.9 mo, p < 0.0001) and remained additive and independent to treatment Line, pre-therapy PSA, and therapy type in multivariate models. Conclusions: Similar to CTCs expressing epithelial markers, CK(-) CTCs have gross genomic aberrations consistent with metastatic prostate cancer and are a negative prognostic factor associated with worse overall survival in both iterative threshold and multivariable analyses. Citation Format: Ryon P. Graf, Yipeng Wang, Nicole Schreiber, Brigit McLaughlin, Stephanie Greene, Angel Rodriguez, Adam Jendrisak, Jerry Lee, Mark Landers, Ryan Dittamore, Howard I. Scher. Phenotypic, genomic, and clinical associations of Circulating Tumor Cells (CTCs) lacking epithelial biomarkers in metastatic Castration Resistant Prostate Cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1740. doi:10.1158/1538-7445.AM2017-1740

5076 Background: Ra-223 prolongs life in mCRPC pts with symptomatic osseous metastasis with inconsistent effects on PSA. Survival times are prolonged further when combined with Abi/Enza. Data from preclinical studies suggest that Ra-223 may sensitize tumors to DDR agents and/or biologic therapies. But predictive biomarkers of benefit to each or both combinationso are lacking. We studied CTC counts and the prevelance of specific CTC subtypes in patients before and following Ra-223 therapy, both as a single agent and in combination, to identify biomarkers of sensitivity and treatement efficacy, and effects of Ra-223 on tumor biology. Methods: Pre and ~4 week post RA-223 therapy blood samples were collected from 35 pts (2 samples each) given as a single agent (n = 20 pts) or in combination with other therapies (n = 15 pts, 9 w/ Enza, 5 w/ Abi, 1 w/ Taxane). Samples were processed and CTCs analyzed using the Epic Sciences platform. Total CTC count and the prevalence of specific CTC phenotypes present pre and post Rx were identified utilizing high content digital pathology and associated with therapy type and post-treatment change. Results: CTC declines were observed in 55% (11/20) and 60% (9/15) of pts treated with single agent and combination respectively. In Ra-223 alone pts, a novel CTC subtype (high N/C ratio, high nuclear area) was identified at baseline 11/20 samples (med = 33% of CTCs). Which was no longer detected in 10 (90%) of the pts treated. This contrasts with a second novel CTC subtype present at baseline in 4 pts (med CTC = 9%) that increased to 9 cases (med CTC = 18%) at follow-up. Conclusions: A subset of pts demonstrate post-therapy CTC declines following Ra-223 alone or in combination. A novel CTC subtype resolved by RA-223 in conjunction with total CTC kinetics may indicate pt benefit from Ra-223. A novel emergent CTC subtype has also been identified in pts already receiving Ra-223. Single CTC sequencing and protein analysys of these CTC subtypes are ongoing, and may help describe tumor evolution and sensitization to novel therapuetics.

110 Background: Approximately 15% of men with newly diagnosed prostate cancer (PCa) have high-risk features, many of these patients will recur despite definitive therapy. Better predictive biomarkers could allow for earlier detection of recurrence and change surveillance paradigms. The role of circulating tumor cells (CTCs) as biomarkers in this context is not well defined. Here, we evaluate the ability to detect CTCs from men with high risk, localized PCa after radical prostatectomy (RP) and correlate their presence with prospective clinical data. Methods: Blood samples from 31 patients with high risk, localized PCa were obtained 2-4 months post RP and sent to Epic Sciences on an IRB approved protocol. Nucleated cells were subjected to immunofluorescent (IF) staining for cytokeratin (CK), CD45, and AR N-terminus. CTCs were identified by fluorescent scanners using algorithmic analysis. Cytokeratin expressing (CK+) CTCs were enumerated and subsequently analyzed for AR expression and individually sequenced for copy number variation (CNV) and large scale transitions (LST, a surrogate of genomic instability). Patients were followed prospectively for biochemical recurrence, defined as detectable PSA. Progression free survival (PFS) was calculated using Kaplan-Meier and Cox proportional hazards. Results: CTCs were detected in 87.1% (27/31) samples with an average of 5.6 CTCs/ml (range: 0 – 22.87) detected per patient. AR expression was detected in 12.9% (4/31) of patients. Ninety-nine CTCs from 14 patients were amenable to LST and CNV analyses. 10.1% (10/99) CTCs from 7 patients exhibited higher ( > = 6) LSTs than control WBCs (95% WBCs had LST < 6). Copy number alterations were detected in CTCs in commonly mutated genes in PCa, including AR, MYC, and TP53 amplification and deletions in PTEN and RB1. Patients with higher CTC burdens exhibited a trend toward shorter PFS (hazard ratio: 1.65; 95% confidence interval: 0.7-3.86; p: 0.13). Conclusions: There was a high incidence of CTC detection after RP in this population using a novel platform. We observed a trend toward shorter PFS in those with higher CTC burden. Genomic alterations were detectable in CTCs and consistent with established CNAs in PCa.

