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Purpose: Men with metastatic castration-resistant prostate cancer (mCRPC) frequently develop resistance to androgen receptor signaling inhibitor (ARSI) treatment; therefore, new therapies are needed. Trophoblastic cell-surface antigen (TROP-2) is a transmembrane protein identified in prostate cancer and overexpressed in multiple malignancies. TROP-2 is a therapeutic target for antibody–drug conjugates (ADC). Experimental Design: TROP-2 gene (TACSTD2) expression and markers of treatment resistance from prostate biopsies were analyzed using data from four previously curated cohorts of mCRPC (n = 634) and the PROMOTE study (dbGaP accession phs001141.v1.p1, n = 88). EPCAM or TROP-2–positive circulating tumor cells (CTC) were captured from peripheral blood for comparison of protein (n = 15) and gene expression signatures of treatment resistance (n = 40). We assessed the efficacy of TROP-2–targeting agents in a mouse xenograft model generated from prostate cancer cell lines. Results: We demonstrated that TACSTD2 is expressed in mCRPC from luminal and basal tumors but at lower levels in patients with neuroendocrine prostate cancer. Patients previously treated with ARSI showed no significant difference in TACSTD2 expression, whereas patients with detectable AR-V7 expression showed increased expression. We observed that TROP-2 can serve as a cell surface target for isolating CTCs, which may serve as a predictive biomarker for ADCs. We also demonstrated that prostate cancer cell line xenografts can be targeted specifically by labeled anti–TROP-2 agents in vivo. Conclusions: These results support further studies on TROP-2 as a therapeutic and diagnostic target for mCRPC.

Background: Triple negative breast cancer (TNBC) represents the most aggressive breast cancer subtype, and despite recent treatment advances, clinical outcomes remain poor, particularly in the metastatic setting. TNBC is a biologically heterogeneous entity, and up to 50% of TNBC express the androgen receptor (AR). Gene expression subtyping has identified a subset of AR-TNBC with a luminal AR-driven (LAR) phenotype. Consequently, there has been great interest in translating androgen receptor signaling inhibitors (ARSIs) approved for use in prostate cancer such as enzalutamide and bicalutamide into the management of LAR-TNBC. However, early phase clinical trials of anti-androgens in metastatic AR-TNBC have had modest results. While multiple mechanisms of anti-androgen resistance have been identified in prostate cancer, including AR amplification and over-expression, emergence of AR splice variants (AR-Vs), and transition to AR independent growth, molecular determinants of anti-androgen resistance in TNBC remain poorly understood, and pre-clinical models to study acquired ARSI resistance in LAR-TNBC have been limited. Methods: A novel pre-clincial model of acquired ARSI resistance was derived from the LAR subtype MDA-MB-453 cell line through serial passaging with increasing concentrations of enzalutamide. Celltiter Blue viability assays were used to determine enzalutamide IC50 of parental and enzalutamide-resistant (EnzR) cell lines. RNA was extracted from the parental and resistant cells, reverse transcribed and expression of AR, splice-variants and canonical AR target genes were evaluated using RT-qPCR. Immunofluorescence was used to investigate the amount and nuclear localization of the AR protein within the parental and enzalutamide resistant cells. Results: EnzR MDA-MB-453 cells were derived through culture in increasing concentrations of enzalutamide for >6 months, and found to have a significant increase in enzalutamide IC50 compared to the parental cell line. EnzR MDA-MB-453 cells had a 4.5 fold increase in full-length AR expression at the transcriptional level compared to the parental cell line. AR protein expression and nuclear localization were also increased in EnzR cells compared to the parental cell line. Canonical AR targets including NKX3.1 were downregulated in response to enzalutamide in parental but not EnzR cells. While pathogenic AR splice variants (AR-Vs) implicated in ARSI resistance in prostate cancer were detected at low levels in MDA-MB-453 cells, no increase in AR-V expression was observed in the EnzR cell line. Conclusions: We have developed a novel pre-clinical model of anti-androgen/ARSI resistance in LAR-TNBC, and identified AR overexpression and persistent AR signaling associated with acquired enzalutamide resistance in LAR-TNBC. While pathogenic AR splice variants have been implicated in ARSI resistance in metastatic prostate cancer and are detected in the MDA-MB-453 cell line model, enzalutamide resistance was not associated with increased AR splice variant expression in this model. Future work will investigate mechanisms leading to AR overexpression as well as combination therapeutic strategies to overcome acquired ARSI resistance. Citation Format: Hannah Krause, Marina N. Sharifi, Serena K. Wolfe, Jamie M. Sperger, Kari B. Wisinski, Ruth O’Regan, Joshua Lang. Increased androgen receptor expression as a mechanism of acquired anti-androgen resistance in androgen receptor-positive triple negative breast cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-14.

Figure S1. Correlation of TACSTD2 with Prostate Cancer Cell Surface Markers.

Purpose:Men with metastatic castration-resistant prostate cancer (mCRPC) frequently develop resistance to androgen receptor signaling inhibitor (ARSI) treatment; therefore, new therapies are needed. Trophoblastic cell-surface antigen (TROP-2) is a transmembrane protein identified in prostate cancer and overexpressed in multiple malignancies. TROP-2 is a therapeutic target for antibody–drug conjugates (ADC).Experimental Design:TROP-2 gene (TACSTD2) expression and markers of treatment resistance from prostate biopsies were analyzed using data from four previously curated cohorts of mCRPC (n = 634) and the PROMOTE study (dbGaP accession phs001141.v1.p1, n = 88). EPCAM or TROP-2–positive circulating tumor cells (CTC) were captured from peripheral blood for comparison of protein (n = 15) and gene expression signatures of treatment resistance (n = 40). We assessed the efficacy of TROP-2–targeting agents in a mouse xenograft model generated from prostate cancer cell lines.Results:We demonstrated that TACSTD2 is expressed in mCRPC from luminal and basal tumors but at lower levels in patients with neuroendocrine prostate cancer. Patients previously treated with ARSI showed no significant difference in TACSTD2 expression, whereas patients with detectable AR-V7 expression showed increased expression. We observed that TROP-2 can serve as a cell surface target for isolating CTCs, which may serve as a predictive biomarker for ADCs. We also demonstrated that prostate cancer cell line xenografts can be targeted specifically by labeled anti–TROP-2 agents in vivo.Conclusions:These results support further studies on TROP-2 as a therapeutic and diagnostic target for mCRPC.

Purpose: Androgen receptor (AR) variant AR-V7 is a ligand-independent transcription factor that promotes prostate cancer resistance to AR-targeted therapies. Accordingly, efforts are under way to develop strategies for monitoring and inhibiting AR-V7 in castration-resistant prostate cancer (CRPC). The purpose of this study was to understand whether other AR variants may be coexpressed with AR-V7 and promote resistance to AR-targeted therapies.Experimental Design: We utilized complementary short- and long-read sequencing of intact AR mRNA isoforms to characterize AR expression in CRPC models. Coexpression of AR-V7 and AR-V9 mRNA in CRPC metastases and circulating tumor cells was assessed by RNA-seq and RT-PCR, respectively. Expression of AR-V9 protein in CRPC models was evaluated with polyclonal antisera. Multivariate analysis was performed to test whether AR variant mRNA expression in metastatic tissues was associated with a 12-week progression-free survival endpoint in a prospective clinical trial of 78 CRPC-stage patients initiating therapy with the androgen synthesis inhibitor, abiraterone acetate.Results: AR-V9 was frequently coexpressed with AR-V7. Both AR variant species were found to share a common 3′ terminal cryptic exon, which rendered AR-V9 susceptible to experimental manipulations that were previously thought to target AR-V7 uniquely. AR-V9 promoted ligand-independent growth of prostate cancer cells. High AR-V9 mRNA expression in CRPC metastases was predictive of primary resistance to abiraterone acetate (HR = 4.0; 95% confidence interval, 1.31–12.2; P = 0.02).Conclusions: AR-V9 may be an important component of therapeutic resistance in CRPC. Clin Cancer Res; 23(16); 4704–15. ©2017 AACR.

This supplementary file contains additional Matierials and Methods detailing the VERSA process. Figure S1. Longitudinal analysis of capture efficiency of Epcam antibody. Figure S2. Multi-parametric analysis of gene expression, genomic profiling and AR protein analysis from captured CTCs. Table S1. Catalog numbers of primers used for quantitative qPCR. Table S2. Patients characteristics for patients 1-17. Table S3. Patient characteristics from patients 18-43

1. Supplementary table 1S describing details of biopsies undergoing WES. 2. Table 2S includes the primers used in the CTC gene expression analysis. 3. Table 3S includes summary of PSA response based on prior treatment. 4. Table 4S includes objective response in the overall cohort and based on patients with measurable and non-measurable disease. 5. Table 5S is the PSA progression and radiographic PFS based on prior lines of treatment. 6. Table 6S is the adverse events in >10% of patients. 7. Table 7S is a listing of the biopsy procedures and outcomes. 8. Table 8S is the frequency of genomic alterations in the baseline and progression samples and PSA response data. 9. Table 9S includes the details for the AR mutations observed in the baseline and progression samples. 10. Figure 1S includes the KM curves for PSA progression and radiographic PFS. 11. Figure 2S includes the Consort Diagram for the study. 12. Figure 3S includes the integrative data of paired biopsy and CTC samples. 13. Figure 4S includes the KM curves for time to PSA progression, radiographic PFS, and overall survival in the CTC biomarker groups.

Purpose: There is a critical clinical need for new predictive and pharmacodynamic biomarkers that evaluate pathway activity in patients treated with targeted therapies. A microscale platform known as VERSA (versatile exclusion-based rare sample analysis) was developed to integrate readouts across protein, mRNA, and DNA in circulating tumor cells (CTC) for a comprehensive analysis of the androgen receptor (AR) signaling pathway.Experimental Design: Utilizing exclusion-based sample preparation principles, a handheld chip was developed to perform CTC capture, enumeration, quantification, and subcellular localization of proteins and extraction of mRNA and DNA. This technology was validated across integrated endpoints in cell lines and a cohort of patients with castrate-resistant prostate cancer (CRPC) treated with AR-targeted therapies and chemotherapies.Results: The VERSA was validated in cell lines to analyze AR protein expression, nuclear localization, and gene expression targets. When applied to a cohort of patients, radiographic progression was predicted by the presence of multiple AR splice variants and activity in the canonical AR signaling pathway. AR protein expression and nuclear localization identified phenotypic heterogeneity. Next-generation sequencing with the FoundationOne panel detected copy number changes and point mutations. Longitudinal analysis of CTCs identified acquisition of multiple AR variants during targeted treatments and chemotherapy.Conclusions: Complex mechanisms of resistance to AR-targeted therapies, across RNA, DNA, and protein endpoints, exist in patients with CRPC and can be quantified in CTCs. Interrogation of the AR signaling pathway revealed distinct patterns relevant to tumor progression and can serve as pharmacodynamic biomarkers for targeted therapies. Clin Cancer Res; 23(3); 746–56. ©2016 AACR.

