Abstract P2-02-19: Somatic genetic profiling of circulating tumor cells (CTC) in metastatic breast cancer (MBC) patients
Authors: Paoletti C , Cani AK , Aung K , Darga EP , Cannell EM , Hovelson DH , Yazdani M , Blevins AR , Tokudome N , Larios JM , Thomas DG , Brown ME , Gersch C , Schott AF , Robinson DR , Chinnaiyan AM , Bischoff F , Hayes DF , Rae JM , Tomlins SA 
Published: May 2016

Abstract
Introduction: Somatic mutations, including those in TP53, PIK3CA, and estrogen receptor alpha (ESR1), are key to the biology of cancer and response to therapy. Recently, somatic cancer-associated mutations have been identified in circulating cell free plasma tumor DNA (ptDNA). Less is known about the mutation profile of DNA extracted from CTC (CTC-DNA). Since CTC-DNA provides mutational information of single cells, we hypothesize CTC-DNA will complement ptDNA to give greater insight into tumor heterogeneity.
Methods: Patients with ER positive MBC who were enrolled in the Mi CTC-ONCOSEQ, a companion trial to Mi-ONCOSEQ (the Michigan Oncology Sequencing Program), and who had ≥5CTC/7.5 ml whole blood were included. CTC were enriched from white blood cells (WBC) with CellSearch© (CXC kit). CTC and WBC were then purified using DEPArrayTM. DNA from individual CTC and WBC was isolated and subjected to whole genomic amplification (Ampli 1TM WGA). Genetic analysis was performed on individual CTC, pooled CTC and pooled WBC DNA by multiplexed PCR based targeted next generation sequencing (NGS) using the Oncomine Comprehensive Panel (targeting ∼130 onco- and tumor suppressor genes) and the Ion Torrent Proton. All patients had exome sequencing performed on research biopsies of metastases using an Illumina HiSeq 2500 platform.
Results: This pilot study was conducted using high quality DNA from two patients assessed to date. Both patients had lobular carcinoma and as expected harbored somatic, deleterious CDH1 (E-cadherin) mutations (frameshift and non-sense) in both research biopsy and CTC-DNA. These data supported our approach. Patient #1 was TP53 wild type in her research biopsy, but multiple CTC harbored somatic TP53 frame-shift mutations (Table). Patient #2 harbored an ESR1 Y537S mutation in her research biopsy. However, only 4 of 7 CTC also harbored this somatic, heterozygous mutation.
Prioritized mutations in CTCPt#Cell Type (CTC vs WBC), numberGeneMutationVariant fraction (expected 1=homozygous; 0.5=heterozygous)Found in research biopsy?1CTC_A2CDH1p.I584fs1YES CTC_A4 1 CTC_A7 0.54 CTC_pool* 0.74 WBC_pool 0 CTC_A2TP53p.152_156del1NO CTC_A4 1 CTC_A7 0.51 CTC_pool* 0.88 WBC_pool 0 2CTC_A9ESR1p.Y537S0.52YES CTC_D1 0.34 CTC_D2 0.46 CTC_D6 0.65 CTC_pool* 0.35 WBC_pool 0 CTC_A12 0 CTC_D3 0 CTC_D7 0 CTC_A12CDH1p.Q641X1YES CTC_A9 1 CTC_D1 1 CTC_D3 1 CTC_D6 1 CTC_pool* 1 WBC_pool 0 * pool of all CTC
Conclusions: We demonstrate the ability to purify CTC, isolate, and amplify DNA of suitable quality for genetic analysis using a comprehensive targeted sequencing panel. Both known and novel alterations were identified in comparison to research biopsy specimens. This approach allows single cell analysis demonstrating heterogeneity of mutational status in different single cells. Studies of CTC-ESR1 and other genetic abnormalities in patients with known tissue mutations who participated in Mi CTC-ONCOSEQ are now underway.
Citation Format: Paoletti C, Cani AK, Aung K, Darga EP, Cannell EM, Hovelson DH, Yazdani M, Blevins AR, Tokudome N, Larios JM, Thomas DG, Brown ME, Gersch C, Schott AF, Robinson DR, Chinnaiyan AM, Bischoff F, Hayes DF, Rae JM, Tomlins SA. Somatic genetic profiling of circulating tumor cells (CTC) in metastatic breast cancer (MBC) patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-19.