5034 Background: Loss of the retinoblastoma tumor suppressor (RB) function was identified as a major means to develop CRPC; the expression of the androgen receptor (AR) is under stringent RB control; and tumors devoid of RB function are hypersensitive to treatment with chemotherapy. Exploratory analysis evaluated baseline N-terminal AR expression in CTCs in men with chemotherapy-naïve CPRC and correlated to changes in PSA, leading us to inquire if this biomarker may identify pts sensitive to chemotherapy. Methods: In a multicenter phase II randomized trial of approved doses of abiraterone acetate/prednisone (AA-Arm 1) or combination AA and standard doses of cabazitaxel (AA/CBZ-Arm 2). Patients on AA received CBZ upon progression. Baseline CTCs were obtained on all pts and expression of N-terminal AR expression was performed by Epic Sciences. Positive AR N-terminal expression (AR+) was based on the presence of at least 1 CTC or CK- cell with AR N-terminal signal expression above the 3.0 positivity threshold. Serial PSAs were determined at baseline and every 3 weeks with routine labs and imaging every 12 weeks. Results: To date, 42 of 80 pts have been enrolled: 22 pts to AA, and 20 pts to AA/CBZ. Both regimens were well tolerated with 8/42 (19%) pts experiencing treatment-related grade 3 or 4 toxicities. Blood from 35 patients underwent CTC analysis. Seventy-seven percent of pts (27/35) had detectable CTCs; 11 of 35 pts (31%) had AR overexpression. Of the pts with AR+ CTCs, 1/5 pts treated with AA, and 5/6 pts treated with AA/CBZ had a PSA decline > 50% from baseline. Conclusions: Real-time CTC analysis of N-terminal AR expression was feasible and data suggests that this may identify a cohort of pts who may benefit from the combination of CBZ with AA. Further studies are ongoing to evaluate whether cellular heterogeneity and RB expression in CTCs play a role in identifying pts who would benefit from chemotherapy. The trial is coordinated by the Prostate Cancer Clinical Trials Consortium, LLC and funded by Sanofi US Services Inc. and Prostate Cancer Foundation. Clinical trial information: NCT02218606.

5070 Background: Prior response to A or E does not predict sensitivity to E following A or A following E. The detection of AR-V7 predicts insensitivity to either drug, but identifies only a portion of non-responders. We previously identified 15 CTC subtypes based on unique phenotypic features in mCRPC pts, each with unique biology and different degree of likelihood of predicting resistance to either drug. Here we explored the relationship between individual subtypes and sensitivity to A vs. E, but not both. Methods: 107 pre-treatment blood samples from mCRPC pts starting A (n = 47) or E (n = 60) as a 1st or 2nd line of Tx were analyzed for CTCs utilizing the Epic Sciences platform. Samples were assayed for CTC subtypes based upon 15 pre-defined phenotypic CTC classifiers (Type A-O). Treatment outcomes were assessed by serial PSA changes and landmarked percent time of therapy progression on radiographs, and overall survival following either A or E. Cell type prevalence was also analyzed in relation to clinical outcomes, and subsets of the CTC subtypes subject to single cell NGS to ascertain genomic drivers common to each subtype. Results: CTCs were identified in 94% (101/107) of pt samples. One, cell Type K, found in 25% (27/107) of pts, was associated with a statistically significant inferior outcomes on E for all measures. Whereas similar outcomes were seen between K+ & K- pts treated with A. The distinct features of Cell Type K include a large nucleus, high nuclear entropy and high Nuclear/cytoplasmic AR terminal ratio; and a unique genomic profile enriched for cell cycle and DNA repair alterations relative to other CTC subtypes. Conclusions: The presence of specific CTC subtypes in pre-Rx phlebotomy samples associated with outcomes on A or E. A CTC subtype (Cell Type K) helped to identify pts with poor outcomes on E but not A vs. those without the cell type. Further biologic interrogation of K cells and ongoing clincial validation of the CTC subtype is planned. [Table: see text]