Supplementary Table 1: Baseline Characteristics of the Patients; Supplementary Table 2: Baseline RNA-seq Expression Levels for AR/ARVs (spliced reads per million); Supplementary Table 3: TaqMan Gene Expression Assays with Circulating Tumor Cells; Supplementary Figure 1: Actinomycin D responses of AR-V9 and AR-V7 mRNA in 22Rv1 cells; Supplementary Figure 2: 3’ rapid amplification of cDNA ends (RACE) for single molecule real-time (SMRT) sequencing; Supplementary Figure 3: Purified AR-V9 polyclonal antibodies detect overexpressed AR-V9 in western blot with overexpressed AR-V9; Supplementary Figure 4: Relationship between tumor RNA p13 purity, AR-V7, and AR-V9 expression levels in CRPC metastases.

Purpose:Enzalutamide is a second-generation androgen receptor (AR) inhibitor that has improved overall survival (OS) in metastatic castration-resistant prostate cancer (CRPC). However, nearly all patients develop resistance. We designed a phase II multicenter study of enzalutamide in metastatic CRPC incorporating tissue and blood biomarkers to dissect mechanisms driving resistance.Patients and Methods:Eligible patients with metastatic CRPC underwent a baseline metastasis biopsy and then initiated enzalutamide 160 mg daily. A repeat metastasis biopsy was obtained at radiographic progression from the same site when possible. Blood for circulating tumor cell (CTC) analysis was collected at baseline and progression. The primary objective was to analyze mechanisms of resistance in serial biopsies. Whole-exome sequencing was performed on tissue biopsies. CTC samples underwent RNA sequencing.Results:A total of 65 patients initiated treatment, of whom 22 (33.8%) had received prior abiraterone. Baseline biopsies were enriched for alterations in AR (mutations, amplifications) and tumor suppression genes (PTEN, RB1, and TP53), which were observed in 73.1% and 92.3% of baseline biopsies, respectively. Progression biopsies revealed increased AR amplifications (64.7% at progression vs. 53.9% at baseline) and BRCA2 alterations (64.7% at progression vs. 38.5% at baseline). Genomic analysis of baseline and progression CTC samples demonstrated increased AR splice variants, AR-regulated genes, and neuroendocrine markers at progression.Conclusions:Our results demonstrate that a large proportion of enzalutamide-treated patients have baseline and progression alterations in the AR pathway and tumor suppressor genes. We demonstrate an increased number of BRCA2 alterations post-enzalutamide, highlighting the importance of serial tumor sampling in CRPC.

PURPOSE Nearly all men with prostate cancer treated with androgen receptor (AR) signaling inhibitors (ARSIs) develop resistance via diverse mechanisms including constitutive activation of the AR pathway, driven by AR genomic structural alterations, expression of AR splice variants (AR-Vs), or loss of AR dependence and lineage plasticity termed neuroendocrine prostate cancer. Understanding these de novo acquired ARSI resistance mechanisms is critical for optimizing therapy. MATERIALS AND METHODS A novel liquid biopsy technology was used to collect mRNA from circulating tumor cells (CTCs) to measure expression of AR-Vs, AR targets, and neuroendocrine prostate cancer markers. An institutional review board–approved prospective cohort (N = 99) was used to identify patterns of gene expression. Two prospective multicenter phase II clinical trials of ARSIs for men with castration-resistant prostate cancer (ClinicalTrials.gov: NCT01942837 [enzalutamide, N = 21] and NCT02025010 [abiraterone, N = 27]) were used to further validate these findings. RESULTS Hierarchical clustering of CTC transcripts identified two distinct clusters. Cluster 2 (C2) exhibited increased expression of AR-regulated genes and was associated with worse overall survival (median 8.6 v 22.4 months; P < .01; hazard ratio [HR] = 3.45 [1.9 to 6.14]). In multivariable analysis, C2 was prognostic independent of other clinicopathologic variables. AR-V status was not significant when accounting for C2. Upon further validation in pooled multicenter phase II trials, C2 was associated with worse overall survival (15.2 months v not reached; P < .01; HR = 8.43 [2.74 to 25.92]), prostate-specific antigen progression-free survival (3.6 v 12 months; P < .01; HR = 4.64 [1.53 to 14.11]), and radiographic progression-free survival (2.7 v 40.6 months; P < .01; HR = 4.64 [1.82 to 17.41]). CONCLUSION We demonstrate that a transcriptional profile detectable in CTCs obtained from liquid biopsies can serve as an independent prognostic marker beyond AR-V7 in patients with metastatic prostate cancer and can be used to identify the emergence of multiple ARSI resistance mechanisms. This is currently being investigated in additional prospective trials.

Although therapeutic options for patients with advanced renal cell carcinoma (RCC) have increased in the past decade, no biomarkers are yet available for patient stratification or evaluation of therapy resistance. Given the dynamic and heterogeneous nature of clear cell RCC (ccRCC), tumor biopsies provide limited clinical utility, but liquid biopsies could overcome these limitations. Prior liquid biopsy approaches have lacked clinically relevant detection rates for patients with ccRCC. This study employed ccRCC‐specific markers, CAIX and CAXII, to identify circulating tumor cells (CTC) from patients with metastatic ccRCC. Distinct subtypes of ccRCC CTCs were evaluated for PD‐L1 and HLA‐I expression and correlated with patient response to therapy. CTC enumeration and expression of PD‐L1 and HLA‐I correlated with disease progression and treatment response, respectively. Longitudinal evaluation of a subset of patients demonstrated potential for CTC enumeration to serve as a pharmacodynamic biomarker. Further evaluation of phenotypic heterogeneity among CTCs is needed to better understand the clinical utility of this new biomarker., Circulating tumor cells (CTCs) were evaluated for their expression of programmed death ligand‐1 (PD‐L1) and human leukocyte antigen class I (HLA‐I), so that biomarkers of therapeutic resistance of clear cell renal cell carcinoma can be developed. CTCs were captured with antibody‐conjugated magnetic beads against EpCAM and CAIX then probed for CAXII and CK expression using microfluidic technology.

Castration-resistant prostate cancer (CRPC) is driven by AR gene aberrations that arise during androgen receptor (AR)-targeted therapy. AR amplification and mutations have been profiled in circulating tumor cells (CTCs), but whether AR gene rearrangements can be assessed in CTCs is unknown. In this study, we leveraged CRPC cell lines with defined AR gene rearrangements to develop and validate a CTC DNA analysis approach that utilized whole genome amplification and targeted DNA-sequencing of AR and other genes important in CRPC. We tested the utility of this approach by analyzing matched CTC DNA and plasma cell-free DNA (cfDNA) from a case series of ten CRPC patients. One of ten CTC samples and two of ten cfDNA samples were positive for AR gene rearrangements. All AR gene rearrangements were discordant between matched liquid biopsy samples. One patient harbored separate AR gene rearrangements in CTC DNA and cfDNA, but concordant AR amplification and AR T878A mutation. This patient also displayed concordant loss of TP53 and PTEN, but the loss of RB1 in cfDNA only. The overall frequency of discordant alterations in these genes between matched CTC DNA and cfDNA was high. This study establishes the technical feasibility of analyzing structural rearrangements, mutations, and copy number variants in AR and other CRPC genes using two different sources of DNA from a single blood sample. Paired CTC DNA and cfDNA analysis may have utility for capturing the heterogeneity of genetic alterations in CRPC patients.

Purpose: Enzalutamide is a second-generation androgen receptor (AR) inhibitor that has improved overall survival (OS) in metastatic castration-resistant prostate cancer (CRPC). However, nearly all patients develop resistance. We designed a phase II multicenter study of enzalutamide in metastatic CRPC incorporating tissue and blood biomarkers to dissect mechanisms driving resistance. Patients and Methods: Eligible patients with metastatic CRPC underwent a baseline metastasis biopsy and then initiated enzalutamide 160 mg daily. A repeat metastasis biopsy was obtained at radiographic progression from the same site when possible. Blood for circulating tumor cell (CTC) analysis was collected at baseline and progression. The primary objective was to analyze mechanisms of resistance in serial biopsies. Whole-exome sequencing was performed on tissue biopsies. CTC samples underwent RNA sequencing. Results: A total of 65 patients initiated treatment, of whom 22 (33.8%) had received prior abiraterone. Baseline biopsies were enriched for alterations in AR (mutations, amplifications) and tumor suppression genes (PTEN, RB1, and TP53), which were observed in 73.1% and 92.3% of baseline biopsies, respectively. Progression biopsies revealed increased AR amplifications (64.7% at progression vs. 53.9% at baseline) and BRCA2 alterations (64.7% at progression vs. 38.5% at baseline). Genomic analysis of baseline and progression CTC samples demonstrated increased AR splice variants, AR-regulated genes, and neuroendocrine markers at progression. Conclusions: Our results demonstrate that a large proportion of enzalutamide-treated patients have baseline and progression alterations in the AR pathway and tumor suppressor genes. We demonstrate an increased number of BRCA2 alterations post-enzalutamide, highlighting the importance of serial tumor sampling in CRPC.

