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

Haiyan E Liu,Natalie H Chan,Stefanie S Jeffrey,Vida Shokoohi,Violet R Hanft,Corinne Renier,Amin Zia,John Coller,Evelyn Kidess-Sigal,Meghah Vuppalapaty,James Che,Vanita S Natu,Elodie Sollier-Christen,Melanie Triboulet

doi:10.1038/s41525-017-0034-3

Haiyan E Liu, Natalie H Chan + Show 12 more

Open Access

https://doi.org/10.1038/s41525-017-0034-3

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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.

Highlights

Circulating tumor cells (CTCs) are cancer cells shed into the blood stream by both primary and metastatic tumors
When the QIAamp DNA Micro kit and standard cell protocol were tested on HCT116 cells fixed with 4% PFA, DNA yield was as low as 0.1–0.5 pg/cell, i.e., around 1% of the yield obtained for fresh cells (Table 1)
Enumeration and mutational profiling of CTCs are both important aspects of CTC research studies

Summary

Introduction

Circulating tumor cells (CTCs) are cancer cells shed into the blood stream by both primary and metastatic tumors. Single-site tumor biopsies may not recapitulate intra-tumor and inter-tumor heterogeneity, if multiple metastases are present, and may fail to reflect the genetic diversity of a patient’s disease. These limitations can be overcome with non-invasive blood tests, called liquid biopsies, and importantly include the isolation and analyses of CTCs. Liquid biopsy facilitates serial sampling to enable real-time and more accurate monitoring of disease during tumor evolution and through assessment of patient response to treatment changes, providing a more personalized and timesensitive treatment of the cancer. Previous studies have shown that CTC enumeration and mutational profiling may be used to monitor cancer disease,[4] and to predict progression and overall survival of cancer patients.[5,6,7] This is important because several studies have suggested that in some tumor subtypes, such as colorectal or lung cancer, some patients’ CTCs and cell-free ctDNA may show different mutational profiles.[8,9,10,11]

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