139 Background: Genomic profiling of metastatic tumors can identify mCRPC pts w/ “actionable” targets. However, responses from this approach are infrequent and not durable, broadly attributed to intra-tumor and intrer-cellular het. We sought to understand relationships between single CTC phenotypic and genomic patterns present when starting ARSi or taxane therapy (Tx) to develop more effective biomarker-directed treatment approaches. Methods: 9225 CTCs from 319 blood samples from 179 mCRPC pts were detected and digital phenotypically profiled using the Epic Sciences platform. Unsupervised clustering revealed 15 phenotypic CTC subtypes based on digital pathology features (Type ‘A’-‘O’). Phenotypic diversity was determined by a Shannon index of these subtypes in pt samples. Next, 856 CTCs representing each subtype were individually whole genome sequenced for copy number variation and genomic instability, to define phenogenomic single cell profile and taxonomy. Tx outcomes were associated with CTC profiles. Results: Pre-therapy identification of certain CTC phenotypic subtypes was associated with poor overall survival (OS) following ARSi and/or taxanes. 5/15 subtypes existed primarily in pts w/ high CTC het (84-94%, Bonferroni adjusted p < 0.05, permutation test), and linked to amp of AR and cMYC, and loss of TP53 and PTEN. Clusters of genomic events were linked to phenotypic features (i.e. small cell CTC w/AuroraK & nMYC amp) and provide single cell phenogenomic associations with pathways such as AR/PTEN feedback and DNA repair machinery. Conclusions: Presence ofspecific CTC subtypes was associated w/ poor survival on ARSi and/or taxanes and may inform Tx selection. Identifying CTC subtypes present in high het samples can provide insight into individual pt disease evolution and potential approaches to maximize benefit. Longitudinal monitoring of CTC phenogenomics is being studied in novel Tx trials.[Table: see text]

Background: A critical decision in the management of patients (pts) with mCRPC is when to administer an androgen receptor signaling (ARS) directed or a taxane therapy. The detection of AR-V7 mRNA in CTCs has been shown to predict for resistance to ARS,but not to taxane chemotherapy. We evaluated the relationship between AR-V7 protein expression and localization on CTCs to treatment outcomes in a separate, larger cohort as a predictive biomarker for clinical decision making. Methods: 193 prospectively collected blood samples from 161 unique pts with progressive mCRPC about to start an ARS or taxane therapy were evaluated with an Epic Sciences CTC immunoflorescent assay that assesses CTC AR-V7 protein expression and localization in individual cells. Associations between the presence AR-V7(+) CTCs pre-therapy and anti-tumor effects post-therapy included prostate-specific antigen (PSA) changes, radiographic progression free survival (rPFS), time on therapy, and overall survival (OS). Results: 130 pre-ARS inhibitor and 63 pre-taxane samples were assessed of which 191 (99%) were evaluable. AR-V7(+) CTCs were found in 34 (18%) samples including 3% of the 1st, 18% of the 2nd and 31% of the 3rd+ line baseline pre-therapy samples. Patients with AR-V7 positive CTCs in a pre-ARS sample showed no PSA response and had shorter rPFS, time on therapy, and OS than those without AR-V7(+) CTCs. PSA resistance to ARS was also seen in 65 of 112 (58%) of the AR-V7(-) samples. There was no association between PSA response, rPFS, and time on therapy between AR-V7(+) and AR-V7(-) pts treated with taxane therapy. In a multivariate model adjusting for age, type of therapy, line of therapy, and pre-therapy LDH, Hgb, and presence of visceral metastasis, AR-V7(+) pts had a superior OS on taxane therapy relative to ARS (HR: 0.242, CI: 0.103 to 0.569, p = 0.0350). Conclusions: The results validate the expression of the AR-V7 protein in the nucleus of CTCs in men with mCRPC as a treatment specific biomarker that predicts resistance to ARS inhibitor therapy and separately, clinical benefit with taxane therapy over ARS-directed therapy in a clinical practice setting. Continued examination of this biomarker in prospective studies will further determine its clinical utility. Citation Format: Howard I. Scher, David Lu, Nicole A. Schreiber, Jessica Louw, Ryon P. Graf, Ann Johnson, Adam Jendrisak, Glenn Heller, Richard Bambury, Herbert A. Vargas Alverez, Brigit McLaughlin, Justin Wahl, Stephanie Greene, Martin Fleisher, Ryan Dittamore. Nuclear localized AR-V7 protein as a predictive biomarker for treatment selection in metastatic castration resistant prostate cancer (mCRPC). [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 4954.