Background: Somatic genomic alterations that activate the phosphatidylinositol-3-kinase (PI3K) pathway signaling are found in 30-50% of breast cancers, and the p110a-specific inhibitor alpelisib is approved for use in combination with fulvestrant in PIK3CA mutated hormone receptor-positive (HR+) metastatic breast cancer. However, preclinical and clinical data has demonstrated significant variability in response to alpelisib and other PI3K inhibitors even in the presence of hotspot PIK3CA activating mutations, likely reflecting functional differences between somatic alterations, bypass signaling mechanisms, and intratumor clonal heterogeneity. Thus, there is an ongoing need for novel predictive biomarkers to help guide patient selection for these therapies. Circulating tumor cells (CTCs) represent an accessible source of tumor derived analytes that allow for the interrogation of protein level readouts of PI3K pathway activation, may better reflect the biologic heterogeneity of metastatic disease than single site solid tumor biopsy, and are amenable to longitudinal analysis. Here, we report the development of an assay to evaluate the expression of activated AKT (AKT pS473) in CTCs as a putative biomarker of sensitivity to PI3K inhibitor therapies in metastatic breast cancer. Methods: Peripheral blood for CTC isolation was collected serially from patients with metastatic breast cancer and known somatic PI3K pathway mutation status. Samples were processed using VERSA (Versatile Exclusion-based Rare Sample Analysis), a microfluidic technology that integrates CTC capture and downstream analysis. Following fixation, CTCs were captured immunomagnetically with antibodies for EpCAM and Trop2, permeabilized and stained on chip for pan-AKT plus AKT pS473. Quantitative fluorescent microscopy was used to enumerate CTCs, defined as cytokeratin positive cells with intact nuclei and negative for a group of normal blood cell markers (CD45/CD11b/CD34/CD66b), and to quantify activated AKT protein expression in CTCs and matched peripheral blood mononuclear cells (PBMCs). Results: 100% of patients with somatic PIK3CA mutations in our pilot cohort had CTCs with detectable phospho-AKT expression, and in 50% of patients, the median phospho-AKT/pan AKT ratio was higher in CTCs compared to matched peripheral blood cells, suggestive of increased PI3K pathway activity. All patients demonstrated heterogeneity in phospho-AKT expression and phospho-AKT/pan-AKT ratio among individual CTCs at a single timepoint and across timepoints in longitudinal analysis. Conclusion: We report here the feasibility of quantitative and longitudinal detection of activated AKT in EpCAM/Trop2 captured CTCs from metastatic breast cancer with somatic PIK3CA mutations. Future work will prospectively evaluate multiple clinical applications of this assay in patients receiving alpelisib plus endocrine therapy for PIK3CA mutated HR+ metastatic breast cancer, as well as expanding the assay to include the detection of additional phospho-protein readouts of PI3K pathway activity in CTCs. In addition to the potential to complement solid biopsy PIK3CA mutation status as a predictive biomarker of sensitivity to PI3K therapies, this assay has the unique potential to provide a pharmacodynamic assessment of PI3K inhibitor activity in CTCs while on treatment, which may serve as an early biomarker of clinical response or resistance. Citation Format: Marina N Sharifi, Jamie M Sperger, Cole Gilsdorf, Serena K Wolfe, Amanda Parkes, Kari B Wisinski, Ruth M O'Regan, Joshua M Lang. Detection of PI3K pathway activation in circulating tumor cells in PIK3CA mutated metastatic breast cancer as a putative predictive biomarker for PI3K inhibitor therapies [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-35.

Background DNA methylation alterations have emerged as hallmarks of cancer and have been proposed as screening, prognostic, and predictive biomarkers. Traditional approaches for methylation analysis have relied on bisulfite conversion of DNA, which can damage DNA and is not suitable for targeted gene analysis in low-input samples. Here, we have adapted methyl-CpG-binding domain protein 2 (MBD2)-based DNA enrichment for use on a semi-automated exclusion-based sample preparation (ESP) platform for robust and scalable enrichment of methylated DNA from low-input samples, called SEEMLIS. Results We show that combining methylation-sensitive enzyme digestion with ESP-based MBD2 enrichment allows for single gene analysis with high sensitivity for GSTP1 in highly impure, heterogenous samples. We also show that ESP-based MBD2 enrichment coupled with targeted pre-amplification allows for analysis of multiple genes with sensitivities approaching the single cell level in pure samples for GSTP1 and RASSF1 and sensitivity down to 14 cells for these genes in highly impure samples. Finally, we demonstrate the potential clinical utility of SEEMLIS by successful detection of methylated gene signatures in circulating tumor cells (CTCs) from patients with prostate cancer with varying CTC number and sample purity. Conclusions SEEMLIS is a robust assay for targeted DNA methylation analysis in low-input samples, with flexibility at multiple steps. We demonstrate the feasibility of this assay to analyze DNA methylation in prostate cancer cells using CTCs from patients with prostate cancer as a real-world example of a low-input analyte of clinical importance. In summary, this novel assay provides a platform for determining methylation signatures in rare cell populations with broad implications for research as well as clinical applications.

PURPOSE Intrapatient treatment response heterogeneity is under-recognized. Quantitative total bone imaging (QTBI) using 18F-NaF positron emission tomography/computed tomography (PET/CT) scans is a tool that allows characterization of interlesional treatment response heterogeneity in bone. Understanding spatial-temporal response is important to identify individuals who may benefit from treatment beyond progression. PATIENTS AND METHODS Men with progressive metastatic castration-resistant prostate cancer (mCRPC) with at least two lesions on bone scintigraphy were enrolled and treated with enzalutamide 160 mg daily (ClinicalTrials.gov identifier: NCT02384382 ). 18F-NaF PET/CT scans were obtained at baseline (PET1), week 13 (PET2), and at the time of prostate-specific antigen (PSA) progression, standard radiographic or clinical progression, or at 2 years without progression (PET3). QTBI was used to determine lesion-level response. The primary end point was the proportion of men with at least one responding bone lesion on PET3 using QTBI. RESULTS Twenty-three men were enrolled. Duration on treatment ranged from 1.4 to 34.1 months. In general, global standardized uptake value (SUV) metrics decreased while on enzalutamide (PET2) and increased at the time of progression (PET3). The most robust predictor of PSA progression was change in SUVhetero (PET1 to PET3; hazard ratio, 3.88; 95% CI, 1.24 to 12.1). Although overall functional disease burden improved during enzalutamide treatment, an increase in total burden (SUVtotal) was seen at the time of progression, as measured by 18F-NaF PET/CT. All (22/22) evaluable men had at least one responding bone lesion at PET3 using QTBI. CONCLUSION We found that the proportion of progressing lesions was low, indicating that a substantial number of lesions appear to continue to benefit from enzalutamide beyond progression. Selective targeting of nonresponding lesions may be a reasonable approach to extend benefit.

Centrosome amplification (CA) is a common phenomenon in cancer, promotes genomic stability and cancer evolution, and has been reported to promote metastasis. CA promotes a stochastic gain/loss of chromosomes during cell division, known as chromosomal instability (CIN). However, it is unclear whether CA is present in circulating tumor cells (CTCs), the seeds for metastasis. Here, we surveyed CA in CTCs from human subjects with metastatic breast cancer. CTCs were captured by CD45 exclusion and selection of EpCAM‐positive cells using an exclusion‐based sample preparation technology platform known as VERSA (versatile exclusion‐based rare sample analysis). Centriole amplification (centrin foci> 4) is the definitive assay for CA. However, determination of centrin foci is technically challenging and incompatible with automated analysis. To test if the more technically accessible centrosome marker pericentrin could serve as a surrogate for centriole amplification in CTCs, cells were stained with pericentrin and centrin antibodies to evaluate CA. This assay was first validated using breast cancer cell lines and a nontransformed epithelial cell line model of inducible CA, then translated to CTCs. Pericentrin area and pericentrin area x intensity correlate well with centrin foci, validating pericentrin as a surrogate marker of CA. CA is found in CTCs from 75% of subjects, with variability in the percentage and extent of CA in individual circulating cells in a given subject, similar to the variability previously seen in primary tumors and cell lines. In summary, we created, validated, and implemented a novel method to assess CA in CTCs from subjects with metastatic breast cancer. Such an assay will be useful for longitudinal monitoring of CA in cancer patients and in prospective clinical trials for assessing the impact of CA on response to therapy., Circulating tumor cells (CTCs) are a rare population of cancer cells released into peripheral circulation from primary and metastatic cancer sites. Centrosome amplification (CA) has been reported in virtually all human cancers and has been shown to make cells more invasive. In this study, we develop an assay to detect CA in CTCs. We find that CA is prevalent in CTCs from subjects with metastatic breast cancer.

Comprehensive analysis of tumor heterogeneity requires robust methods for the isolation and analysis of single cells from patient samples. An ideal approach would be fully compatible with downstream analytic methods, such as advanced genomic testing. These endpoints necessitate the use of live cells at high purity. A multitude of microfluidic circulating tumor cell (CTC) enrichment technologies exist, but many of those perform bulk sample enrichment and are not, on their own, capable of single-cell interrogation. To address this, we developed an affordable semiautomated single-cell aspirator (SASCA) to further enrich rare-cell populations from a specialized microwell array, per their phenotypic markers. Immobilization of cells within microwells, integrated with a real-time image processing software, facilitates the detection and precise isolation of targeted cells that have been optimally seeded into the microwells. Here, we demonstrate the platform capabilities through the aspiration of target cells from an impure background population, where we obtain purity levels of 90%-100% and demonstrate the enrichment of the target population with high-quality RNA extraction. A range of low cell numbers were aspirated using SASCA before undergoing whole transcriptome and genome analysis, exhibiting the ability to obtain endpoints from low-template inputs. Lastly, CTCs from patients with castration-resistant prostate cancer were isolated with this platform and the utility of this method was confirmed for rare-cell isolation. SASCA satisfies a need for an affordable option to isolate single cells or highly purified subpopulations of cells to probe complex mechanisms driving disease progression and resistance in patients with cancer.

