Abstract 1922: Target mutation comparasion of WGA circulating tumor cell and primary tumor in epithelial ovarian cancer cell line using NGS
Authors: Sung Pi-Lin , Huang Chi-Ying F. , Hsiao Michael , Wen Kuo-Chang , Yen Ming-Shyen , Lee Mong-Hong 
Published: August 2015

Introduction

Epithelial ovarian cancer is the third most common gynecologic cancer in the United States but a leading cause of gynecologic cancer deaths. Previous finding that numbers of circulating tumor cells (CTCs) in the blood of ovarian cancer patients are prognostically significant. Moreover, comprehensive analyses of ovarian cancer genomes may provide more information for precise cancer treatments. Genomic analysis of circulating tumor cells (CTC) can provide possible approach for EOC patients who lack primary tumor tissue or present with un-resectable metastatic diseases. The discordance between whole genome amplification of circulating tumors and primary tumor should be validated firstly and so we conducted a pilot analysis of CTC capture and targeted NGS in EOC cell lines.

Methods and materials

Under IRB approval, EOC cell lines, different numbers of SKOV-3 (serous adenocarcinoma) and ES-2 (clear cell carcinoma) were spiked in fresh blood samples (20 ml) of healthy donor and captured by CytoQuestTM CR system or negative enrichment method (Abnova, Taipei, Taiwan). CTCs were identified by pan-CK, CD45 and DAPI expression and stained with CEP8 FISH for aneuploidy. Spiked CTCs of two cell lines were captured and underwent whole genome amplification by PicoPLEX™ WGA kit (Rubicon Genomic, MI, USA). The WGA DNA product and DNA of two primary cell lines were prepared libraries for TruSight(TM) Tumor sequencing panel (Illumina Inc.MI,USA) according to manufacturer's instruction. Sequencing procedure were performed on MiSEQ system(Illumina Inc.) by a local service provider.

Results

SKOV-3 and ES-2 can be stained and captured with these antibodies. Mean recovery of these cells were more than 80% by both systems. However, ES-2 is less Pan-CK stained but still could be captured by this system. WGA products of each one, three and five ES-2 cells and one SKOV3 cell were prepared well for library. We choose one cell WGA product and DNA extracted from primary SKOV-3 and ES2 were subjected to targeted 26 genes mutational analysis composed in TruSight Tumor panel. Average coverage depth for each amplicon is minimum 1000x with 7000x means. We would like to show if any discordance appear due to the acquisition of DNA WGA in the single cells, of which the analysis is currently ongoing.

DISCUSSION

Our study would like to demonstrate the condition of variants calls of single tumor cells isolated from whole blood samples and primary tumor in a targeted mutation analysis using NGS after a highly automated isolation workflow. This approach provides a pilot workflow for studying the heterogeneity within the CTC population in blood samples of patients with tumor and their primary tumor and the results can also document the possibility of WGA-induced bias of a recently commercialized WGA kit.

Citation Format: Pi-Lin Sung, Chi-Ying F. Huang, Michael Hsiao, Kuo-Chang Wen, Ming-Shyen Yen, Mong-Hong Lee. Target mutation comparasion of WGA circulating tumor cell and primary tumor in epithelial ovarian cancer cell line using NGS. [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 1922. doi:10.1158/1538-7445.AM2015-1922