Background: Androgen receptor (AR) is a nuclear transcription factor and a member of the steroid hormone receptor superfamily that can be detected by immunohistochemical staining in all three subtypes of invasive breast cancer with frequency of >70% of estrogen receptor-positive tumors (ER+), >60% of HER2-positive tumors (HER2+), and 40-50% of triple-negative breast cancers (TNBC) respectively. AR-target therapies are showing early activity in AR+ breast cancer. Given early evidence of activity with agents targeting AR, it is desirable to improve our understanding of the AR biology in breast cancer. Although circulating tumor cells (CTCs) have been proposed as a liquid biopsy for biomarker detection in multiple cancer types, the rate and heterogeneity of AR expression in CTCs in breast cancer is still unclear. In this study, we determine the rate and heterogeneity of AR expression in CTCs of patients with metastatic breast cancer. Methods: Blood samples from patients with metastatic breast cancer were smeared onto glass slides and subjected to nuclear staining with DAPI and reacted with fluorescent-labeled antibodies to detect CD45 on leukocytes and cytokeratin and AR on epithelial cells; the slides were scanned on the Epic Sciences CTC platform and the data analyzed using established algorithms. Results: Among the 59 patients analyzed, 25 (42.4%) had ER+/HER2-, 12 (20.3%) had ER+/HER2+, 6 (10.2%) had ER-/HER2+, and 16 (27.1%) had TNBC based on the most recent staining. CTCs were detected in 50 of 59 (84.7%) patients. Characterization of CTCs identified CK+ CTCs (45 of 59, 76.3%), CK-negative CTCs (16 of 59, 27.1%), CK+ CTC clusters (16 of 59, 27.1%), and apoptotic CTCs (45 of 59, 76.3%), respectively. Number of CTCs per patient ranged from 0 to 282.1 per mL (median = 4.4). Six of the 50 patients (12%) with CTCs had AR+ CTCs. The number of AR+ CTCs per patient ranged from 0.3 to 52.6 per mL (median = 1.15). Among the patients with AR+ CTCs, the percentage of CTCs that were AR+ ranged from 5% to 50% (median = 21%). Among the six patients with AR+ CTCs, five patients had ER+ breast cancer and one had TNBC. Conclusions: We demonstrated that the Epic Sciences non-enriching or selecting platform can detect AR expression in CTCs in breast cancer. These preliminary results suggest the need for clinical validation of CTC AR expression as a potential test to stratify and identify patients who might benefit from AR therapy. The heterogeneity of intra-patient CTC AR expression leads us to another hypothesis that patients with AR+ CTCs might have heterogeneous disease with multiple drivers. Further studies are warranted to allow serial monitoring of changes in AR and to investigate the clinical applicability of AR+ CTCs and their heterogeneity. Citation Format: Takeo Fujii, Yipeng Wang, James M. Reuben, Rachel Krupa, Ryon Graf, Lyndsey Dugan, Jessica Kouw, Dena Marrinucci, Bora Lim, Carlos H. Barcenas, Angela N. Marx, Debu Tripathy, Ryan Dittamore, Naoto T. Ueno. Androgen receptor expression on circulating tumor cells in metastatic breast 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 496.

8524Background: Lung cancer pts currently require biopsies to diagnose and profile tumors prior to treatment. There is a need for tumor profiling from circulating biomarkers to avoid invasive proce...