247 Background: The mainstay of therapy in metastatic prostate cancer is androgen receptor (AR) signaling inhibition. However, the emergence of early castration resistance or neuroendocrine transformation is associated with poor prognosis. Reliable biomarkers are needed to identify these patients and guide selection of clinical therapy. Methods: mRNA was isolated from EpCAM-positive circulating tumor cells (CTCs) isolated from patients with CSPC, CRPC, or NEPC to measure expression of KLK2, KLK3 (PSA), TMPRSS2, FOLH1 (PSMA), synaptophysin ( SYP), and chromogranin ( CHGA). Post-hoc retrospective analysis of an institutional review board–approved prospective cohort (N = 98) was performed to identify patterns of gene expression. Samples were considered AR+ if 3 of 4 AR pathway genes ( TMPRSS2, KLK2, KLK3, and FOLH1) were positive, and were considered NE+ if either or both SYP or CHGA were positive. Blood samples from two prospective clinical trials of men with mCRPC treated with abiraterone and enzalutamide, respectively, were analyzed to confirm results. Longitudinal samples were collected from 17 patients (6 NEPC and 11 Adenocarcinoma) and cell free DNA was isolated and sequenced using a novel targeted exon panel. Results: AR and/or NE positive patients were found to have a median overall survival (OS) of 8.58 months as compared to a median OS of 29.6 months in the AR and NE negative population (p

Background: Phosphatidylinositol-3-kinase (PI3K) activating mutations are found in 30-40% of breast cancers, and the PI3K inhibitor alpelisib is FDA-approved for PIK3CA-mutated hormone-receptor positive metastatic breast cancer (MBC). However, rates of intrinsic alpelisib resistance are as high as 40%, and there is an ongoing need for novel biomarkers to help guide patient selection. Circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) may better reflect the heterogeneity of metastatic disease than tissue biopsy, and are amenable to longitudinal analysis. By combining targeted ctDNA sequencing with CTC transcriptional profiling and quantitation of PI3K pathway phospho-proteins, we have developed the first multiplex assay to simultaneously assess genomic alterations and CTC PI3K pathway activity via liquid biopsy. Methods: Peripheral blood was collected from MBC patients with known PIK3CA mutation status serially prior to starting new standard therapies and on treatment. Cell-free DNA was extracted from plasma and sequenced using a custom capture panel (IDT). CTCs were isolated with VERSA microfluidic technology integrating capture with downstream analysis. CTCs were captured immunomagnetically followed by RNA isolation on chip and RNA-seq, or staining on chip for phospho-AKT pS473 and total AKT or phospho-rpS6 pS235/S236 and total rpS6, followed by quantification of single cell phospho/total protein mean fluorescence ratios. Results: In a pilot cohort of 14 patients, mean CTC phospho-AKT/total AKT protein expression ratio was higher in patients with PIK3CA mutations compared to patients without mutations (0.37 vs 0.26, p Conclusion: We have demonstrated the feasibility of a comprehensive liquid biopsy for simultaneous monitoring of PI3K pathway mutations in ctDNA and PI3K pathway signaling in CTCs via transcriptional profiling and phospho-protein expression in patients with MBC. Future work will prospectively evaluate this assay in patients receiving alpelisib for PIK3CA-mutated MBC. This has the potential to complement PIK3CA mutation status as a biomarker of sensitivity to PI3K therapies, and may also provide a pharmacodynamic assessment of PI3K inhibitor activity in CTCs on treatment. Citation Format: Marina N. Sharifi, Kyle T. Helzer, Jamie M. Sperger, Matthew L. Bootsma, Hannah Krause, Cole S. Gilsdorf, Serena K. Wolfe, Zachary Kauffman, Amye J. Tevaarwerk, Mark E. Burkard, Amanda M. Parkes, Ruth M. O'Regan, Kari B. Wisinski, Shuang G. Zhao, Joshua M. Lang. Simultaneous longitudinal assessment of PIK3CA genomic mutations and PI3K pathway activity in circulating tumor cells in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1955.

Purpose: Prostate circulating tumor cells escape into peripheral blood and enter bone marrow as disseminated tumor cells, representing an early step before conventionally detectable metastasis. It is unclear how frequently this occurs in localized disease and existing detection methods rely on epithelial markers with low specificity and sensitivity. We used multiple methodologies of disseminated tumor cell detection in bone marrow harvested at radical prostatectomy.Materials and Methods: Bone marrow was harvested from 208 clinically localized cases, 16 controls and 5 metastatic cases with peripheral blood obtained from 37 metastatic cases. Samples were evaluated at 4 centers with 4 distinct platforms using antibody enrichment with the AdnaTest (Qiagen®) or VERSA (versatile exclusion based rare sample analysis), or whole sample interrogation with the RareCyte platform (Seattle, Washington) or HD-SCA (high definition single cell assay) using traditional epithelial markers and prostate specific markers. We i...

Summary: Tumor cells initiate platelet activation leading to the secretion of bioactive molecules, which promote metastasis. Platelet receptors on tumors have not been well-characterized, resulting in a critical gap in knowledge concerning platelet-promoted metastasis. We identify a direct interaction between platelets and tumor CD97 that stimulates rapid bidirectional signaling. CD97, an adhesion G protein-coupled receptor (GPCR), is an overexpressed tumor antigen in several cancer types. Purified CD97 extracellular domain or tumor cell-associated CD97 stimulated platelet activation. CD97-initiated platelet activation led to granule secretion, including the release of ATP, a mediator of endothelial junction disruption. Lysophosphatidic acid (LPA) derived from platelets induced tumor invasiveness via proximal CD97-LPAR heterodimer signaling, coupling coincident tumor cell migration and vascular permeability to promote transendothelial migration. Consistent with this, CD97 was necessary for tumor cell-induced vascular permeability in vivo and metastasis formation in preclinical models. These findings support targeted blockade of tumor CD97 as an approach to ameliorate metastatic spread. : Tumor-initiated platelet activation promotes tissue invasion of cancer cells and metastasis. Ward et al. demonstrate that a common tumor-associated antigen, CD97, accounts for platelet activation and participates directly in LPA-mediated signal transduction leading to tumor cell invasion. CD97 promotes vascular extravasation and metastasis in pre-clinical models. Keywords: platelets, metastasis, transendothelial migration, circulating tumor cells, CD97, adhesion GPCR, LPA

Tumor metastasis depends on the dynamic regulation of cell adhesion through β1-integrin. The Cub-Domain Containing Protein-1, CDCP1, is a transmembrane glycoprotein which regulates cell adhesion. Overexpression and loss of CDCP1 have been observed in the same cancer types to promote metastatic progression. Here, we demonstrate reduced CDCP1 expression in high-grade, primary prostate cancers, circulating tumor cells and tumor metastases of patients with castrate-resistant prostate cancer. CDCP1 is expressed in epithelial and not mesenchymal cells, and its cell surface and mRNA expression declines upon stimulation with TGFβ1 and epithelial-to-mesenchymal transition. Silencing of CDCP1 in DU145 and PC3 cells resulted in 3.4-fold higher proliferation of non-adherent cells and 4.4-fold greater anchorage independent growth. CDCP1-silenced tumors grew in 100% of mice, compared to 30% growth of CDCP1-expressing tumors. After CDCP1 silencing, cell adhesion and migration diminished 2.1-fold, caused by loss of inside-out activation of β1-integrin. We determined that the loss of CDCP1 reduces CDK5 kinase activity due to the phosphorylation of its regulatory subunit, CDK5R1/p35, by c-SRC on Y234. This generates a binding site for the C2 domain of PKCδ, which in turn phosphorylates CDK5 on T77. The resulting dissociation of the CDK5R1/CDK5 complex abolishes the activity of CDK5. Mutations of CDK5-T77 and CDK5R1-Y234 phosphorylation sites re-establish the CDK5/CDKR1 complex and the inside-out activity of β1-integrin. Altogether, we discovered a new mechanism of regulation of CDK5 through loss of CDCP1, which dynamically regulates β1-integrin in non-adherent cells and which may promote vascular dissemination in patients with advanced prostate cancer.

Rare cell populations provide a patient-centric tool to monitor disease treatment, response, and resistance. However, understanding rare cells is a complex problem, which requires cell isolation/purification and downstream molecular interrogation - processes challenged by non-target populations, which vary patient-to-patient and change with disease. As such, cell isolation platforms must be amenable to a range of sample types while maintaining high efficiency and purity. The multiplexed technology for automated extraction (mTAE) is a versatile magnetic bead-based isolation platform that facilitates positive, negative, and combinatorial selection with integrated protein staining and nucleic acid isolation. mTAE is validated by isolating circulating tumor cells (CTCs) - a model rare cell population - from breast and prostate cancer patient samples. Negative selection yielded high efficiency capture of CTCs while positive selection yielded higher purity with an average of only 95 contaminant cells captured per milliliter of processed whole blood. With combinatorial selection, an overall increase in capture efficiency was observed, highlighting the potential significance of integrating multiple capture approaches on a single platform. Following capture (and staining), on platform nucleic acid extraction enabled the detection of androgen receptor-related transcripts from CTCs isolated from prostate cancer patients. The flexibility (e.g. negative, positive, combinatorial selection) and capabilities (e.g. isolation, protein staining, and nucleic acid extraction) of mTAE enable users to freely interrogate specific cell populations, a capability required to understand the potential of emerging rare cell populations and readily adapt to the heterogeneity presented across clinical samples.

Introduction: The androgen receptor (AR) is expressed in up to 50% of patients with triple negative breast cancer (TNBC), and a luminal androgen receptor (LAR) subtype has been identified that is dependent on androgen receptor signaling, but AR protein expression by immunohistochemistry (IHC) has been a suboptimal biomarker for response to anti-androgen therapies in AR-TNBC and there remains an unmet need for biomarkers to identify patients likely to benefit from anti-androgens. We have developed a multiplex liquid biopsy encompassing AR protein expression and downstream gene expression analysis in circulating tumor cells (CTCs). We report here the longitudinal evaluation of this liquid biopsy as part of a phase I trial (NCT03090165) of the anti-androgen bicalutamide with selective CDK4/6 inhibitor ribociclib in metastatic AR-TNBC. Methods: Peripheral blood was collected (n=11) prior to treatment, after two cycles of treatment, and at progression and processed using the VERSA (Versatile Exclusion-based Rare Sample Analysis) microfluidic chip that integrates CTC capture and downstream analysis. For protein expression, fixed CTCs were captured immunomagnetically and stained on chip for AR followed by quantitative fluorescent microscopy. For gene expression analysis, live CTCs were captured immunomagnetically followed by RNA isolation on chip, reverse transcription, and RT-qPCR for a panel of 40 luminal AR genes. Results: AR protein and transcript were detected in CTCs from 10/11 patients, and AR target gene expression in 8/11 patients. The pathologic ligand independent AR splice variant AR-V7 was detected in 2/11 patients. Pretreatment CTC number was higher in patients with anti-androgen resistant (range 3-753) than anti-androgen sensitive (range 0-30) disease. Among patients with low AR IHC (50%) on biopsy, AR-V7 was detected in CTCs of 2/3 of patients with resistant disease and 0/2 of patients with sensitive disease. Conclusions: This study demonstrates the feasibility of a longitudinal multiplex liquid biopsy as a pharmacodynamic biomarker of AR pathway activity in AR-TNBC, and suggests that AR pathway activity in CTCs may provide additional information to solid tumor biopsy AR expression in predicting sensitivity to anti-androgens. This work also demonstrates for the first time the detection of AR-V7 in CTCs in anti-androgen resistant AR-TNBC, suggesting that AR-V7 expression may be a mechanism of anti-androgen resistance in AR-TNBC as it is in advanced prostate cancer. Citation Format: Marina N. Sharifi, Serena K. Wolfe, Jamie M. Sperger, Jennifer L. Schehr, Saswati Bhattacharya, Kari B. Wisinski, Joshua M. Lang, Ruth M. O'Regan. Multiplex liquid biopsy for AR pathway activity in metastatic androgen receptor-positive triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 590.

