Evaluation of a next-generation sequencing (NGS) platform for genomic analysis in circulating tumour cells (CTCs)

Abstract

Circulating tumour cells (CTCs) are the potential seeds of metastases travelling in the bloodstream of cancer patients. Both their frequency in blood and the gene mutations they carry are believed to have great prognostic impact. Their potentials in being a minimally invasive tool for cancer screening, prognosis prediction, and disease monitoring have been widely studied in recent decades. Yet, their scarcity in blood poses significant challenges in their detection and sequencing. Most existing studies suffer from selection bias for epithelial-like cells in the CTC enrichment. In order to facilitate the translation of CTCs from bench to bedside, this study conducted a systematic comparison of CTC counts across 10 cancer types with standardized size-based enrichment and detection approaches, evaluated the sensitivity and positive predictive value (PPV) of the SeqCap sequencing platform in rare variant detection, and streamlined the sequencing benchwork and analyses procedures of the genomic profiling of clinical CTC samples. In this study, ovarian cancer was detected with the highest proportion of CTC-positive samples and the highest number of somatic mutations by SeqCap sequencing. Under the current analysis pipeline, SeqCap sequencing has a PPV and sensitivity of over 90% and 28%, respectively, on detecting variants with AF at 2.5%. This platform successfully detected deleterious missense, splicing or stop-gain variants on putative driver genes and/or potential therapeutic targets, such as ATM and BRAF, from 85% of CTC samples sequenced. Parallel sequencing with another NGS platform, HEAT-Seq, validated 73% of SeqCap-called variants and identified 32 potential false negatives from SeqCap sequencing platform. The results demonstrated that SeqCap can be used for confident detection of the rare variants from CTCs on putative driver genes and potential druggable gene targets, although further studies are required to confirm the biological functions of novel variants detected. Parallel HEAT-Seq sequencing may be a fast and cost-efficient way to validate the large number of variants called by SeqCap and may potentially help to salvage false negative variants rejected by SeqCap. To further improve the sensitivity of SeqCap sequencing, the use of dual index systems in library preparation may also be explored. In short, the current study has illuminated the potential of the SeqCap sequencing platform in detecting clinically relevant mutations and potentially druggable targets from CTCs. This study provides further insights to understand the prognostic value of CTCs in cancer management, established the role of CTC sequencing in personalized treatment, and facilitates the real-time acquisition of biological information from CTCs.