Hepatocellular Carcinoma–Circulating Tumor Cells Expressing PD‐L1 Are Prognostic and Potentially Associated With Response to Checkpoint Inhibitors

Abstract

Hepatocellular carcinoma (HCC) is a leading cause of mortality. Checkpoint inhibitors of programmed cell death protein-1 (PD-1) and programmed death-ligand 1 (PD-L1) have shown great efficacy, but lack biomarkers that predict response. Circulating tumor cells (CTCs) have promise as a liquid-biopsy biomarker; however, data on HCC CTCs expressing PD-L1 have not been reported. We sought to detect PD-L1-expressing HCC-CTCs and investigated their role as a prognostic and predictive biomarker. Using an antibody-based platform, CTCs were enumerated/phenotyped from a prospective cohort of 87 patients with HCC (49 early-stage, 22 locally advanced, and 16 metastatic), 7 patients with cirrhosis, and 8 healthy controls. Immunocytochemistry identified total HCC CTCs (4′,6-diamidino-2-phenylindolepositive [DAPI+]/cytokeratin-positive [CK+]/clusters of differentiation 45-negative [CD45−]) and a subpopulation expressing PD-L1 (DAPI+/CK+/PD-L1+/CD45−). PD-L1+ CTCs were identified in 4 of 49 (8.2%) early-stage patients, but 12 of 22 (54.5%) locally advanced and 15 of 16 (93.8%) metastatic patients, accurately discriminating early from locally advanced/metastatic HCC (sensitivity = 71.1%, specificity = 91.8%, area under the receiver operating characteristic curve = 0.807; P < 0.001). Compared to patients without PD-L1+ CTCs, patients with PD-L1+ CTCs had significantly inferior overall survival (OS) (median OS = 14.0 months vs. not reached, hazard ratio [HR] = 4.0, P = 0.001). PD-L1+ CTCs remained an independent predictor of OS (HR = 3.22, P = 0.010) even after controlling for Model for End-Stage Liver Disease score (HR = 1.14, P < 0.001), alpha-fetoprotein (HR = 1.55, P < 0.001), and overall stage/tumor burden (beyond University of California, San Francisco, HR = 7.19, P < 0.001). In the subset of 10 patients with HCC receiving PD-1 blockade, all 5 responders demonstrated PD-L1+ CTCs at baseline, compared with only 1 of 5 nonresponders, all of whom progressed within 4 months of starting treatment. Conclusion: We report a CTC assay for the phenotypic profiling of HCC CTCs expressing PD-L1. PD-L1+ CTCs are predominantly found in advanced-stage HCC, and independently prognosticate OS after controlling for Model for End-Stage Liver Disease, alpha-fetoprotein, and tumor stage. In patients with HCC receiving anti-PD-1 therapy, there was a strong association with the presence of PD-L1+ CTCs and favorable treatment response. Prospective validation in a larger cohort will better define the utility of PD-L1+ CTCs as a prognostic and predictive biomarker in HCC. (Hepatology Communications 2020;4:1527-1540). H epatocellular carcinoma (HCC) is the fourth leading cause of cancer mortality worldwide and remains an important global health burden, with an estimated one million deaths attributable to HCC by 2030. (1) In the United States, the age-adjusted incidence of HCC has increased

Liquid Biopsy in Hepatocellular Carcinoma: Opportunities and Challenges for Immunotherapy

“…Winograd and colleagues analyzed the expression of PD-L1 on CTCs and found that PD-L1-positive CTCs are mainly found in the advanced stages of disease and that they represent an independent prognostic factor for overal survival. Moreover, of 10 patients receiving anti-PD-1 treatment, 6 had PD-L1positive CTCs at baseline, and of these, 5 had a favorable treatment response, whereas 4 patients did not have PD-L1+ CTCs and were non-responders [136]. They definitely unraveled the prognostic and potentially predictive value of CTCs in the context of HCC.…”

Epigenetic Biomarkers for the Diagnosis and Treatment of Liver Disease

“…However, the results also suggest that the reliability and utility of liquid biopsy tests most likely rely on the use of multi-marker analyses. Further studies are needed to evaluate the performance of combination of methylated cfDNA biomarkers with ctDNA mutations and/or protein biomarkers in nucleosomes [ 116 ], extracellular vesicles [ 117 ], or CTCs [ 118 ], together with markers of tissue of origin [ 113 , 114 ]. Moreover, the combination of these emerging epigenetic biomarkers with currently used serological markers can be envisioned to be used to improve HCC diagnosis, patient stratification, and evaluation of treatment response.…”

Liquid Biopsy in Hepatocellular Carcinoma: Where Are We Now?

“…Moreover, it is clear from several studies that CTCs are a heterogeneous population and that they may reflect tumoral heterogeneity better than a tissue biopsy [ 187 , 195 ]. The CTC pERK/pAkt phenotype has recently been reported to predict sensitivity to sorafenib [ 222 ], while the presence of CTCs positive for PD-L1 is associated with response to checkpoint inhibitors [ 223 ]. Considered that result, it could be imagined that phenotyped CTCs will be useful surrogates for guiding enrichment trials with molecular targeted therapies.…”