5060 Background: Tumor-associated calcium signal transducer 2 (TROP-2, TACSTD2) is a transmembrane glycoprotein that is highly expressed in many epithelial cancers. Overexpression of TROP-2 is postulated to mediate cancer cell growth, invasion, and is associated with more aggressive disease. TROP-2 has emerged as a therapeutic target for antibody-drug conjugates in clinical trials including sacituzumab govitecan and DS-1062. Here, we evaluated the expression of TROP-2 in tumor biopsies and circulating tumor cells (CTCs) in men with metastatic castration resistant prostate cancer (mCRPC) to evaluate TROP-2 as a clinically relevant target. Methods: RNA-seq data from the SU2C-PCF database and PROMOTE clinical trial (NCT#01953640) was assessed for TACSTD2 and androgen receptor (AR) splice variant ( AR_V7/AR_V9) expression. Prostate cancer ChIP-seq data was analyzed to identify binding of the AR to the TROP-2 promoter. EpCAM and TROP-2 captured CTCs were isolated from patients with mCRPC using the VERSA (Versatile Exclusion-based Rare Sample Analysis) platform and assessed for splice variant, neuroendocrine (NE), and AR-regulated gene signatures, in addition to CTC enumeration and TROP-2 protein expression. Results: TROP-2 expression was detectable in 90% of patients, in both bone and visceral metastatic biopsies (SUC2-PCF). Although TROP-2 low biopsies were infrequent (10%), 58% of these samples showed high levels of NE markers, as compared with 5% in all other patients. In the PROMOTE study, elevated TROP-2 gene expression was significantly higher in biopsies with high AR_V7 expression than in those with low (p = 0.04) or negative (p

// Erika Heninger 1, 3 , Timothy E.G. Krueger 3 , Stephanie M. Thiede 1, 3 , Jamie M. Sperger 1, 3 , Brianna L. Byers 3 , Madison R. Kircher 3 , David Kosoff 1, 3 , Bing Yang 2, 3 , David F. Jarrard 2, 3 , Douglas G. McNeel 1, 3 , Joshua M. Lang 1, 3 1 Department of Medicine, University of Wisconsin, Madison, Madison, WI 53705, USA 2 Department of Urology, University of Wisconsin, Madison, Madison, WI 53705, USA 3 University of Wisconsin Carbone Cancer Center, Madison, Madison, WI 53705, USA Correspondence to: Joshua M. Lang, email: jmlang@medicine.wisc.edu Keywords: cancer testis antigen, prostate cancer, epigenetics, tumor immunotherapy, methylation Received: April 22, 2016 Accepted: October 11, 2016 Published: October 17, 2016 ABSTRACT Immune tolerance to self-antigens can limit robust anti-tumor immune responses in the use of tumor vaccines. Expression of novel tumor associated antigens can improve immune recognition and lysis of tumor cells. The cancer-testis antigen (CTA) family of proteins has been hypothesized to be an ideal class of antigens due to tumor-restricted expression, a subset of which have been found to induce antibody responses in patients with prostate disease. We demonstrate that CTA expression is highly inducible in five different Prostate Cancer (PC) cell lines using a hypomethylating agent 5-Aza-2′-deoxycytidine (5AZA) and/or a histone deacetylase inhibitor LBH589. These CTAs include NY-ESO1, multiple members of the MAGE and SSX families and NY-SAR35. A subset of CTAs is synergistically induced by the combination of 5AZA and LBH589. We developed an ex vivo organ culture using human PC biopsies for ex vivo drug treatments to evaluate these agents in clinical samples. These assays found significant induction of SSX2 in 9/9 distinct patient samples and NY-SAR35 in 7/9 samples. Further, we identify expression of SSX2 in circulating tumor cells (CTC) from patients with advanced PC. These results indicate that epigenetic modifying agents can induce expression of a broad range of neoantigens in human PC and may serve as a useful adjunctive therapy with novel tumor vaccines and checkpoint inhibitors.

Purpose: Androgen receptor (AR) variant AR-V7 is a ligand-independent transcription factor that promotes prostate cancer resistance to AR-targeted therapies. Accordingly, efforts are under way to develop strategies for monitoring and inhibiting AR-V7 in castration-resistant prostate cancer (CRPC). The purpose of this study was to understand whether other AR variants may be coexpressed with AR-V7 and promote resistance to AR-targeted therapies. Experimental Design: We utilized complementary short- and long-read sequencing of intact AR mRNA isoforms to characterize AR expression in CRPC models. Coexpression of AR-V7 and AR-V9 mRNA in CRPC metastases and circulating tumor cells was assessed by RNA-seq and RT-PCR, respectively. Expression of AR-V9 protein in CRPC models was evaluated with polyclonal antisera. Multivariate analysis was performed to test whether AR variant mRNA expression in metastatic tissues was associated with a 12-week progression-free survival endpoint in a prospective clinical trial of 78 CRPC-stage patients initiating therapy with the androgen synthesis inhibitor, abiraterone acetate. Results: AR-V9 was frequently coexpressed with AR-V7. Both AR variant species were found to share a common 3′ terminal cryptic exon, which rendered AR-V9 susceptible to experimental manipulations that were previously thought to target AR-V7 uniquely. AR-V9 promoted ligand-independent growth of prostate cancer cells. High AR-V9 mRNA expression in CRPC metastases was predictive of primary resistance to abiraterone acetate (HR = 4.0; 95% confidence interval, 1.31–12.2; P = 0.02). Conclusions: AR-V9 may be an important component of therapeutic resistance in CRPC. Clin Cancer Res; 23(16); 4704–15. ©2017 AACR.

Background We report secondary and exploratory analysis with 18F-NaF PET/CT and CTCs to determine how tumour burden and RNA expression of androgen receptor (AR) splice variants and neuroendocrine (NE) features predict duration of treatment (DoTx) with ENZA. Methods Men with progressive mCRPC with ≥ 2 lesions on bone scintigraphy were enrolled and treated with ENZA 160 mg daily at 3 US sites. 18F-NaF PET/CT scans were obtained at baseline (BL) (PET1), week 13 (PET2), and at the time of PSA progression (per PCWG2 criteria), standard radiographic or clinical progression, or at 2 years without progression (PET3) using QTBI. CTCs were obtained at BL and at PET3. PSA decline and DoTx were compared with BL QTBI, CTC metrics, and change in QTBI. Results 23 men (median age, 72 years [range, 51-93]; median PSA, 20.5 ng/mL [range, 3.9-133.6]) were enrolled; 22 of which completed planned imaging. The mean number of bone lesions on PET1 was 58 (range 9-168), with a median total standardized uptake value (SUVtotal) of 3886.5 (range 506.7-22852.8). DoTx ranged from 1.4 to 31.8+ mo. In general, SUV metrics decreased while on ENZA (PET2) and increased by PET3. Change in lesion heterogeneity (PET1 to PET3) was the most significant predictor of time to PSA progression (hazard ratio, 3.88; 95% confidence interval, 1.24-12.1). ARV7/9 was found in CTCs in 3/20 men and NE features were found in 4/20 men at BL and in 4/19 and in 11/19 men, respectively, at PET3. Conclusions Although PSA response with ENZA is high, DoTx is variable. SUVtotal at BL did not correlate with DoTx; however, decrease in SUVtotal/mean was associated with increased DoTx, with less heterogeneity correlating with a longer time to PSA progression. ARV7/9 or NE expression in CTCs did not predict lack of benefit from ENZA; however, at the time of progression, the observed increase in ARV7/9 and NE expression are consistent with tumor evolution to more aggressive phenotypes. While BL heterogeneity may be prognostic, it may not predict response or DoTx. Assessing response heterogeneity may predict response duration, thus, should be explored as a possible surrogate of benefit in future studies. Clinical trial identification NCT02384382. Editorial acknowledgement Medical writing and editorial assistance funded by Pfizer Inc. and Astellas Pharma, Inc. was provided by Stephanie Vadasz, PhD, and Michele Salernitano from Ashfield Healthcare Communications. Legal entity responsible for the study Pfizer Inc. and Astellas Pharma, Inc. Funding Pfizer Inc. and Astellas Pharma, Inc. Disclosure G. Liu: Leadership role, Shareholder / Stockholder / Stock options, Employee: AIQ Solutions; Advisory / Consultancy, Research grant / Funding (self), Research grant / Funding (institution): Novartis; Advisory / Consultancy: Exelixis; Advisory / Consultancy, Research grant / Funding (self), Research grant / Funding (institution): Janssen; Advisory / Consultancy, Research grant / Funding (self), Research grant / Funding (institution): TRACON Pharma; Research grant / Funding (self), Research grant / Funding (institution): Madison Vaccines Inc.; Research grant / Funding (self), Research grant / Funding (institution): Pfizer Inc. C.E. Kyriakopoulos: Advisory / Consultancy, Travel / Accommodation / Expenses: Exelixis; Speaker Bureau / Expert testimony: The France Foundation; Research grant / Funding (self), Research grant / Funding (institution): Sanofi. J.M. Lang: Shareholder / Stockholder / Stock options, Licensing / Royalties, I am listed on the patent on a technology for rare cell capture and analysis. This technology has been licensed by Salus Discovery, LLC though no commercial products are available: Salus Discovery; Advisory / Consultancy: Sanofi; Research grant / Funding (institution): Agensys; Research grant / Funding (institution): Pfizer Inc.; Research grant / Funding (institution): Novartis. E.I. Heath: Advisory / Consultancy, Research grant / Funding (institution): Agensys; Honoraria (self), Speaker Bureau / Expert testimony: Sanofi; Honoraria (self): Bayer; Honoraria (self), Research grant / Funding (institution): Dendreon; Honoraria (self), Research grant / Funding (institution): Seattle Genetics; Research grant / Funding (institution): Tokai Pharmaceuticals; Research grant / Funding (institution): Genentech/Roche; Research grant / Funding (institution): Millennium; Research grant / Funding (institution): Celldex; Research grant / Funding (institution): Inovio Pharmaceuticals; Research grant / Funding (institution): Celgene; Research grant / Funding (institution): Zenith Epigenetics; Research grant / Funding (institution): Merck; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Esanik; Research grant / Funding (institution): Oncolys BioPharma; Research grant / Funding (institution): CureMeta; Research grant / Funding (institution): Bristol-Myers Squibb; Research grant / Funding (institution): eFFECTOR Therapeutics; Research grant / Funding (institution): Fortis. S. Perlman: Research grant / Funding (institution): GE Healthcare Progenics; Advisory / Consultancy: Pfizer Inc. T. Mayer: Advisory / Consultancy: Beacon Biosciences/ICON Medical; Honoraria (self): Novartis; Honoraria (self): AstraZeneca; Research grant / Funding (institution): Sotio; Research grant / Funding (institution): Merck; Research grant / Funding (self): Pfizer Inc. K. Modelska: Travel / Accommodation / Expenses, Shareholder / Stockholder / Stock options, Employee: Pfizer Inc. A. Porcari: Travel / Accommodation / Expenses, Shareholder / Stockholder / Stock options, Employee: Pfizer Inc. W. Duggan: Travel / Accommodation / Expenses, Shareholder / Stockholder / Stock options, Employee: Pfizer Inc. R. Jeraj: Licensing / Royalties, Patents: Wisconsin Alumni Research Foundation; Shareholder / Stockholder / Stock options: AIQ Solutions; Research grant / Funding (institution): GE Healthcare. All other authors have declared no conflicts of interest.

