A novel multimarker assay for the phenotypic profiling of circulating tumor cells in hepatocellular carcinoma

Liver Transpl. 2018 Jul;24(7):946-960. doi: 10.1002/lt.25062.


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. Using 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 was optimized for HCC CTC capture using the NanoVelcro CTC Assay. The ability of HCC CTCs and vimentin (VIM)-positive CTCs (a subpopulation expressing an epithelial-to-mesenchymal phenotype) to accurately discriminate tumor stage, recurrence, progression, and overall survival (OS) 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 < 0.001). HCC CTCs were identified in 59/61 (97%) patients, and HCC (median, 6 CTCs) and non-HCC patients (median, 1 CTC; area under the receiver operating characteristic curve [AUROC] = 0.92; P < 0.001; sensitivity = 84.2%; specificity = 88.5%) were accurately discriminated. VIM-positive 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 = 0.001; sensitivity = 87.1%; specificity = 90.0%), and predicted OS for all patients (hazard ratio [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 VIM-positive CTCs appears to signify the presence of aggressive underlying disease and occult metastases and may have important implications for treatment selection. Liver Transplantation 24 946-960 2018 AASLD.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aged
  • Asialoglycoprotein Receptor / analysis
  • Asialoglycoprotein Receptor / metabolism
  • Biological Assay / methods*
  • Biomarkers, Tumor / analysis*
  • Biomarkers, Tumor / metabolism
  • Carcinoma, Hepatocellular / blood*
  • Carcinoma, Hepatocellular / mortality
  • Carcinoma, Hepatocellular / pathology
  • Cell Line, Tumor
  • Epithelial Cell Adhesion Molecule / analysis
  • Epithelial Cell Adhesion Molecule / metabolism
  • Female
  • Glypicans / analysis
  • Glypicans / metabolism
  • Healthy Volunteers
  • Humans
  • Immunoassay / methods
  • Kaplan-Meier Estimate
  • Liquid Biopsy / methods
  • Liver Cirrhosis / blood
  • Liver Neoplasms / blood*
  • Liver Neoplasms / mortality
  • Liver Neoplasms / pathology
  • Male
  • Microfluidics / methods
  • Middle Aged
  • Neoplasm Recurrence, Local / diagnosis*
  • Neoplasm Recurrence, Local / mortality
  • Neoplastic Cells, Circulating / metabolism*
  • Prognosis
  • Prospective Studies
  • Sensitivity and Specificity
  • Tissue Array Analysis
  • Vimentin / metabolism


  • Asialoglycoprotein Receptor
  • Biomarkers, Tumor
  • Epithelial Cell Adhesion Molecule
  • GPC3 protein, human
  • Glypicans
  • VIM protein, human
  • Vimentin