Stroma-driven inter- and intratumoral heterogeneity of PD-L1 expression in cholangiocarcinoma: Implications for biomarker development and prognostic evaluation

BACKGROUND AND AIMS: Immune checkpoint inhibitors are now considered as part of standard of care in biliary tract cancer, based on a statistically significant but overall modest survival benefit in recent pivotal trials. Unlike in other gastrointestinal malignancies, PD-L1-based scores were not significantly associated with survival. Intrahepatic cholangiocarcinomas (iCCA) are morphologically heterogeneous tumors, with a subset of iCCA dominated by stroma rich areas. We aimed at understanding the potential impact of the inter- and intratumoral heterogeneity on PD-L1 expression in iCCA. METHODS: Bulk transcriptome analysis and multiplex immunohistochemistry were performed on 141 clinically annotated resected iCCA. RESULTS: Molecular signatures are critically driven by the relative stroma content with higher inflammatory gene expression scores in tumor microenvironment (TME)-rich compared to TME-poor tumors. In addition to this inter-tumoral heterogeneity we observed a striking intra-tumoral heterogeneity reflected by an enrichment of PD-L1 expressig cells in stroma-dominant areas. The effect of intra-tumoral heterogeneity on PD-L1 based scores was confirmed by analysing "virtual biopsies", i.e. randomly selected adjacent tumor regions designed to mimic clinical biopsies, further highlighting the broad challenges associated with biomarker development using needle biopsies. By integrating clinical data, we provide evidence that the prognostic value of PD-L1-expressing tumor cells is overall modest and may depend not only on the magnitude of expression but also on their localization within the tumor. CONCLUSION: Our data indicate that stroma content may be a surrogate for inflamed iCCA. Moreover, the inter- and intratumoral heterogeneity of iCCA poses a significant challenge to interpreting the PD-L1 signal. To establish tissue-based biomarkers in iCCA, a more granular annotation of their expression in different intratumoral compartments will be critical.