Resistance to targeted therapies can be caused by acquired genomic alterations in genes that code for the targeted protein. Circulating tumor cells (CTCs) provide an accessible source of tumor cells from diverse metastatic lesions from which to evaluate the frequency of genomic alterations that can be matched to distinct phenotypes, including therapeutic resistance. Successful analysis of the diversity of molecular mechanisms of therapeutic resistance requires isolation of live tumor cells for nucleic acid extraction. We have developed a semi-automated single cell aspirating platform to enrich and isolate live, rare cell populations for downstream molecular analysis. Using immortalized cell lines, we were able to enrich a target population of cells from a mixed population resulting a high purity sample (71.7-83.8%) to interrogate. Target cells were captured for nucleic acid extraction for downstream gene expression to validate enrichment. We demonstrate the ability to achieve high quality whole genome and transcriptome endpoints from low cell numbers in cell lines: the lower threshold for cellular input required to attain whole genome amplification (WGA) products reliable for downstream sequencing applications is now being validated. We also isolated CTCs from patient blood samples and showed phenotypic heterogeneity of EpCAM expression among individual CTCs. EpCAMhigh and EpCAMlow CTCs were detected, with a broad range of fluorescent intensities (141.2-5804.56 MFI). Using the phenotypic characterization of EpCAM expression, individual live CTCs were identified and captured for evaluation of their genomic heterogeneity. We have identified molecular heterogeneity in these cells that may reflect heterogeneity driving treatment resistance in CRPC. Early identification of treatment resistant clones may help develop intervention strategies that prolong the efficacy of molecular targeted therapies. Citation Format: Charlotte N. Stahlfeld, Jake J. Tokar, David Quigley, David Niles, Jamie M. Sperger, Felix Feng, Joshua M. Lang. Single cell capture and molecular analysis of live CTCs using integrated microwells and single cell aspirator [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 2291.

Background: Metastatic prostate cancer is the second leading cause of cancer-related deaths in US men. Nearly one-third of men develop recurrence after curative intent treatment. Bone metastases are common, and cause significant morbidity in addition to driving mortality rates. Early dissemination of tumor cells to the bone marrow has been hypothesized as a contributing factor to recurrence. However, the factors that promote early dissemination and survival in the bone microenvironment are not clearly elucidated. Key components of immune regulation such as cytotoxic T cells and regulatory T cells can regulate growth of cancer cell lines and in mouse models. We hypothesize that an immunosuppressive signature results in a permissive environment allowing the growth of disseminated prostate cancer cells. Therefore, the goal of our project is to evaluate mechanisms of immune evasion that promote the development of metastatic prostate cancer. Methods: Matched blood and bone marrow aspirates were collected from patients undergoing prostatectomy. Samples from five healthy donors were collected to establish a normal baseline. Using multicolor flow cytometry, samples were subjected to multi-parameter panels designed to assess the composition and function of lymphoid and myeloid immune compartments. These panels include markers of known immune subtypes, co-stimulatory signals, and activation signals. Results were analyzed with Prism software. Results: Preliminary results show variable T cell to B cell ratios ranging from as low as 1:1 to as high as 9:1. A high CD4 to CD8 T cell ratio was identified in 38% of samples, which corresponded with a higher occurrence of regulatory T cells. These data are consistent with an immunosuppressed bone marrow signature, which importantly was not present in the matched peripheral blood samples. Furthermore, our preliminary data demonstrates altered lymphoid and myeloid subsets in patients with prostate cancer as compared to healthy donors. Discussion: Overall, these results support the hypothesis that an immunosuppressive signature in bone marrow may promote survival of disseminated prostate cancer cells by protecting them from immune eradication. Furthermore, this immune signature within the microenvironment may exist at the time of prostatectomy and can be correlated with clinical biomarkers to identify factors that promote dissemination and metastatic recurrence. Future work involves using descriptive statistics to correlate the immune signatures identified in this project with patient outcomes, including PSA, Gleason grade, recurrence rates and time to recurrence. Ultimately, the goal is to identify an immune signature that accurately predicts risk of developing recurrent prostate cancer, potentially serving as a biomarker for patients that would benefit from aggressive upfront adjuvant systemic therapy. Citation Format: Nan Sethakorn, Erika Heninger, Jamie M. Sperger, Kenneth J. Pienta, Joshua M. Lang. An immunosuppressive signature in bone marrow as a potential biomarker for recurrence of metastatic prostate cancer after prostatectomy [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 1078.

Purpose: There is a critical clinical need for new predictive and pharmacodynamic biomarkers that evaluate pathway activity in patients treated with targeted therapies. A microscale platform known as VERSA (versatile exclusion-based rare sample analysis) was developed to integrate readouts across protein, mRNA, and DNA in circulating tumor cells (CTC) for a comprehensive analysis of the androgen receptor (AR) signaling pathway. Experimental Design: Utilizing exclusion-based sample preparation principles, a handheld chip was developed to perform CTC capture, enumeration, quantification, and subcellular localization of proteins and extraction of mRNA and DNA. This technology was validated across integrated endpoints in cell lines and a cohort of patients with castrate-resistant prostate cancer (CRPC) treated with AR-targeted therapies and chemotherapies. Results: The VERSA was validated in cell lines to analyze AR protein expression, nuclear localization, and gene expression targets. When applied to a cohort of patients, radiographic progression was predicted by the presence of multiple AR splice variants and activity in the canonical AR signaling pathway. AR protein expression and nuclear localization identified phenotypic heterogeneity. Next-generation sequencing with the FoundationOne panel detected copy number changes and point mutations. Longitudinal analysis of CTCs identified acquisition of multiple AR variants during targeted treatments and chemotherapy. Conclusions: Complex mechanisms of resistance to AR-targeted therapies, across RNA, DNA, and protein endpoints, exist in patients with CRPC and can be quantified in CTCs. Interrogation of the AR signaling pathway revealed distinct patterns relevant to tumor progression and can serve as pharmacodynamic biomarkers for targeted therapies. Clin Cancer Res; 23(3); 746–56. ©2016 AACR.

Background Expression of programmed-death ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC) is typically evaluated through invasive biopsies; however, recent advances in the identification of circulating tumor cells (CTCs) may be a less invasive method to assay tumor cells for these purposes. These liquid biopsies rely on accurate identification of CTCs from the diverse populations in the blood, where some tumor cells share characteristics with normal blood cells. While many blood cells can be excluded by their high expression of CD45, neutrophils and other immature myeloid subsets have low to absent expression of CD45 and also express PD-L1. Furthermore, cytokeratin is typically used to identify CTCs, but neutrophils may stain non-specifically for intracellular antibodies, including cytokeratin, thus preventing accurate evaluation of PD-L1 expression on tumor cells. This holds even greater significance when evaluating PD-L1 in epithelial cell adhesion molecule (EpCAM) positive and EpCAM negative CTCs (as in epithelial-mesenchymal transition (EMT)). Methods To evaluate the impact of CTC misidentification on PD-L1 evaluation, we utilized CD11b to identify myeloid cells. CTCs were isolated from patients with metastatic NSCLC using EpCAM, MUC1 or Vimentin capture antibodies and exclusion-based sample preparation (ESP) technology. Results Large populations of CD11b+CD45lo cells were identified in buffy coats and stained non-specifically for intracellular antibodies including cytokeratin. The amount of CD11b+ cells misidentified as CTCs varied among patients; accounting for 33–100% of traditionally identified CTCs. Cells captured with vimentin had a higher frequency of CD11b+ cells at 41%, compared to 20% and 18% with MUC1 or EpCAM, respectively. Cells misidentified as CTCs ultimately skewed PD-L1 expression to varying degrees across patient samples. Conclusions Interfering myeloid populations can be differentiated from true CTCs with additional staining criteria, thus improving the specificity of CTC identification and the accuracy of biomarker evaluation.

Background Donor brain death (BD) triggers a systemic inflammatory response that reduces organ quality and increases immunogenicity of the graft. We characterized the early innate immune response induced by BD in the liver and peripheral blood of hemodinamically stable non-human primates (NHP). Methods Rhesus macaques were assigned to either brain death or control group. BD was induced by inflation of a subdurally placed catheter and confirmed clinically and by cerebral angiography. Animals were monitored for 6 h after BD and managed to maintain hemodynamic stability. Results Cortisol, epinephrine, nor-epinephrine, and IL-6 levels were elevated immediately after BD induction. Neutrophils and monocytes significantly increased in circulation following BD induction, while dendritic cells were decreased at 6 h post-induction. Flow cytometry revealed increased expression of chemokine receptors CxCR1, CxCR2, CCR2, and CCR5 in peripheral blood leukocytes from NHP subjected to BD. Microarray analysis demonstrated a significant up-regulation of genes related to innate inflammatory responses, toll-like receptor signaling, stress pathways, and apoptosis/cell death in BD subjects. Conversely, pathways related to glucose, lipid, and protein metabolism were down-regulated. In addition, increased expression of SOCS3, S100A8/A9, ICAM-1, MHC class II, neutrophil accumulation, and oxidative stress markers (carboxy-methyl-lysine and hydroxynonenal) were detected by immunoblot and immunohistochemistry. Conclusions Activation of the innate immune response after BD in association with a down-regulation of genes associated with cell metabolism pathways in the liver. These findings may provide a potential explanation for the reduced post-transplant function of organs from brain dead donors. In addition, this work suggests potential novel targets to improve donor management strategies.

