Abstract
Single-cell profiling of circulating tumor cells (CTCs) as part of a minimally invasive liquid biopsy presents an opportunity to characterize and monitor tumor heterogeneity and evolution in individual patients. In this study, we aimed to compare single-cell copy number variation (CNV) data with tissue and define the degree of intra- and inter-patient genomic heterogeneity. We performed next-generation sequencing (NGS) whole-genome CNV analysis of 125 single CTCs derived from seven patients with neuroendocrine neoplasms (NEN) alongside matched white blood cells (WBC), formalin-fixed paraffin-embedded (FFPE), and fresh frozen (FF) samples. CTC CNV profiling demonstrated recurrent chromosomal alterations in previously reported NEN copy number hotspots, including the prognostically relevant loss of chromosome 18. Unsupervised hierarchical clustering revealed CTCs with distinct clonal lineages as well as significant intra- and inter-patient genomic heterogeneity, including subclonal alterations not detectable by bulk analysis and previously unreported in NEN. Notably, we also demonstrated the presence of genomically distinct CTCs according to the enrichment strategy utilized (EpCAM-dependent vs size-based). This work has significant implications for the identification of therapeutic targets, tracking of evolutionary change, and the implementation of CTC-biomarkers in cancer.
Highlights
The molecular characterization of tumors has advanced our understanding of the major somatic driver mutations and informed the development of targeted therapies, which have transformed outcomes in selected patient populations (Vogel et al 2002, Sharma et al 2007, Sosman et al 2012)
Seven neuroendocrine neoplasms (NEN) patients were included with primary tumor sites comprising the small intestine (SINET) (n = 4), pancreas (n = 1), gastro-esophageal junction (GOJ) (n = 1), and kidney (n = 1)
Copy number analysis of NEN circulating tumor cells (CTCs) confirmed a wide range of genomic aberrations making them readily distinguishable from white blood cells (WBC)
Summary
The molecular characterization of tumors has advanced our understanding of the major somatic driver mutations and informed the development of targeted therapies, which have transformed outcomes in selected patient populations (Vogel et al 2002, Sharma et al 2007, Sosman et al 2012). Whilst tissue biopsy remains central to diagnostic work-up, it is invasive, limited by the overall percentage of tumor cells, and subject to heterogeneity exhibited in primary and metastatic tumors (Navin et al 2010, Gerlinger et al 2012, Walter et al 2018). Bulk genomic analysis cannot provide resolution at the single-cell level, which is required to fully define the extent of tumor heterogeneity. Characterization of single-circulating tumor cells (CTCs) as part of a minimally invasive 'liquid biopsy' provides an opportunity to explore tumor biology and identify therapeutic targets. Molecular analysis of single CTCs has been used to identify predictive biomarkers, such as the T790M resistance allele in NSCLC (Maheswaran et al 2008). In SCLC, a pretreatment CTCbased biomarker has been shown to predict sensitivity to first-line chemotherapy (Carter et al 2017)
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