4527Background: Recent studies indicate that PD-1 and PD-L1 checkpoint inhibitors have activity in chemotherapy refractory patients with muscle invasive (MIBC) and metastatic (mBCa) bladder cancer....

5013Background: An unmet need in the management of mCRPC is how to use approved AR signaling inhibitors (AR tx) and taxanes (T) to maximize survival for the individual patient. Pts with AR-V7+ CTCs...

5044Background: Prediction of pt response to AR Tx or T is an unmet medical need, as pts may respond to both classes of drug, to one but not the other, or have no response to either. Molecular prof...

446 Background: Recent studies indicate that PD-1 and PD-L1 checkpoint inhibitors have activity in chemotherapy refractory patients with muscle invasive (MIBC) and metastatic (mBCa) bladder cancer. PD-L1 expression on tumor cells or lymphocytes may correlate with response to therapy. To identify potential patients who may benefit from PD-1/PD-L1 targeted immunotherapeutics, we utilized a non-invasive blood test to evaluate PD-L1 protein expression in CTCs and WBCs of bladder cancer patients. Methods: Whole blood from 20 patients with MIBC or mBCa were collected and shipped to Epic Sciences. All nucleated cells were plated onto glass slides and subjected to immunofluorescent (IF) staining and CTC identification using scanners and algorithmic analysis. CTCs, defined as traditional (CK+ CD45- w/ intact DAPI+ nuclei and morphologically distinct) or CK- (CK-, CD45-, DAPI+, intact and distinct) were identified. PD-L1 biomarker characterization was assessed by IF staining. UroVysion FISH testing was used to assess genomic abnormalities in a subset of patient samples. Additionally, WBCs (CD45+ cells) were assessed for PD-L1 expression, relative to healthy controls. Results: Eighty percent of patients had mBCa and 20% had MIBC, 70% percent were men, and themedian age was 67 years, (range 43 to 88). Traditional CTCs were detected in 11/20 (55%) patients. Seven out of 20 (35%) patients had PD-L1+ cells, 4 of these patients had exclusively CK-/PD-L1+ CTCs, a subset of which were confirmed as malignant via FISH. CK- CTCs were detected in 14/20 (70%) patients. Five patients had greater than 4-fold PD-L1 positivity in WBCs as compared to healthy donors. Conclusions: Patients with MIBC and mBCa patients have detectable, heterogeneous CTCs with a population of CK-/PD-L1+ CTCs. Utilization of a liquid biopsy to identify patients with PD-L1+ CTCs and PD-L1+ WBCs may enable patient selection or short term therapeutic monitoring for measuring therapeutic efficacy. Further work will investigate association of PD-L1+ CTCs and WBCs with clinical response to PD-1 checkpoint immunotherapy.

163 Background: AR directed Tx, including Abiraterone (A) and Enzalutamide (E), prolong survival in patients with mCRPC and are FDA approved. After primary Tx with either A or E, the response to E or A is decreased as is the duration of response. NGS tissue sequencing of mCRPC shows a range of disease phenotypes and genotypes which are associated with increased tumor heterogeneity. Single site biopsy, may not represent the diversity of disease. Our objective is to develop biomarkers of heterogeneity from the analysis of single cells from patients with mCRPC. Methods: 221 blood samples from 179 unique patients (pts) were collected from pts about to begin AR Tx (n = 150) or Taxanes (n = 71). Samples were analyzed with the Epic Sciences platform. Analysis included digital pathology of 20 discrete phenotypic cell features inclusive of AR, CK, size and shape measures. 9225 single CTCs were characterized, data standardized, features clustered and categorized into 15 phenotypically distinct CTC subtypes. Individual pt samples were then analyzed for the frequency and heterogeneity (Shannon Index) of CTC subtypes and monitored for clinical endpoints. A subset of CTCs (n = 350) were individually sequenced and analyzed for clonality and CNV to assess genomic heterogeneity. Results: A diversity of CTC phenotypes were observed at each line of therapy. In AR Tx pts, high heterogeneity predicts 6mo PFS (OR = 2.57, p = 0.01) and shorter OS (HR = 4.5, p < 0.001). Separately, specific CTC phenotypes also predict 6mo PFS (OR = 5, p < 0.001) and shorter OS (HR = 5.8, p < 0.001). Heterogeneity scores were not associated with taxane resistance. NGS analysis identified subclonal drivers of disease progression including multiple private and/or clustered genomic drivers. Conclusions: Single CTC characterization of phenotype and genotype supports the concept of tumor heterogeneity as a driver of disease resistance. The heterogeneity observed supports the potential value of single cell characterization to identify rare somatic sub-clonal alterations and can aid in the development of rational therapeutic trials.

