A Novel Multimarker Assay for the Phenotypic Profiling of Circulating Tumor Cells in Hepatocellular Carcinoma

摘要

Current clinicopathologic staging systems and serum biomarkers poorly discriminate tumor biology in hepatocellular carcinoma (HCC), with high recurrence rates following curative-intent surgical resection and liver transplantation (LT). Identification of accurate biomarkers for improved prognostication and treatment selection is a critical unmet need. We sought to develop a novel “liquid-biopsy” assay capable of detecting HCC circulating tumor cells (CTCs), and characterizing phenotypic subpopulations with prognostic significance. Utilizing HCC cell lines, a tissue microarray, and human blood samples, an antibody cocktail targeting the cell-surface markers asialoglycoprotein receptor (ASGPR), Glypican-3, and epithelial cell adhesion molecule (EpCAM) was optimized for HCC-CTC capture utilizing the NanoVelcro microfluidic assay. The ability of HCC-CTCs and vimentin(+)-CTCs (a subpopulation expressing an epithelial-to-mesenchymal phenotype) to accurately discriminate tumor stage, recurrence, progression, and overall survival was evaluated in a prospective study of 80 patients. Multimarker capture detected greater numbers of CTCs than any individual antibody alone for both cell line and patient samples (p<0.05). HCC-CTCs were identified in 59/61 patients (97%), and accurately discriminated HCC (median: 6 CTCs) and non-HCC patients (median: 1 CTC; AUROC=0.92, p<0.0001; sensitivity=84.2%, specificity=88.5%). Vimentin(+)-CTCs accurately discriminated early-stage, LT eligible patients (median: 0 CTCs) from locally advanced/metastatic, LT ineligible patients (median: 6 CTCs; AUROC=0.89, p<0.0001; sensitivity=87.1%, specificity=90.0%), and predicted overall survival for all patients (HR 2.21, p=0.001), and faster recurrence after curative-intent surgical or locoregional therapy in potentially curable early stage HCC (HR 3.14, p=0.002). In conclusion, we developed a novel multimarker CTC enrichment assay that detects HCC-CTCs with high efficiency and accuracy. A phenotypic subpopulation of vimentin(+)-CTCs appears to signify the presence of aggressive underlying disease and occult metastases, and may have important implications for treatment selection.