Drivers and breaks in the cholangiocarcinoma immune microenvironment

Clinically effective immune checkpoint inhibitors (ICI) have transformed cancer treatment over the last decade. This progress was built on a foundation of understanding tumor immunology, immune checkpoint biology, and biomarkers for sensitivity to ICI (1). Monoclonal antibodies disrupting the PD-L1/PD-1 axis or CTLA-4 are highly effective in tumors that are primed for an anti-tumor immune response but suppressed by immune checkpoints (1). This is the case for cancer types with abundant neoantigens, such as melanoma and lung cancer, and in tumors regardless of histology that have elevated tumor mutation burden (TMB) or microsatellite instability (MSI) due to mismatch repair deficiency (dMMR). Elevated expression of PD-L1 is a biomarker of immunosuppression and predicts sensitivity to PD-(L)1 blockade in several tumor types, including lung and gastroesophageal cancer. In cholangiocarcinoma (CCA), the immune landscape and factors predicting response to immunotherapy are less well understood (2). CCA generally lacks a high TMB, and MSI-high status is present in less than 5% of cases. Moreover, CCAs have a desmoplastic and immunosuppressive microenvironment (3). Congruently, early clinical trials of PD-1 blockade showed limited response rates in the range of 6–14% in biliary tract cancers (4,5). Combination immunotherapy targeting PD-1 and CTLA-4 yielded a modestly higher response rate of 23% (6). Tumor PD-L1 expression was associated with a higher response rate and PFS in the nivolumab trial (4) while the difference was not statistically significant for pembrolizumab in KEYNOTE-158 (5). This fact underscores the need to profile biomarkers beyond PD-L1 to identify factors that predict response to existing therapies, and to better understand the unique immunobiology of CCA to develop novel immunotherapy strategies.

In this context, Carapeto et al. (7) sought to delineate the immunogenomic landscape of CCA by defining spatially-refined, IHC-based profiles of tumor infiltrating lymphocytes, macrophages and multiple immune checkpoint molecules in a publication in Hepatology. Expression profiles were correlated with genetic drivers, clinical features, and patient survival. A cohort of 96 patients with intrahepatic CCA (iCCA) that underwent resection at a single institution were included to limit clinical heterogeneity and facilitate identification of significant correlations. Notably, only 4% of the tumors evaluated had PD-L1 expression, indicating that this cohort of patients represents those with a low response rate to ICI monotherapy in prior clinical trials (4,5). Sample clustering based on expression of immune cell markers (CD3, CD4, CD8, and CD68) and immune checkpoints (PD1, LAG3, ICOS, OX40, TIM3, and VISTA) identified 4 subgroups. Subgroup 3 had significantly shorter overall survival, and tumors characterized by high PD-1/LAG3 and low CD3/CD4/ICOS expression, particularly in the central tumor, suggesting a “cold” immune microenvironment. Accordingly, immune cells and checkpoint molecule expression were decreased in the tumor center compared to the invasive margin (Figure 1). These findings are consistent with observations in other malignancies including colorectal cancer, where a cold immune microenvironment is a marker of poor prognosis (1). Additionally, correlation of immune markers and genetic drivers identified a novel relationship between BAP1 loss-of-function mutations and increased expression of the immune checkpoints B7H3 and B7H4. A causal relationship for B7H4 was confirmed in vitro. These finding are an intriguing advancement of our understanding of the immunobiology of CCA, yet there remain many open questions regarding how to operationalize these findings for effective immunotherapy.

Figure 1 Immunohistochemistry-based profiling of immune cells (CD4⁺ and CD8⁺ T-cells and macrophages) and checkpoint molecule expression (ICOS, LAG3, OX40, PD-1, TIM3, and VISTA) in resected iCCA demonstrated that both immune cells and most checkpoint molecules were decreased in the tumor center compared to the invasive margin. A signature of low T-cell infiltration and high PD-1 or LAG3 expression was associated with lower overall survival. Figure created with BioRender.com.

Are these findings generalizable beyond resectable iCCA?

The inclusion of only resectable iCCA in the US in this study limits the applicability to CCA arising in other anatomical locations or in association with etiologic factors which are more common globally, including HBV and liver fluke infections. Additionally, the applicability to unresectable and metastatic disease is unknown. This was evaluated in a recent abstract from Mody et al. (8), which correlated tumor genetic drivers with RNA-estimated immune infiltrates and immune-related gene expression scores in a cohort of predominantly metastatic biliary tract cancers, including extrahepatic, intrahepatic, and gallbladder subsets. They found a more “inflamed” immunophenotype in gallbladder cancer versus iCCA and identified correlations of interest between genetic drivers and immune profiles. These results suggest that immune biomarkers will vary by anatomic subtype and genetic drivers.

Do the identified immunophenotype clusters predict response to existing immunotherapies?

In current clinical practice, only expression of PD-L1, TMB, and dMMR/MSI-high status are used to predict response to ICI, as monotherapy or combined with other systemic therapies. As this study included only resected CCA without adjuvant treatment data, immunophenotype could not be correlated with response to systemic therapy. In future studies, it will be critical to identify which immune cell and checkpoint profiles predict response to ICI monotherapy and combination strategies. Such studies could reveal the immune profile of PD-L1 negative responders to PD-1 blockade and further refine use of existing immunotherapies in CCA.

How can unique features of the immune microenvironment in CCA be exploited for novel therapeutic interventions?

Carapeto et al. make a meaningful contribution to the field by profiling a broad set of immune checkpoints and correlating their expression with genetic drivers. This work raises the question of whether immune checkpoints beyond PD-(L)1 and CTLA-4 can be targeted in CCA. Many such clinical trials are ongoing (2). Given that a subset of CCA with poor prognosis appear to be characterized by a cold immune microenvironment, combination strategies to increase immune priming may be warranted in this subset (1). Combinations of immunotherapies and cytotoxic therapy, targeted therapy, or locoregional therapies are being tested to overcome the limited response rates with single agent immunotherapy. These approaches have the potential to be rationally designed based on immunoprofiling, for example, co-targeting LAG3 and PD-1 (NCT03219268).

Beyond T-cells, the immune microenvironment in CCA is shaped by immunosuppressive macrophages, myeloid-derived suppressor cells, and other cell types (3,9). Additionally, while histologic profiling of immune checkpoint molecules on the surface of tumor and immune cells is highly informative, it is blind to extracellular sources of immune checkpoints including soluble and extracellular vesicle-associated molecules which also regulate the tumor immune response (10). Future studies are needed to profile these elements of the immune microenvironment as well. Ultimately, clinical profiling of immune and genetic features may be used to select rational combinations of systemic therapies, with the goal of improving quality of life and survival for patients with CCA.

Acknowledgments

Funding: The work of the authors was supported by the National Institute of Health (1K08CA236874-01) and the Hepatobiliary Cancer SPORE (P50 CA210964).

Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, Hepatobiliary Surgery and Nutrition. The article did not undergo external peer review.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://hbsn.amegroups.com/article/view/10.21037/hbsn-21-572/coif). SII reports receiving consulting fee from AstraZeneca BTC Consultancy. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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