Analysis of programmed death-ligand 1 (PD-L1) expression, transforming growth factor (TGF)-β gene expression signatures (GES) and tumor-infiltrating immune cells (IC) in hepatocellular carcinoma (HCC): Rationale for targeting PD-L1- and TGF-β

Background: HCC evades antitumor immune responses via multiple mechanisms, including the PD-L1 and TGF-β pathways. PD-L1 expression correlates with tumor aggressiveness and recurrence. Increased TGF-β activity corresponds with poor clinical outcomes. Using immunohistochemistry (IHC), we previously showed that PD-L1 expression in HCC stems primarily from IC. To further assess the HCC immune milieu, we measured IC, TGF-β-associated GES, and PD-L1 expression using IHC/RNAseq. Methods: We assessed protein expression in 50 resected HCC specimens by quantitative (Q) IHC (primary antibodies: PD-L1, CD8, CD68) using standard techniques and automated software. For RNAseq, we prepared strand-specific libraries from extracted RNA, which were sequenced and compared to GES from published papers, CIBERSORT and Ingenuity Pathway Analysis. Results: All cases had typical morphology (low- to high-grade trabecular, pseudoglandular, or solid with common cytoplasmic features). Q CD8 IHC significantly correlated with CD8 mRNA expression and CD8 T cell GES, supporting the utility of RNAseq to evaluate the role of CD8+ T cells in HCC. RNAseq identified TGF-β1 as the main TGF-β isoform in HCC. Predefined TGF-β GES correlated strongly with EMT GES. There was a trend toward increased TGF-β1 activity and EMT marker expression in the S1 molecular subtype, which has previously been associated with TGF-β-driven aberrant Wnt signaling. Q CD8 IHC correlated with PD-L1 mRNA and protein levels in IC. In samples with high CD8, there was a trend of increased tumor-associated macrophages (TAMs); the presence of TAMs strongly correlated with TGF-β GES. Interestingly, few tumor cells displayed membranous PD-L1 staining as confirmed by PD-L1/pan-cytokeratin double labeling. Conclusions: We used RNAseq and IHC to better understand the immunosuppressive environment in HCC driven by TGF-β and PD-L1, which may mediate different mechanisms to inhibit preexisting CD8+ IC. Clinical trial identification: N/A Legal entity responsible for the study: Funding was provided by Merck KGaA, Darmstadt, Germany. Funding: Funding was provided by Merck KGaA, Darmstadt, Germany. Disclosure: Y. Zhang, B.J. Naughton, P.A. Rolfe, I. Dussault: Employee of EMD Serono Research & Development Institute, Billerica, MA, USA. E. Frick-Krieger, C. Ihling: Employee of Merck KGaA, Darmstadt, Germany. L. Terracciano: Consulting/advisory role to Merck AG