Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection

Abstract

Genomic characterization of circulating tumor cells (CTCs) may prove useful as a surrogate for conventional tissue biopsies. This is particularly important as studies have shown different mutational profiles between CTCs and ctDNA in some tumor subtypes. However, isolating rare CTCs from whole blood has significant hurdles. Very limited DNA quantities often can’t meet NGS requirements without whole genome amplification (WGA). Moreover, white blood cells (WBC) germline contamination may confound CTC somatic mutation analyses. Thus, a good CTC enrichment platform with an efficient WGA and NGS workflow are needed. Here, Vortex label-free CTC enrichment platform was used to capture CTCs. DNA extraction was optimized, WGA evaluated and targeted NGS tested. We used metastatic colorectal cancer (CRC) as the clinical target, HCT116 as the corresponding cell line, GenomePlex® and REPLI-g as the WGA methods, GeneRead DNAseq Human CRC Panel as the 38 gene panel. The workflow was further validated on metastatic CRC patient samples, assaying both tumor and CTCs. WBCs from the same patients were included to eliminate germline contaminations. The described workflow performed well on samples with sufficient DNA, but showed bias for rare cells with limited DNA input. REPLI-g provided an unbiased amplification on fresh rare cells, enabling an accurate variant calling using the targeted NGS. Somatic variants were detected in patient CTCs and not found in age matched healthy donors. This demonstrates the feasibility of a simple workflow for clinically relevant monitoring of tumor genetics in real time and over the course of a patient’s therapy using CTCs.

VTX‐1 Liquid Biopsy System for Fully‐Automated and Label‐Free Isolation of Circulating Tumor Cells with Automated Enumeration by BioView Platform

“…Here, CTC capture is based on cell size, cell shape, and deformability and is the agnostic of tumor biomarkers (Fig. Numerous workflows have been optimized for various CTC research applications, such as immunofluorescence staining and CTC enumeration (23)(24)(25)(26)29), cytopathology and cytogenetics (24), Sanger sequencing (26), next-generation sequencing (27,29), and even Western-blotting on single cells (28) (Fig. The Vortex chip has been characterized and validated for CTC isolation from blood samples of metastatic cancer patients with breast, colon, lung and prostate cancer (23)(24)(25)(26)(27)(28)(29).…”

“…Workflows for Sanger sequencing (26), qPCR, or next-generation sequencing (27) have been previously published or presented. Workflows for Sanger sequencing (26), qPCR, or next-generation sequencing (27) have been previously published or presented.…”

“…1A). The Vortex chip has been characterized and validated for CTC isolation from blood samples of metastatic cancer patients with breast, colon, lung and prostate cancer (23)(24)(25)(26)(27)(28)(29). Numerous workflows have been optimized for various CTC research applications, such as immunofluorescence staining and CTC enumeration (23)(24)(25)(26)29), cytopathology and cytogenetics (24), Sanger sequencing (26), next-generation sequencing (27,29), and even Western-blotting on single cells (28) ( Fig.…”

“…The VTX-1 system makes the molecular analysis of CTCs simpler by first isolating the CTCs with high purity and then presenting them in a format where DNA and RNA extraction can be easily accomplished, as needed for the assay selected. Workflows for Sanger sequencing (26), qPCR, or next-generation sequencing (27) have been previously published or presented. Furthermore, the plasma can be collected prior to isolation of the CTCs for the isolation of ctDNA.…”

Toward Microfluidic Label‐Free Isolation and Enumeration of Circulating Tumor Cells from Blood Samples

“…1B), while blood cells pass through the main channels. Isolated cells are thus unbiased by molecular characteristics, enabling the isolation of both epithelial and mesenchymal CTCs (19). At the outlet, CTCs are collected in suspension at high purity, and different downstream assays can be performed such as, immunofluorescence, cytology, cytogenetics, nextgeneration sequencing, qPCR, live cell assays, RNA sequencing, or protein assays (20)(21)(22)(23).…”