Whole-genome sequencing of single circulating tumor cells from neuroendocrine neoplasms

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.

Keywords: circulating tumor cells; copy number variation; neuroendocrine tumors; single cell; whole-genome sequencing.

Figure 1

Experimental design of the study. Workflow used in the study to enrich for CTCs and CNV profiling using Ampli1 WGA and LowPass kit for Illumina. Following enrichment (EpCAM-dependent vs size-based platforms), single NEN CTCs and matched WBC are selectively recovered in dynamically controlled dielectrophoretic cages using the DEPArray Image-Assisted Digital Cell Sorter. CTC samples undergo WGA and QC prior to low-resolution whole-genome sequencing for CNV profiling. Where surgical resection or biopsy specimens are available, samples are processed for bulk LPWGS and single-cell LPWGS as per CTCs.

Figure 2

Comparison of low-resolution whole-genome copy number profiles for CTCs and bulk tissue reveals reproduction of the majority of the CNV from the formalin-fixed paraffin-embedded (FFPE) and fresh frozen (FF) tissue in CTC samples. Unsupervised hierarchical clustering heat map of each analyzed individual CTC and tissue sample based on CNV from three SINET patients. Each patient is depicted with one color as shown on the phenobar at the bottom of the heat map. Individual CTCs are categorized according to enrichment method and tissue into bulk vs single-cell FFPE (see key). Chromosomal CNV is shown from top to bottom for each individual cell or sample; copy number gains are depicted in blue, losses in orange.

Figure 3

Individual CTC CNV data depicting complex intrapatient and interpatient genomic diversity. Unsupervised hierarchical clustering heat map of all analyzed CTCs based on CNV across seven patients. Each patient is depicted with one color as shown on the phenobar at the bottom of the heat map along with the cell sorting method and primary NET site.

Figure 4

Relationship between CTCs from all seven NEN patients is revealed through TSNE analysis. (A) Single CTCs from all seven patients are visualized and can be identified by color in the phenobar at the top of the figure. Cells are also depicted according to enrichment strategy (see key). (B) TSNE of all analyzed CTCs and WBC.

Figure 5

Distribution plot describing the impact of enrichment strategy in patient 7 (A) and all patients (B) on the proportion of the genome that is aberrant and CNV diversity as quantified by Shannon Index. Each small line represents the described value for a single CTC. Large bars represent mean values.

Figure 6

Frequency of genomic amplifications and deletions across all CTCs. Profiles demonstrated for SINET patients (A), patient 5; pancreatic NEN (B), patient 6; GOJ NEN (C), and patient 7; renal NEN (D).