HHLA2 in intrahepatic cholangiocarcinoma: an immune checkpoint with prognostic significance and wider expression compared with PD-L1

Abstract

Background: Intrahepatic cholangiocarcinoma (ICC) is a highly mortal malignancy with limited therapeutic options. Immunotherapies targeting PD-1/PD-L1 pathway represent a promising treatment for ICC. However, PD-L1 expression and microsatellite instability are not common in ICC. This study aimed to investigate whether HHLA2, a newly identified B7 family immune checkpoint for T cells, could be a therapeutic target next to PD-L1 in ICC.

Methods: Expression levels of PD-L1 and HHLA2 as well as infiltrations of CD3+, CD8+, CD4 + Foxp3+, CD68+, CD163+ and CD20+ cells were evaluated by immunohistochemistry in 153 resected ICC samples. Comprehensive comparisons were made between PD-L1 and HHLA2 in terms of the expression rates, clinicopathological features and infiltrations of different immune cells. The expression level and prognostic significance of HHLA2 were further validated in an independent cohort.

Results: Expression of HHLA2 is more frequent than PD-L1 in ICC (49.0% vs 28.1%). Co-expression of both immune checkpoints was infrequent (13.1%) and 50% PD-L1 negative cases were with elevated HHLA2. HHLA2 overexpression was associated with sparser CD3+ tumor infiltrating lymphocytes (TILs), CD8+ TILs and a higher CD4 + Foxp3+/CD8+ TIL ratio, whereas PD-L1 expression was associated with prominent T cells and CD163+ tumor associated macrophages infiltrations. PD-L1 failed to stratify overall survival (OS) but HHLA2 was identified as an independent prognostic indicator for OS in two independent cohorts.

Conclusions: Compared with PD-L1, HHLA2 is more prevalent and possesses more explicit prognostic significance, which confer the rationale for HHLA2 as a potential immunotherapeutic target next to PD-L1 for ICC patients.

Keywords: HHLA2; Immunotherapy; Intrahepatic cholangiocarcinoma; PD-L1; Prognosis; Tumor associated macrophages; Tumor infiltrating lymphocytes.

Fig. 1

PD-L1 and HHLA2 expression in ICC tissue samples. Representative micrographs of PD-L1 (a) and HHLA2 (c) expression within tumor (scale bar, 50 μm). The positive rate of PD-L1 on tumor cells and immune cells were 28.1 and 17.0%, respectively (b). HHLA2 was elevated in 49.0 and 67.7% of cases in training and validation cohort, respectively (d). No significant correlation was found between HHLA2 and PD-L1 expression (e)

Fig. 2

Kaplan Meier survival curves for OS of patients with ICC according to HHLA2 and PD-L1 expression. High HHLA2 expression was significantly associated with poor overall survival (OS) in the training cohort (a) and the significance was validated in an independent validation cohort (b). PD-L1 expression on TC (c) and IC (d) both failed to stratify OS in the training cohort. The P-values were determined via log-rank test

Fig. 3

Tumor infiltrating T cells, cytotoxic T cells (CTLs) and regulatory T cells (Tregs) and their correlation between HHLA2 and PD-L1 expression. Images of positive CD3 (a), CD8 (b) staining and the corresponding intra-tumor negative controls. Magnification × 50 for full views and × 400 for zoomed-in views (scale bar, 200 μm). Cells with double staining of CD4 and Foxp3 were identified as Tregs (Arrow) (c). Original magnification × 500 (scale bar, 50 μm). Scatter plot depicted the correlation between classic subsets of T cells and HHLA2 expression (d). High HHLA2 expression was significantly correlated with lower intra-tumor counts of T cells and cytotoxic T cells as well as increased ratio of Tregs to CTLs. PD-L1 expression was associated with higher intra-tumor counts of T cells and cytotoxic T cells (e and f). P -values were generated by Mann-Whitney U test. Error bars indicate median and interquartile range

Fig. 4

CD68+ tumor associated macrophages (TAMs), CD163+ TAMs and CD20+ tumor infiltrating lymphocytes (TILs) and their correlation between HHLA2 and PD-L1 expression. Representative images of CD68 and CD163 staining (a; scale bar, 50 μm). No significant differences were found on the infiltrations of CD68+ TAMs, CD163+ TAMs as well as CD163+/CD68+ TAMs ratio between ICC with different HHLA2 expression levels (b). PD-L1 expression on TC was significantly correlated with a higher density of CD163+ TAMs and a higher CD163+/CD68+ TAMs ratio (c). PD-L1 expression on IC was significantly correlated with prominent infiltrations of CD68+ TAMs and CD163+ TAMs as well as a higher CD163+/CD68+ TAMs ratio (d). Images of positive CD20 staining and the corresponding intra-tumor negative control (e; scale bar, 50 μm). No significant differences were found on CD20+ TIL counts between ICC with different HHLA2 and PD-L1 expression levels (f). P -values were generated by Mann-Whitney U test. Error bars indicate median and interquartile range