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Abstract
Background Cholangiocarcinoma (CC) is a rare malignancy with an increasing incidence and poor prognosis. Immunotherapy represents one potential treatment for CC, however identification of immunotherapeutic targets requires a thorough characterization of the tumor immune microenvironment (TIME). Mesothelin, a tumor associated antigen, is abundantly expressed in other malignancies, though its expression in CC has not been well characterized. We hypothesized that (1) the TIME of CC would vary by primary tumor location and between primary and metastatic lesions, (2) high tumor infiltration by CD8+ T cells and low infiltration by M2 macrophages would be associated with improved survival, and (3) most CC would express mesothelin.
Methods 99 CC tumors from unique stage I-IV patients were included, of which 89 were primary tumors (24 intrahepatic (ICC), 65 extrahepatic (ECC - 30 hilar (H-ECC) and 35 distal (D-ECC))) and 10 were metastatic lesions. Tissue microarrays were constructed and immunohistochemistry (IHC) was performed for lymphoid and myeloid markers, as well as for PD-L1 and mesothelin. IHC+ cells were quantified by automated image analysis. Expression of mesothelin and PD-L1 by tumors cells were evaluated on a semiquantitative scale (0, +1, +2, or +3). Hypothesis testing was performed using Kruskal-Wallis test and survival analyses were performed with Univariate and Multivariate Cox Hazard Models.
Results Most tumors were infiltrated by myeloid cells in addition to CD4+, CD8+, and FoxP3+ T-cells. Mesothelin was expressed (≥1+) in 68% of tumors (figure 1), while PD-L1 was expressed (≥1%) in only 16% of tumors. Higher densities of M1 macrophages (CD68+) were present in D-ECC relative to ICC and H-ECC (figure 2). M1 macropahges were also found in higher densities in metastatic tumors. Mesothelin and granzyme-B expression was significantly higher in D-ECC. Increasing density of myeloid cells (CD14+) and M2 macrophages (CD163+) was associated with worse survival (p= 0.02, 0.03, respectively) (figure 3). Intraepithelial and intratumoral T cell infiltration did not correlate with OS.
Mesothelin expression by primary tumor locationA+C) Representative low Mesothelin expression at low (X10) (A) and higher power (X20) (C). B+D) Representative high Mesothelin expression at low (X10)(B) and higher power (X20)(D). E) Log(x+1) transformed Mesothelin Expression as determined by automated cell counting, median and IQR, all data points shown. Median: 5.5, 79.5, 146.0 for ICC, H-ECC, D-ECC, respective, p-value = 0.025. F-H) Mesothelin Expression determined by visual inspection and scoring for ICC (F), H-ICC (G), and D-ECC (D).
Immune infiltration based on primary tumor locationIncrease in immune infiltrate in primary tumors as distance from liver increases. P-values determined by Jonckheere-Terpstra Test with FDR corrections
CD14 and CD163 Correlate with OSA+C) Kaplan Meier Curve of OS for (A) CD14 (Median OS: 20 vs. 90 months, log-rank p-value <0.01) and (C) CD163 (Median OS: 15 vs. 32 months, log-rank p-value<0.01). B+D) Multivariate Cox Hazard Models. Assumptions of Cox Hazard Model were checked with Schoenfeld residual values, significance level <0.01
Conclusions The TIME of CC varies significantly by primary tumor location and between primary and metastatic lesions. D-ECC has a favorable immune profile compared to ICC and H-ECC, with a better milieu for antigen presentation including increased mesothelin and less suppressive macrophages, which may support better response to checkpoint blockade. The data supported the hypothesis that higher densities of intra-tumoral M2 macrophages and myeloid cells correlated with worse OS, even after controlling for clinical variables, suggesting that these cell populations may represent promising immunotherapeutic targets in CC.
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