Abstract 3151: Genetic profiling of circulating tumor cells (CTC) in metastatic breast cancer (MBC) patients
Authors: Paoletti Costanza , Cani Andi K. , Aung Kimberly , Darga Elizabeth P. , Cannell Emily M. , Hovelson Daniel H. , Yazdani Maryam , Blevins Allen R. , Tokudome Nahomi , Baratta Paul J. , Larios Jose’ M. , Thomas Dafydd G. , Brown Martha E. , Gersch Christina , Schott Anne F. , Robinson Daniel , Chinnaiyan Arul M. , Bischoff Farideh , Hayes Daniel F. , Rae James M. , Tomlins Scott A. 
Published: July 2016

Abstract
Introduction: Cancer-associated mutations are present in circulating cell free plasma tumor DNA (ptDNA). We have previously reported mutation profiles of DNA extracted from CTC (CTC-DNA) from two patients with MBC (#2 and 24 in table). Here, we report an expanded cohort with an updated gene panel.
Methods: We studied seven patients (two previously reported, along with five additional patients) with MBC who were enrolled in Mi-CTC-ONCOSEQ, had ≥5 CTC/7.5 ml whole blood (WB), and had at least one CTC with high quality DNA determined by the Ampli1™ quality control kit. CTC were enriched from WB with CellSearch© and purified from white blood cells (WBC) (DEPArray™). DNA from individual CTC and WBC was isolated and subjected to whole genomic amplification (Ampli 1™ WGA) and genotyped by multiplexed PCR-based next generation sequencing with the Oncomine Comprehensive Panel (OCP) on the Ion Torrent Proton. Exome sequencing of research biopsies of metastatic tissue was performed using an Illumina HiSeq 2500 platform. Previously reported patients (#2 and 24) sequenced with a beta version of the OCP were re-run, and updated results are provided.
Results: Six of seven patients were ER positive. Patients #2, 12, and 24 had CTC with mutations also found in the research biopsy (table). Novel alterations were found in comparison to research biopsy in five of the seven patients (table). In two patients (#19, 24), two potential actionable mutations (PTCH1 and NOTCH1) were found in CTC-DNA but not in tissue-DNA. No mutations were detected in any WBC.
Conclusions: We demonstrate the ability to purify CTC, and to isolate and amplify DNA of suitable quality for genetic analysis using a comprehensive targeted sequencing panel. Mutations found in tissue as well as novel mutations were found in CTC-DNA. Two potential actionable mutations were identified in CTC, but not in tissue, opening potentially new therapeutic opportunities. We conclude that mutational analysis of CTC-DNA and of tissue may be complementary. Prioritized mutations in CTCsPt #Gene (Mutation)# CTC Single (S) Pooled (P)# with mutation (variant fraction)# without mutation# not evaluable (insufficient coverage)# WBC (all pooled)# with mutationPresent in Biopsy?2CDH1 (p.Q641X)7 (S)5 (1.00)NA230YCDH1 (S70F)7 (S)5 (1.00)NA230YESR1 (p.Y537S)7 (S)4 (0.46)2130YESR1 (unreported mutation)7 (S)1 (0.56)5130N8NA3 (P)*NANA3 (P)40NA12PIK3CA (H1047R)1 (S)1 (0.85)NANA10YTP53 (p.R248Q)1 (S)1 (0.72)NANA10Y14HNF1A (p.W206C)3 (P)+ (0.17)NANA40N17BRCA2 (p.Q1931X)4 (P)+ (0.10)NANA40N19PTCH1 (p.E1242X)3 (P)+ (0.28)NANA30N24CDH1 (p.I584fs)5 (P)+ (0.79)NANA40Y4 (P)+ (0.68)4 (P)+ (0.77)CDH1 (p.E841X)5 (P)0NANA40N4 (P)+ (0.14)4 (P)0TP53 (p.152_156del)5 (P)+ (0.94)NANA40Y4 (P)+ (0.29)4 (P)+ (0.36)NOTCH1 (p.S2492X)5 (P)0 +NANA40N4 (P)(0.17)4 (P)0Legend: NA = not applicable; + = mutation present in pooled CTC; Y = Yes; N = No; *CTC-DNA from the pool of 3 CTC had low and high quality.
Citation Format: Costanza Paoletti, Andi K. Cani, Kimberly Aung, Elizabeth P. Darga, Emily M. Cannell, Daniel H. Hovelson, Maryam Yazdani, Allen R. Blevins, Nahomi Tokudome, Paul J. Baratta, Jose’ M. Larios, Dafydd G. Thomas, Martha E. Brown, Christina Gersch, Anne F. Schott, Daniel Robinson, Arul M. Chinnaiyan, Farideh Bischoff, Daniel F. Hayes, James M. Rae, Scott A. Tomlins. Genetic profiling of circulating tumor cells (CTC) in metastatic breast cancer (MBC) patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3151.