EpCAM Circulating Tumor Cells Research Articles

Intra-patient heterogeneity in urothelial cancer (UC) circulating tumor cells (CTC) and PDL1 expression to identify biomarkers of response and new therapeutic targets: A pilot study.

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.

Trop-2 expression on treatment resistant cancer cells in castrate-resistant prostate cancer (CRPC) as a predictive biomarker for targeted therapy.

5045 Background: Trophoblastic cell-surface antigen (Trop-2) is a transmembrane glycoprotein that is highly expressed in many solid tumors. Sacituzumab govitecan (IMMU-132) is an antibody-drug conjugate of an anti-Trop-2 humanized antibody with SN-38. Early clinical trials have shown high response rates in a broad range of diseases including triple negative breast and urothelial cancers. We evaluated Trop-2 expression in tumor biopsies and circulating tumor cells (CTCs) from men with mCRPC (metastatic castrate-resistant prostate cancer). Methods: Trop-2 expression was evaluated from mCRPC biopsies from patients (Pts) treated with abiraterone acetate (AA) on the PROMOTE clinical trial, CTCs from a separate cohort treated with either enzalutamide or AA. Trop-2 CTCs were compared with EpCAM captured CTCs using a microscale technology termed the VERSA (Vertical Exclusion-based Rare Sample Analysis) platform to compare protein and gene expression signatures of resistance to these agents. Results: RNA sequencing identified Trop-2 gene expression in > 70% of metastatic biopsies. The AR splice variant V7 was found in 48 biopsies that also expressed Trop-2. Trop-2 expression was not altered by treatment with AA at 12 weeks. The number of CTCs captured from 25 pts with Trop-2 or EpCAM were closely correlated (R2= 0.84). Gene expression analysis showed similar patterns of expression for the TROP-2 and EPCAM captured cells. AR splice variant expression (AR-V7, AR-V9) in Trop-2 and EpCAM CTCs was detected in 33% of patients. Expression of neuroendocrine markers was identified in 40% of Trop-2 CTCs. Conclusions: Trop-2 is frequently expressed in mCRPC and co-expressed in tumors that express AR splice variants. Trop-2 CTCs are detected in CRPC pts previously treated with AA or Enzalutamide that also express multiple AR splice variants and neuroendocrine markers. The results support Trop-2 expression as predictive biomarker of sensitivity to targeted therapies tumors resistant to AA or Enzalutamide. Men with mCRPC are being enrolled on a Phase I trial with IMMU-132, and multi-site Phase II clinical trial in men who have progressed on AA or Enzalutamide is being finalized.

Abstract 1602: Development and validation of an NGS-based assay to detect all classes of genomic alterations in circulating tumor cells (CTCs) from patients with solid tumors

Abstract Purpose With increased use of targeted therapeutics, the ability to perform a ‘liquid biopsy’ on circulating tumor cells (CTCs) holds potential to address the need for a more representative and longitudinal means to monitor tumor burden in patients. However, the analytic and clinical validity of such an assay has proved difficult to establish, as the cell population in circulation is complex, and the CTC portion is heterogeneous, evolving, and highly dependent on clinical factors as well as CTC isolation methods. No studies to date have characterized CTCs for the full range of relevant genomic alterations in the context of patient-matched primary and metastatic tissue biopsies. We set out to develop a method to perform comprehensive NGS-based genomic profiling on phenotypically-isolated CTCs, and to compare CTC analysis with a comprehensive tissue-based assessment of paired primary and metastatic tissue in patients. Methods An integrated platform (VERSA) was employed for capture of EpCAM CTCs, enumeration and DNA extraction. Extracted DNA was amplified using a modified version of MDA-based whole genome amplification (WGA), followed by standard adaptor-ligated library construction and solution hybridization using a custom DNA baitset before sequencing on Illumina HiSeq. Data was analyzed using a computational workflow developed to detect base substitutions, indels, high-level copy number alterations, and select gene rearrangements. We performed extensive analytic validation of the assay using over 65 cell line mixtures, constructed to contain all classes of known variants across all allele frequencies (0-100%). We used the assay to analyze CTC DNA from 20 lung cancer patients, paired with analysis of temporally-matched primary and/or metastatic FFPE samples analyzed by FoundationOne, an FFPE tissue-based assay. Results Validation experiments showed that for samples meeting minimum acceptance criteria (50 cells at 50% purity), assay sensitivity exceeded 95% for detections of base substitutions, 85% for indels and rearrangements, and 80% for high-level copy number alterations, while maintaining tight specificity control. WGA-induced coverage bias limited sensitivity to low-level copy number alterations. Comparison of driver alterations across CTC samples and paired primary/metastatic tissue for 20 patients with lung cancer is ongoing. Conclusions A comprehensive NGS-based approach to genomic profiling is feasible on CTCs isolated from lung cancer patients, and demonstrated accuracy sufficient to characterize actionable targets for clinical decision-making. However, more extensive tumor-type specific studies with a similar matched-tissue approach must be performed in order to establish full analytic and clinical validation, and assay specifications will need to be optimized to enable use on broader patient populations. Citation Format: Allison Welsh, Garrett M. Frampton, Zachary R. Chalmers, Jamie Sperger, Roman Yelensky, Doron Lipson, Geoff Otto, Lindsay Strotman, Scott Berry, Hannah Pezzi, Anne Traynor, David J. Beebe, Vincent A. Miller, Joshua M. Lang, Philip J. Stephens. Development and validation of an NGS-based assay to detect all classes of genomic alterations in circulating tumor cells (CTCs) from patients with solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1602. doi:10.1158/1538-7445.AM2015-1602