Abstract 3788: Monitoring of multimodality immune checkpoint inhibitor treatment efficacy in metastatic melanoma patients through molecular analysis of circulating tumor cells
Authors: Lin Selena , Lam Stella , Ohe Shuichi , Tran Kevin , Ramos Irene , Bhagat Ali Asgar , Lim Chwee Teck , O'Day Steven J. , Foshag Leland , Hoon Dave 
Published: July 2017

Abstract
There is a dire need for molecular tools that can monitor therapy efficacy and prognosis in melanoma patients who suffer from distant metastasis. Blood biopsies offer a minimally invasive platform for clinicians to monitor melanoma patients. In particular, the molecular analysis of circulating tumor cells (CTCs) would be advantageous as they reflect both mutation and transcriptome profile of the tumor. However, the isolation and assessment of a minimal number of CTCs present in 10 ml of blood still remains challenging. To address the needs for more informative molecular CTC assays, our group used well-established blood-based melanoma mRNA CTC biomarkers to demonstrate utility in several multicenter phase II/III clinical trials through a direct quantitative RT-PCR approach. Here, we employed the microfluidics-based ClearCell® FX system (Clearbridge BioMedics) system, non-antibody based approach to develop a dual molecular assay to characterize CTCs for our previously established melanoma mRNA biomarkers (MART1, TRP2, MAGEA3, PAX, and GALNCT) and the clinically-actionable BRAF V600E mutation (mt). The assay was optimized for CTC isolation from whole blood using three BRAF V600E (+) melanoma cell lines and demonstrated sensitivity for 25 melanoma cells in 10 ml (107 cells) of blood. Immunofluorescence (IF) staining of the melanoma associated antigens verified CTCs were of melanoma origin post-isolation in stage IV patients. Next, the assay was subjected to 100 blood samples collected from 56 melanoma stage III/IV patients. We identified CTCs in 94% of pre-operative (pre-op) patients (n=15/16) as determined by detection of ≥2 mRNA markers. BRAF V600E mt was detected in 100% of pre-op patient (n=5/5) CTCs with BRAF V600E (+) tumors using a sensitive droplet digital PCR (ddPCR) assay. Bloods from patients undergoing active mono/combination immunotherapy (ipilimumab, nivolumab, pembrolizumab) were also evaluated by our CTC biomarker panel. Serial bleeds (2-5 bleeds) from 22 patients were profiled and correlated with clinicopathological factors. To facilitate further biomarker identification predictive of therapy response, CTCs isolated from stage IV patients undergoing immunotherapy were cultured to develop spheres and eventually primary cell lines, and further analyzed by deep RNA sequencing. This study developed a unique platform to analyze patient CTCs for melanoma biomarkers, and provides a potential platform to identify novel CTC-derived melanoma biomarkers indicative of multimodality treatment efficacy.
Citation Format: Selena Lin, Stella Lam, Shuichi Ohe, Kevin Tran, Irene Ramos, Ali Asgar Bhagat, Chwee Teck Lim, Steven J. O'Day, Leland Foshag, Dave Hoon. Monitoring of multimodality immune checkpoint inhibitor treatment efficacy in metastatic melanoma patients through molecular analysis of circulating tumor cells [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 3788. doi:10.1158/1538-7445.AM2017-3788