Abstract 1724: Genomic profiling of Vortex-enriched CTCs using whole genome amplification and multiplex PCR-based targeted next generation sequencing
Authors: Liu Haiyan E. , Triboulet Melanie , Zia Amin , Vuppalapaty Meghah , Kidess-Sigal Evelyn , Coller John , Natu Vanita S. , Shokoohi Vida , Che James , Renier Corinne , Chan Natalie , Hanft Violet , Sollier-Christen Elodie , Jeffrey Stefanie S. 
Published: July 2017

Abstract
Background: Genomic characterization of circulating tumor cells (CTCs) provides insights into cancer genetic changes, and might be utilized for cancer prognosis, diagnosis, as well as monitoring of therapeutic efficacy. Targeted Panel Next Generation Sequencing (NGS) enables analyzing CTC genetic variants of a focused gene panel at a relatively lower cost1. However, CTCs are rare, often resulting in very limited DNA quantities available that require whole genome amplification (WGA). In previous studies, we introduced the Vortex technology, a platform enabling label-free enrichment of CTCs from blood samples of colorectal cancer (CRC) patients and their use for genomic assays downstream2. In this study, we developed a simple and efficient NGS workflow for CTC samples collected by this technology.
Method: An optimized workflow using the Qiagen GeneRead DNAseq targeted panel and Illumina MiSeq NGS was first verified on HCT116 CRC cell line before being applied on patient CTCs. For patient blood samples, CTCs were collected with the Vortex technology, immunostained (CK, Vimentin, CD45) and enumerated. Matched white blood cell (WBC) DNA was included to subtract germline background. Fresh frozen liver metastasis tissue was collected and analyzed using the same NGS workflow. DNA from CTCs was extracted and amplified using Qiagen REPLI-g single cell WGA kit. Mutation detection on the WGA amplified DNA was performed using the GeneRead DNAseq CRC targeted panel of 38 genes and MiSeq sequencing. The sequencing data were analyzed by QIAGEN NGS Data Analysis Web Portal and Ingenuity Variant Analysis software.
Results: The Vortex technology was validated for the capture of CTCs from CRC patients. REPLI-g performed a uniform, unbiased amplification on fresh rare cells with a coverage of 97.7%, which enabled further targeted panel NGS. Blood from 3 CRC patients (P1, P2, P3) and 2 healthy donors (HD1, HD2) was processed with Vortex platform. Less than 1 CTCs/mL blood were found in HD1 and HD2. P1 and P2 had 66 and 20 CTCs/ mL of blood respectively, with many vimentin positive CTC clusters. P3 had 2 CTCs/mL of blood. No somatic mutation was found in healthy donors. Somatic variants were only detected in the CTCs from patient samples that were not present in matched germline WBCs. For P1, more mutations were found in the CTCs than in the liver metastasis while it was the opposite for P2 and P3.
Conclusion: For each patient, variants in CTCs and germline WBCs were analyzed from one blood sample using an optimized targeted NGS workflow and compared to liver mets. Our optimized workflow, using the Qiagen REPLIg and GeneRead DNAseq Targeted Panel NGS enabled the detection of CTC mutations for 38 CRC-focused genes. The inclusion of a germline WBC control in the workflow allowed the detection of mutations from pooled CTC samples collected using the Vortex technology.
Altmüller J, et al. (2014). Biol Chem.
Kidess-Sigal E, et al. (2016). Oncotarget.
Citation Format: Haiyan E. Liu, Melanie Triboulet, Amin Zia, Meghah Vuppalapaty, Evelyn Kidess-Sigal, John Coller, Vanita S. Natu, Vida Shokoohi, James Che, Corinne Renier, Natalie Chan, Violet Hanft, Elodie Sollier-Christen, Stefanie S. Jeffrey. Genomic profiling of Vortex-enriched CTCs using whole genome amplification and multiplex PCR-based targeted next generation sequencing [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 1724. doi:10.1158/1538-7445.AM2017-1724