Characterization of Tumor-Infiltrating Lymphocytes in Hepatocellular Carcinoma

Levels of tumor-infiltrating lymphocytes (TILs) in the hepatocellular carcinoma (HCC) microenvironment are correlated with prognosis in patients with early-stage HCC following surgical resection, but levels in patients with more advanced stage or infiltrative disease, and their association with outcomes after locoregional therapy (LRT), have not been characterized. We retrospectively identified 124 consecutive patients with 132 HCC diagnosed by percutaneous biopsy between 2006-2017 prior to LRT. Of these, 36 tumor biopsies in 35 patients (23 men, 12 women; median age 58 y) were found to have sufficient tissue available for analysis. Baseline Barcelona Clinic Liver Cancer (BCLC) stage were 0 (n=3, 8.3%), A (n=10, 27.8%), B (n=7, 19.4%), C (n=8, 22.2.9%) and D (n=8, 22.2%). One 5 μm section was obtained per biopsy specimen for immunohistochemical analysis. A novel multiplex immunolabeling protocol based on tyramide signal amplification with Opal fluorophores was utilized to characterize the location and levels of TIL populations of CD3+, CD4+ helper, CD8+ cytotoxic, CD45RO+ memory, and FoxP3+ regulatory T cells. Levels of TIL subtypes were determined for regions of tumor, tumor stroma, adjacent dysplasia, and adjacent non-tumor liver tissue for each sample. This characterization study provides the first comprehensive assessment of the HCC immune microenvironment in patients who were not candidates for curative surgical resection.