Abstract LB-B09: Somatic mutation detection via sequencing using circulating tumor cell samples from patients with renal cell and prostate cancer

Abstract

Abstract Introduction: Tumor genotyping using fluid samples such as blood can potentially allow tracking of dynamic changes in mutational profiles over time and allow better access than biopsies. We present a method to detect somatic mutations from a blood draw, where circulating tumor cell (CTC) enrichment above 10% of total cell numbers allows the use of standard gene panels typically used to analyze tissue-based biopsies. Methods: Clinical samples were obtained from 9 prostate cancer (PC) patients and 6 renal cell cancer (RCC) patients, followed by CTC enrichment using the IsoFluxTM System. Cells were lysed and DNA amplified by whole genome amplification (WGA) using the NGS Kit (Fluxion Biosciences) and quantified via qPCR. CTCs defined as CK+, CD45- nucleated cells (DAPI+) for cell enumeration. Analytical samples were prepare by spiking tumor derived cell lines into whole blood and parallel analysis. Next-generation sequencing was performed using 3 targeted cancer panels on the Ion torrentTM PGM platform: the Ion ampliseqTM cancer hot spot panel (50 genes; 6 PC samples), Oncomine (143 genes; 3 PC samples), and a 29-gene panel of actionable mutations in RCC (6 samples). Data was analyzed using a customized variant calling/filtering pipeline. Variant filtering and functional interpretation was performed using VarSeqTM. All data was analyzed in a blinded manner. Results: Our method was able to isolate CTCs from all patient samples. WGA DNA concentrations were at a range of 25-164 ng/μL (median, 69) in PC and 29-180 ng/μL (median, 69) in RCC. CTC purity after the first enrichment step was in 2.9-33.7% (median, 10.5%) of PC samples and 1.9-33% (median, 14.5%) of RCC; final CTC purity is estimated at 5-40%. We found 1 variant/sample using hotpot, 12/sample using Oncomine, and 3/sample using the RCC panel. Conclusions: Our assay consistently detected somatic variants from blood draw using standard gene panels in both PC and RCC. Obtaining repeat tumor biopsies from patients during treatment and/or at time of progression is both challenging and impractical from a clinical perspective. Our assay provides molecular characterization using standard blood draws and will be prospectively validated in clinical trials. Citation Format: Robert J. Amato, Reynolds Brobey, Mehdi Dehghani, Kevin Rosenblatt, Glauco Souza, Hubert Tseng, Jeff Jensen, Cristian Ionescu-Zanetti. Somatic mutation detection via sequencing using circulating tumor cell samples from patients with renal cell and prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B09.