302 Background: CellSearch is an FDA cleared assay that enumerates intact CK+, DAPI+, CD45- cells via EpCAM capture. CTC number is prognostic for survival pre- and post- systemic therapy. These cells are a subset of confirmed genomically altered CTC’s in blood. We report the prognostic significance for OS in mCRPC of CTC subtypes not enumerated by CellSearch including apoptotic CTCs, CK- CTCs, small CTCs, and CTC clusters. Methods: 221 blood samples from 179 unique patients were collected from pts about to begin AR directed (n = 150) or taxane (n = 71) therapy for progressive CRPC. Samples were analyzed utilizing the Epic Sciences platform to enumerate traditional (CK+, abnormal morph), apoptotic (CK+, fragmented nuclei), CK- (CK-/abnormal morph), small (CK+, normal morph), and CTC clusters. Patients were followed for up to 2.3 yrs. Paired CellSearch and Epic traditional CTC counts were collected on 173 patient samples. Results: In paired analysis, Epic traditional CTCs were identified in 86% of patients vs. 67% by CellSearch, and any Epic CTC subtype was found in 96% of the patient samples. Increased frequency of any CTC subtype was a univariate predictor of OS (see table). Conclusions: Epic identified CTC subtypes not identified by CellSearch or other technologies that select or sort based upon size or epithelial expression. Cells were detected in 96% of the patient samples and all 5 subtypes were found to be prognostic for survival. The biologic characterization of CTC subtypes as a biomarker of drug sensitivity is ongoing. The increased clinical sensitivity of subtypes and prevalence of CTCs identified on the Epic platform provides a greatly expanded array of clinically relevant biomarkers to develop predictive liquid biopsy signatures in the metastatic setting. [Table: see text]

Background: Mostly asymptomatic until late stage, colorectal cancer is driven by the successive accumulation of genetic alterations resulting in genomic instability within subclonal tumor populations. mCRC often progresses as a subclonally diverse multifocal disease due to selective therapeutic pressure, the surrounding tumor microenvironment, and underlying genomic heterogeneity. Targeted therapies against EGFR, VEGF or BRAF have shown increased response in a subset of patients; however, patient stratification using standard population analysis of DNA markers from tumor biopsy, (i.e. chromosomal instability, microsatellite instability, promoter methylation, resistance mutations), is problematic due to tumor heterogeneity. CTCs reflect the active metastatic subclonal populations at any given time, making single cell analysis of DNA markers a more accurate, real-time picture of cumulative metastatic diversity. Using Epic's enrichment-free CTC analysis platform, we characterized individual CTCs from a mCRC patient to understand the extent of intra-patient genomic heterogeneity, including the presence genomic instability and point mutations. We compared the prevalence of clinically relevant subclonal alterations within patient CTCs to CRC TCGA data, offering insights into identification of therapeutic opportunities and potential mechanisms of resistance. Methods: Blood was collected from a heavily pretreated mCRC patient and was processed for CTC analysis using the Epic Platform. 34 CTCs were individually recovered, lysed, whole genome amplified, constructed into shotgun libraries and target enriched for all coding regions of 500 pan-cancer genes. Enriched libraries were sequenced to an average depth of 697X coverage by 2×150 PE sequencing. Sequences were aligned and somatic mutations were determined using VarScan with the patient's WBC as germline reference. Variants were filtered for functional gain- or loss-of-function mutations by SIFT/PolyPhen2 and selected based on low frequency in 1000g database. Genomic instability and loss of heterozygosity (LOH) was also assessed. Somatic variants deriving from the patient CTC cohort and TCGA CRC cohort of 302 patients were annotated, analyzed, and compared using GenePool™ software (Station X). Results: MLL3 alterations, frequently observed in primary CRC biopsies (14%), were identified in 70% of all CTCs sequenced. Previously cited somatic variants were detected in minor subclonal populations of CTCs, including APC (12%), BRCA1/2 (8%), KRAS (6%), PI3KCA (6%) and TP53 (6%). A wide range of genomic instabilities and LOH was also observed across CTCs. Conclusions: The Epic CTC platform is suited to identify subclonal population of CTCs harboring clinically relevant genomic alterations on a single cell level, which can inform clonal drift, identify rare clonal populations, and enable patient stratification at higher resolution. Citation Format: Stephanie Greene, Jerry Lee, Mark Landers, Sandeep Sanga, Adam Jendrisak, Ryon Graf, Jessica Louw, Shannon Werner, Yipeng Wang, Ryan Dittamore, Dena Marrinucci. Single cell genomic profiling of circulating tumor cells (CTCs) from metastatic colorectal cancer (mCRC) identify tumor heterogeneity and rare somatic driver alterations. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A35.