6S RNAs function through interaction with housekeeping forms of RNA polymerase holoenzyme (Eσ(70) in Escherichia coli, Eσ(A) in Bacillus subtilis). Escherichia coli 6S RNA accumulates to high levels during stationary phase, and has been shown to be released from Eσ(70) during exit from stationary phase by a process in which 6S RNA serves as a template for Eσ(70) to generate product RNAs (pRNAs). Here, we demonstrate that not only does pRNA synthesis occur, but it is an important mechanism for regulation of 6S RNA function that is required for cells to exit stationary phase efficiently in both E. coli and B. subtilis. Bacillus subtilis has two 6S RNAs, 6S-1 and 6S-2. Intriguingly, 6S-2 RNA does not direct pRNA synthesis under physiological conditions and its non-release from Eσ(A) prevents efficient outgrowth in cells lacking 6S-1 RNA. The behavioral differences in the two B. subtilis RNAs clearly demonstrate that they act independently, revealing a higher than anticipated diversity in 6S RNA function globally. Overexpression of a pRNA-synthesis-defective 6S RNA in E. coli leads to decreased cell viability, suggesting pRNA synthesis-mediated regulation of 6S RNA function is important at other times of growth as well.

Background: Tumor metastasis requires dynamic changes in cell adhesion mediated through beta 1-integrin and focal adhesion kinase (FAK). In particular, FAK is responsible for counteracting anoikis and promoting survival of CTCs. The Cub-Domain Containing Protein–1 (CDCP1) is a transmembrane glycoprotein expressed in epithelial cells that regulates cell adhesion. CDCP1 loss leads to activation of FAK in nonadherent cancer cells (na-PCC) (Spassov, 2011). As we demonstrated, another consequence of loss of CDCP1 is the inhibition of inside-out activation of beta 1-integrin in na-PCC, suggesting that FAK activation can occur in the absence of active beta 1-integrin. The main goal of this project is to determine how FAK becomes activated in na-PCC upon CDCP1 silencing and to establish that CDCP1 loss sensitizes to treatment with FAK inhibitors. Methods: CTCs were collected from nine patients with castrate-resistant prostate cancer (CRPC) (Schehr, 2016). CTC mRNA was eluted and used for CDCP1 qPCR. CDCP1 was stably silenced in DU145 with short hairpins (Spassov, 2013). In the anoikis assay, 5 x 105 DU145 shControl and shCDCP1 cells were suspended on HEMA coated plates for up to 6 hrs. Western blot membranes were probed for pMAPK1/2, MAPK1/2, FAKpY397, FAKpY925, and FAK. For MTT assays, 5,000 DU145 shControl and shCDCP1 were incubated for 24 hrs on HEMA-coated 96-well plates in regular culture medium +/- Saracatinib, FAKi-PF573228 (kinase domain inhibitor) and FAKi-LGA (FAT domain inhibitor) and/or MEKi-PD98059. A kinase dead mutant of PIP5K-D316A from the Addgene PIP5K plasmid was generated by site directed mutagenesis. Results: We find reduced CDCP1 expression in CTCs from patients with CRPC. Six patients did not express detectable CDCP1 mRNA in CTCs, 1 patient demonstrated CDCP1 expression at the limit of detection, and in 2 patients CDCP1 mRNA levels were above the limit of detection. Silencing CDCP1 in DU145 and PC3 cells results in 3.4-fold higher proliferation rates and 4.4-fold greater anchorage-independent growth rates. CDCP1-negative tumors grew in 100% of mice, versus 30% growth of CDCP1 positive control tumors. In na-PCC, CDCP1 silencing triggered FAK activation, as demonstrated by de novo pY397 and pY925. In addition, 73% of CDCP1-negative cells expressed FAK-stimulated pMAPK. FAK activation required a factor in human plasma and involved a trypsin-sensitive receptor in na-PCC. CDCP1 loss also conferred increased sensitivity to treatment with the PF573228 FAK-kinase inhibitor. At the 4uM concentration 98.1% of CDCP1-positive cells survived compared to 32.9% of CDCP1-silenced cells. An inhibitor treatment matrix showed a synergistic treatment effect of dual targeting of SRC, FAK, and MEK with up to 3-fold increased cytotoxicity of FAKi+SRCi or FAKi+MEKi. The largest treatment effect was observed when all 3 inhibitors were combined. To better understand the role of the FAK-MEK pathway in anoikis, a novel C-terminal FAT domain inhibitor was investigated and compared to PF573228. To determine the mechanism of FAK activation in na-PCC, we expressed a dominant negative PIP5K-D316A mutant. Expression of this mutant is expected to reduce CDCP1-induced FAK activation and also diminish SRC activity. Conclusion: We discovered a synergistic treatment effect between FAK and SRC and/or MEK inhibition in CDCP1-negative nonadherent cells. Expression of pFAK and pMAPK downstream of CDCP1 loss overcomes anoikis and depends on a factor in human plasma. By silencing beta 1-integrin and activating FAK, CDCP1-negative cancer cells may gain easy access to the circulation and survive during the CTC phase of cancer dissemination. CDCP1 loss may serve as a biomarker to identify patients who benefit from treatment with FAK inhibitors. Funding: This work was supported by Department of Defense Synergistic Idea Development Award W81XWH-08-1-0268. Citation Format: Sara Pollan, Jamie Sperger, Joshua Lang, Fangjin Huang, Jie Zheng, Colm Morrissey, Anne E. Cress, Danislav Spassov, Mark Moasser, William Carter, Beatrice Knudsen. CDCP1, a potential noninvasive biomarker in circulating tumor cells for treatment of prostate cancer with FAK inhibitors [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B096.

Background: Genomic instability and evolution of cancers is linked to chemotherapy sensitivity and resistance in patients with metastatic cancer. Taxane-based chemotherapies operate by promoting chromosomal instability (CIN) by increasing multipolar mitoses. Breast tumors with CIN are more sensitive to paclitaxel compared to tumors without CIN. There is a critical need for new biomarkers based on biological evaluation of mechanisms of response and resistance in metastatic cancer. Although CIN is difficult to detect directly, centrosome amplification (CA) is a common mechanism of CIN. In this study, we develop an assay to detect CA in circulating tumor cells (CTCs) as a minimally invasive assay to evaluate sensitivity to taxane-based chemotherapies. Methods: Blood samples from patients with metastatic breast and prostate cancer were collected and processed for nucleated cells using a Ficoll gradient and magnetic depletion of CD45-positive cells. EpCAM-positive CTCs were captured using an exclusion-based sample preparation technology known as the VERSA (Versatile Exclusion-based Rare Sample Analysis) platform. CTCs were defined as cells positive for Hoechst, cytokeratin and negative for multiple immune and endothelial cell markers (CD45/CD11b/CD14 and CD34). EpCAM-captured CTCs were also stained with pericentrin and centrin to identify CA and evaluated for pericentrin (whole centrosome marker) and centrin (centriole marker) protein-staining intensity, number and size. Results: CA was detected in a subset of CTCs from patients with metastatic breast and prostate cancer patients. As a control, centrosomes were also analyzed in matched peripheral blood mononuclear cells from the same patients. CA was heterogeneous with 100% of patients demonstrating some degree of CA (>4 centrioles), ranging from 15-48% of CTCs. Conclusion: In conclusion, we report the presence of CA in EpCAM-captured CTCs from metastatic cancer patients. Evaluation of CA in CTCs using pericentrin and centrin staining may serve as a predictive biomarker of taxane based chemotherapy response and resistance. We are recruiting taxane-naïve metastatic cancer patients to test the hypothesis that CA and/or CIN predict taxane sensitivity. Citation Format: Ashok Singh, Ryan A. Denu, Jamie M. Sperger, Beth A. Weaver, Mark E. Burkard, Joshua M. Lang. Centrosome amplification in epcam-captured circulating tumor cells from metastatic 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 3604.

Purpose: Circulating biomarkers are an emerging tool to monitor treatment response and the emergence of resistant phenotypes. However, studies of circulating biomarkers, including circulating tumor cells (CTCs), in patients with clear cell renal cell carcinoma (ccRCC) have been limited due to difficulty in biomarker identification. Platforms relying on EpCAM and cytokeratin to identify CTCs have been limited due to significant phenotypic and intrapatient heterogeneity in renal cancer. Carbonic anhydrase IX (CAIX) and XII (CAXII) are more broadly expressed in ccRCC and recently been shown to capture CTCs from patients with ccRCC. However, downregulation of these targets can also occur; EpCAM is also expressed on a subset of cells that could go undetected if only CAIX was used to capture these cells. The aim of this study is to optimize multi-marker capture and analysis of ccRCC CTCs using EpCAM, CAIX and CAXII for further molecular analysis. Methods: We utilized the VERSA platform, an integrated CTC capture and analysis technology, to optimize capture of multiple ccRCC cell lines using antibodies to CAIX and/or EpCAM. To maximize the capture efficiency of ccRCC CTCs, we altered the magnetic particle type, antibody concentration, and tested both direct and indirect capture methods. Once an optimal method of capture was determined, we captured CTCs in an initial cohort of ten patients with ccRCC. CTCs were identified as cells that were captured by either CAIX or EpCAM, had an intact nucleus, were negative for CD45/CD34/CD66b, and positive for cytokeratin. Results: Capture of cell lines show that a combined CAIX + EpCAM capture was more efficient than single antibody capture using either EpCAM or CAIX alone. The type of magnetic particle used in the assay also affected capture efficiency. Sera-Mag beads (GE Healthcare) captured significantly more cells than FlowComp Dynabeads (Life Technologies) (94% vs 76%). Further increases in efficiency were made by incubating with antibody prior to bead conjugation (indirect binding, 94%) when compared to incubating the cells with antibody-conjugated beads (direct binding, 98%). This may result from increased accessibility of free antibody to partially obstructed antigens that is unique to renal cell carcinoma. This optimized assay has now been applied to ccRCC patients and has identified CTCs in up to 90% of patients with metastatic disease. Conclusions: We have increased the capture efficiency and identification of ccRCC cells by capturing with a combination of CAIX and EpCAM antibodies and optimizing bead and binding conditions. In doing so, we are able to identify and interrogate populations of CTCs that would be lost in capture methods that rely on EpCAM alone. These assays are now being utilized in multiple biomarker and therapeutic trials for patients with clear cell renal cell carcinoma. Citation Format: Rory M. Bade, Benjamin K. Gibbs, Jamie M. Sperger, Christos Kyriakopolous, Hamid Emamekhoo, Rana R. McKay, Toni K. Choueiri, Joshua M. Lang. Development of multi-marker capture and analysis of circulating tumor cells in renal cell carcinoma [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 4585.

