C. M. Court1,5, S. Hou1,3, S. Sho1,5, P. Winograd1,5, Q. Li1, S. Sadeghi4, R. S. Finn4, F. W. Samuel3,6, B. V. Naini6, F. M. Kaldas1,2, R. W. Busuttil1,2, J. S. Tomlinson1,5, H. R. Tseng3, V. G. Agopian1,2 1University Of California – Los Angeles,Surgery,Los Angeles, CA, USA 2University Of California – Los Angeles,Liver Transplantation,Los Angeles, CA, USA 3University Of California – Los Angeles,Molecular And Medical Pharmacology,Los Angeles, CA, USA 4University Of California – Los Angeles,Hematology/Oncology,Los Angeles, CA, USA 5VA Greater Los Angeles,Surgery,Los Angeles, CA, USA 6University Of California – Los Angeles,Pathology,Los Angeles, CA, USA
Introduction: Treatment options available to patients with hepatocellular carcinoma (HCC) include potentially curative surgical therapy (resection and liver transplantation) for early stage patients and molecular targeted therapy for advanced/metastatic patients. Currently, treatment selection is based on clinical and radiological staging of disease, partly due to the lack of available biomarkers to inform treatment decisions. Circulating tumors cells (CTCs) are one such potential biomarker with proven efficacy in many cancers, but have not been well characterized in HCC. We sought to develop a novel, blood-based assay capable of detecting HCC CTC phenotypes and genotypes with prognostic importance that may impact treatment selection.
Methods: Utilizing the microfluidic NanoVelcro platform which allows for multichannel immunocytochemistry as well as single cell isolation and sequencing, we prospectively evaluated 4 milliliters of blood from 69 patients (50-HCC, 11-benign liver disease, 8-healthy controls) and developed assays to enumerate and characterize CTC phenotype (epithelial-type cytokeratin (CK+) CTCs, mesenchymal-type Vimentin (Vim+) CTCs, programmed death-ligand 1 (PDL1+) CTCs) and identify CTC genotype by sequencing for actionable somatic mutations associated with targeted drug therapy. CTC number and phenotype was correlated with clinicopathologic data.
Results: CK+ CTCs were detected in 96% of HCC patients (Mean=6.3, range=1-27) and discriminated HCC and non-HCC patients (≥2 CTCs, sensitivity=90.0%, specificity=89.5%, AUROC=0.946, p<0.001). Mesenchymal-type Vim+ CTCs were detected nearly exclusively in advanced (stage III/IV) patients (Mean = 6.5, range = 1-20), with excellent discrimination between early, transplant-eligible and advanced stage, transplant-ineligible patients (AUROC = 0.970, p < 0.001; Fig). In advanced HCC patients, PDL1+ CTCs were detected in 5/8 (63%) of patients, and were noted to decrease following treatment with nivolumab, a PD-1 inhibitor. Using our single CTC isolation and sequencing method, we were able to perform gene panel sequencing in CTCs from 10 patients. In two of those patients, potentially actionable mutations (PIK3CA and TP53) were found in CTC-DNA.
Conclusion:CTCs are a promising serum biomarker in HCC with potential treatment implications for both early and late stage patients. Vim+ CTCs accurately discriminated early and late stage HCC patients and may improve transplant candidate selection. For advanced stage patients, both CTC phenotype (PDL1+) and genotype analysis may allow for selection of specific targeted therapies. Longitudinal follow-up with evaluation of cancer-specific outcomes is necessary to establish whether CTCs may improve current treatment algorithms.