Introduction: CTCs hold promise as prognostic and predictive biomarkers for cancer management. Many CTC technologies enrich CTCs based on cell surface markers or physical parameters such as cell size. However, genomic and phenotypic intra-tumor heterogeneity is well documented and similarly observed in CTCs. We sought to validate the Epic Platform, an enrichment-free approach, for CTC identification and biomarker characterization. In addition, we sought to enumerate and molecularly characterize CTCs in Metastatic Castrate Resistant Prostate Cancer (mCRPC) patients. Experimental Procedures: To assess intra/inter-assay and -operator reproducibility, cell line cells (CLCs) were spiked into healthy donor (HD) blood and processed onto slides (∼0.5mL/slide) in dilutions ranging from 6-300 CLCs/slide and assessed by%CV of CLC counts in replicate samples. Accuracy and linearity were assessed via linear regression and slope of all dilutions. CLCs were also molecularly characterized by immunofluorescence (IF), FISH, and NGS. To test clinical sensitivity, ∼1mL of blood from 28 mCRPC patients was processed. CTCs (CK+, CD45-, DAPI+) and CTC subtypes including apoptotic (CK+, CD45-, fragmented DAPI), and CK- (CK-, CD45-, DAPI+) were enumerated. Androgen receptor (AR) expression was quantitated on each CTC. A subset of samples were further analyzed for ERG rearrangements via FISH and whole genome copy number variation (CNV) by NGS. In addition, 18 HDs were tested to assess clinical specificity. Results: Intra/inter-assay and operator reproducibility across 3 runs each produced ≤ 20% CV at 300 CLC/slide and ≤ 30% CV at 25 CLC/slide. The CTC enumeration assay demonstrated accuracy, linearity and sensitivity throughout all dilutions tested, 6-300 CLC/mL, (x = 1.15, r2 = 0.9987,%CV ≤ 30%). CLCs exhibited expected biomarker expression (IF) and genetic aberrations (FISH and NGS). No CTCs or CTC subtypes were detected in HD samples. At least 1 CTC/mL was detected in 82% of mCRPC patient samples. Including CK- and apoptotic CTCs, 100% of mCRPC patients had at least 1 CTC/mL. Analysis of AR expression/localization, ERG rearrangements by FISH, and whole genome CNV analyses were consistent with malignant origin. Conclusions: We established the reproducibility, linearity, accuracy, and sensitivity of the Epic Platform. CTCs and CTC subtypes were absent in HDs and present in all mCRPC samples tested. We also demonstrated integrated IF, FISH, and NGS characterization of single CTCs, which has powerful implications for improved patient selection for targeted therapy and could enable a better understanding of intra-patient heterogeneity, clonal evolution, and drug resistance and response over time. Citation Format: Ryon P. Graf, David T. Valenta, Mark Landers, Bryan Taggart, Stephanie Greene, Shannon L. Werner, Ryan Dittamore, Dena Marrinucci. Analytical and clinical validation of the Epic Circulating Tumor Cell (CTC) Platform: enrichment-free CTC detection and biomarker characterization. [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 1584. doi:10.1158/1538-7445.AM2015-1584