Defining A Liquid Biopsy Profile of Circulating Tumor Cells and Oncosomes in Metastatic Colorectal Cancer for Clinical Utility

SIMPLE SUMMARY: Metastatic colorectal cancer (mCRC) is typified by its tumor heterogeneity and changing disease states, suggesting that personalized medicine approaches could be vital to improving clinical practice. As a minimally invasive approach, the liquid biopsy has the potential to be a powerful longitudinal prognostic tool. We investigated mCRC patients’ peripheral blood samples using an enrichment-free single-cell approach to capture the broader rare-event population beyond the conventionally detected epithelial-derived circulating tumor cell (CTC). Our analysis reveals a heterogenous profile of CTCs and oncosomes not commonly found in normal donor samples. We identified select rare cell types based on their distinct immunofluorescence expression and morphology across multiple assays. Lastly, we highlight correlations between enumerations of the blood-based analytes and progression-free survival. This study clinically validates an unbiased rare-event approach in the liquid biopsy, motivating future studies to further investigate these analytes for their prognostic potential. ABSTRACT: Metastatic colorectal cancer (mCRC) is characterized by its extensive disease heterogeneity, suggesting that individualized analysis could be vital to improving patient outcomes. As a minimally invasive approach, the liquid biopsy has the potential to longitudinally monitor heterogeneous analytes. Current platforms primarily utilize enrichment-based approaches for epithelial-derived circulating tumor cells (CTC), but this subtype is infrequent in the peripheral blood (PB) of mCRC patients, leading to the liquid biopsy’s relative disuse in this cancer type. In this study, we evaluated 18 PB samples from 10 mCRC patients using the unbiased high-definition single-cell assay (HDSCA). We first employed a rare-event (Landscape) immunofluorescence (IF) protocol, which captured a heterogenous CTC and oncosome population, the likes of which was not observed across 50 normal donor (ND) samples. Subsequent analysis was conducted using a colorectal-targeted IF protocol to assess the frequency of CDX2-expressing CTCs and oncosomes. A multi-assay clustering analysis isolated morphologically distinct subtypes across the two IF stains, demonstrating the value of applying an unbiased single-cell approach to multiple assays in tandem. Rare-event enumerations at a single timepoint and the variation of these events over time correlated with progression-free survival. This study supports the clinical utility of an unbiased approach to interrogating the liquid biopsy in mCRC, representing the heterogeneity within the CTC classification and warranting the further molecular characterization of the rare-event analytes with clinical promise.