Remarkably little is known about the transcriptional profiles of human embryonic stem (ES) cells or the molecular mechanisms that underlie their pluripotency. To identify commonalties among the transcriptional profiles of different human pluripotent cells and to search for clues into the genesis of human germ cell tumors, we compared the expression profiles of human ES cell lines, human germ cell tumor cell lines and tumor samples, somatic cell lines, and testicular tissue samples by using cDNA microarray analysis. Hierarchical cluster analysis of gene expression profiles showed that the five independent human ES cell lines clustered tightly together, reflecting highly similar expression profiles. The gene expression patterns of human ES cell lines showed many similarities with the human embryonal carcinoma cell samples and more distantly with the seminoma samples. We identified 895 genes that were expressed at significantly greater levels in human ES and embryonal carcinoma cell lines than in control samples. These genes are candidates for involvement in the maintenance of a pluripotent, undifferentiated phenotype.

The Cub-Domain Containing Protein - 1, CDCP1, is a transmembrane glycoprotein, which is able to sequester Src and PKCδ into unique microdomains within the plasma membrane. CDCP1 can either promote or suppress tumor metastasis, dependent on the cancer type and experimental system. How the loss of CDCP1 leads to tumor metastasis is not well understood. We demonstrated for the first time in patients with prostate cancer (PCA), a significant reduction of CDCP1 expression in circulating cancer cells (CTC) and tumor metastasis relative to primary tumors and concordant analytical results. To investigate how the loss of CDCP1 facilitates PCA metastasis, we determined the consequences of CDCP1 loss in non-adherent cancer cells, which provide an experimental model system of CTCs. In this system, CDCP1 silenced cells exhibit 3-fold higher proliferation, 4-fold greater anchorage independent growth with colonies exceeding 5 microns in diameter, and a 2-fold reduction in migration ability. In 10% human plasma, these cells up-regulate p-FAK, p-SRC, p-AKT and p-MAPK expression, and at the same time loose expression of activated β1-integrin. The loss of inside-out activation of β1-integrin occurs through prevention of TALIN phosphorylation. Upon loss of CDCP1, Talin is no longer phosphorylated by CDK5 and this causes the disassembly of the β1 integrin - Talin complex. In addition, β1-integrin dissociates from CDK5 and from the CDK5-regulatory subunit, p35, but remains bound to p-FAK. We determined the pathway by which the loss of CDCP1 arrests CDK5 kinase activity. Upon loss of CDCP1, SRC phosphorylates p35. This generates a binding site for the C2 domain of PKCδ and phosphorylation of CDK5-T77 by PKCδ. The subsequent dissociation of the regulatory subunit abolishes the activity of CDK5. We show that both SRC inhibition and silencing of PKCδ reestablish the inside-out activation of β1-integrin. Altogether we discovered a new mechanism of regulation of CDK5 in prostate cancer cells, which leads to the uncoupling of β1-integrin and FAK. The potential biological and clinical consequences of this mechanism are (1) a switch from cell-matrix to cell-cell adhesion, (2) increased sensitivity of CTCs to FAK inhibitors and (3) improved adaptation and survival of cancer cells in the circulation and at the metastatic sites. Citation Format: Sara G. Pollan, Fangjin Huang, Joshua M. Lang, Jamie M. Sperger, Kavita Shah, Beatrice S. Knudsen. Loss of CDCP1 in patient prostate cancer metastasis leads to uncoupling of beta-1 integrin from its cytoplasmic signaling through FAK [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 5795. doi:10.1158/1538-7445.AM2017-5795

Purpose: Therapeutic resistance to Androgen Receptor (AR) targeted therapies in patients with Castration Resistant Prostate Cancer (CRPC) is complex and originates from a wide range of molecular alterations including AR point mutations, gene rearrangements and splice variant expression. An aggressive subtype of CRPC involves neuroendocrine de-differentiation that is often found in patients with visceral metastases. To evaluate neuroendocrine signatures in patients progressing on AR targeted therapies, we measured gene expression in circulating tumor cells (CTCs) as a minimally-invasive approach to examine putative resistance mechanisms to AR targeted therapies. Methods: We utilized the VERSA platform, an integrated CTC capture and analysis technology, to capture CTCs using antibodies for EpCAM or TROP2 in parallel. mRNA was extracted on chip from live cells for comparison of gene expression profiles for markers of neuroendocrine de-differentiation (SYP, CHGA, GHGB, MYCN, NCAM) and the AR signaling pathway. Fixed PBMC were stained and imaged with fluorescence microscopy and CTCs were enumerated by defining CTCs as EpCAM or TROP2 captured, intact nuclei, negative for CD45/CD11b/CD14/CD34 and cytokeratin positive. Synaptophysin or Chromogranin A protein expression was quantified in individual CTCs and compared to tumor biopsy results where available. Results: Expression of the Synaptophysin (SYP) gene was found in 39% of the patients in our initial 18 patient cohort. These patients could be split into two groups. Group 1 patients had low AR expression with minimal detection of AR target genes. Of these four patients, three had visceral metastases. Group 2 patients had high levels of AR expression including expression of the splice variants ARV7 and ARV9, and robust expression of KLK3 (PSA) and PSMA. Of these three patients, two had visceral metastases. Subsets of the patients in both groups also exhibited expression of genes identified in neuroendocrine prostate cancer including Chromogranin B, MYCN, and NCAM. A subset of the patients tested for gene expression are being screened for CTC detection of protein markers of neuroendocrine phenotypes including Chromogranin A and Synaptophysin. Conclusions: We report the first identification of neuroendocrine gene expression signatures in CTCs from patients with CRPC experiencing disease progression on advanced AR targeted therapies. These signatures may serve as predictive biomarkers of resistance to AR targeted therapies. Future studies will evaluate these signatures in prospective clinical trials. Citation Format: Jamie M. Sperger, Anupama Singh, David Niles, Charlotte N. Stahlfeld, David J. Beebe, Joshua M. Lang. Expression of neuroendocrine markers in circulating tumor cells from patients with prostate cancer visceral metastases [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 3781. doi:10.1158/1538-7445.AM2017-3781

Background: Breast cancer (BC) is the second leading cause of cancer related death in American women. There is a critical need for new biomarkers to evaluate treatment response and emerging mechanisms of resistance. The identification and characterization of new biomarkers such as Circulating Tumor Cells (CTCs) may be helpful tools for these purposes as well as understanding the biology of metastatic BC. Methods: Blood samples were processed with a Ficoll gradient to isolate nucleated cells. CTCs were isolated from these samples using an exclusion-based sample preparation technology known as the VERSA (Versatile Exclusion-based Rare Sample Analysis) platform. EpCAM positive CTCs were captured in the VERSA followed by mRNA extraction for gene expression profiling for the estrogen receptor (ER) and other breast cancer relevant targets. CTCs were defined as Hoechst positive, CD45/CD11b/CD14 negative, and cytokeratin positive. Captured CTCs were evaluated for ER protein staining intensity and localization. Results: The VERSA platform was validated to capture and enumerate CTCs from patients with breast cancer using EpCAM capture and immunofluorescent staining for intact nucleus, ER, and cytokeratin while negative for multiple immune markers (CD45, CD11b, CD14). The VERSA can capture cells with >90% capture efficiency with EpCAM antibody in MCF7 and T47D cell lines. ER specific transcriptome analysis in BC patient CTCs and cell lines was performed. We evaluated expression of ER signaling pathway, prognostic, metastatic/EMT; stemness; epithelial markers with concordant expression of RPLPO and HPRT1. We identified expression of ER in 6 out of 7 BC samples. Expression of PR and HER2 in a subset of samples confirms capture of breast cancer cells for further molecular interrogation. Conclusion: In conclusion, we report the expression profiles of ER signaling genes in EpCAM captured CTCs from BC patients. These tools will be used to compare subpopulations of CTCs from patients with BC as predictive and pharmacodynamic biomarkers of response to targeted therapies or chemotherapies. Citation Format: Ashok Singh, Jamie M. Sperger, Jennifer Schehr, Tessa Witkowski, Beth A. Weaver, Mark E. Burkard, Joshua M. Lang. Expression of estrogen receptor specific signaling transcriptome in epithelial cell adhesion molecule (EpCAM) captured circulating tumor cells from patients with breast cancer [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 3784. doi:10.1158/1538-7445.AM2017-3784

4537 Background: Recent use of immune checkpoint inhibitors (CI) has improved overall survival (OS) in a subset of patients (pts) with metastatic UC. Intra-patient tumoral heterogeneity and epithelial-to-mesenchymal transition has been hypothesized as a driver of treatment resistance to both chemotherapy and CI in UC. There is a critical need to evaluate heterogeneity in UC for biomarkers of treatment response and new therapeutic targets. Trop2 is hypothesized to play a role in UC progression and is the target of a new antibody-drug conjugate, IMMU-132 that is being tested in UC trials. We report the phenotypic comparison of PDL1 expression among CTC subpopulations in UC pts. Methods: Peripheral blood samples were collected from pts with UC treated at Cleveland Clinic and U. of Wisconsin. Immunomagnetic capture and CTC enumeration using both EpCAM and Trop2 from matched blood samples was performed in the VERSA platform. Protein expression for PDL1 in these CTC populations was quantified. Longitudinal analysis of UC pts treated with chemotherapy or CI is ongoing. Results: CTC were captured using EpCAM and Trop2 in all 10 pts in our initial cohort. The frequency of Trop2 CTC was higher than EpCAM CTC with a mean of 248 Trop2 CTC (range 2-1885) compared to 76 EpCAM CTC (range 1-632). PDL1 expression was more frequent in Trop2 CTC than EpCAM CTC. In two pts progressing on Atezolizumab, Trop2 CTC had a higher frequency of PDL1 expression compared to EpCAM CTC (85% vs 2% in pt 1 and 2% vs 0% in pt 2). In pts followed longitudinally, Trop2 CTC dropped from 1885 to 1 in a pt with response to Atezolizumab and PDL1+ CTC declined from 4 to 0. After 1 cycle of Carbo/Gem in another pt, EpCAM CTC declined from 46 to 3 while Trop2 CTC from 116 to 47. Conclusions: This is the first report of CTC heterogeneity in pts with UC identifying high frequency of Trop2 CTCs with variable expression of PDL1 across different pts. Early results from longitudinal analysis suggest CTC as potential predictive / pharmacodynamic biomarkers of treatment response. Prospective data validation in larger cohort is ongoing, while IMMU-132 clinical trials in UC may provide context for future validation.