In this study, we observed that the expression of HHLA2 was similar with the expression of PD-L1 in HCC, and there was no correlation between the expression of these two proteins. Moreover, either HHLA2 or PD-L1 on TC (or IC) was identified as an independent prognostic factor for OS. Meantime, combined the expression of HHLA2 and PD-L1 on TC (or IC) was also an independent prognostic factor for OS. The presence of PD-L1 and HHLA2 was associated with differential infiltration of immune cells: PD-L1 positive tumors were observed to have higher densities of stromal TILs, CD3 + and CD8 + TILs, whereas HHLA2 over-expression was correlated with lower densities of stromal TILs.
It is relatively common for members of the B7 family to have dual functions depending on the immune environment, as well as their interaction with different receptors or receptor engagement or blockade [28]. As a member of B7 family, Xiao et al. showed that HHLA2 had either co-inhibitory or co-stimulatory properties, depending on the malignancy type [29]. Recently study indicated that HHLA2 was a significant prognostic factor in a portion of tumors, but obvious heterogeneity prognostic value was observed on different kinds of tumors, part of them were protective and others were unfavorable or no significant prognostic factors. Luo et al. showed that HHLA2 was upregulated in 55 HCC tissues, and may play a major role in the development and progression of HCC, supporting a tumor progression role in HCC, meantime, the expression of HHLA2 was associated with poor prognosis of HCC patients (202 cases) in her study [24]. However, according to our data, elevated expression of HHLA2 was frequently associated with good prognosis and good clinicopathological features, such as with low AFP level, and well tumor differentiation in a larger cohort of 547 HCC cases. The basis for these results may mainly on the positive correlations between HHLA2 expression and low serum AFP levels, well tumor differentiation. AFP that generally reflect tumor burden both at primary sites and in the circulation for HCC, and well tumor differentiation suggested less tumor aggressively, indicating that patients with HHLA2 overexpression are less likely to suffer from recurrence and metastasis [29]. In view of the different conclusions, there are several situations need to be considered, different antibody source manufacturers, different HCC cases, different statistical analysis, most importantly, the cutoff value of HHLA2 expression was different. Moreover, our data was in line with other studies indicating that HHLA2 was overexpressed and associated with better prognosis, such as in another HCC study [25], in epithelial ovarian cancer [20], in pancreatic and ampullary cancers [21] and in kidney renal clear cell carcinoma patients [22]. Collectively, we consider our data are more representative to show the role of HHLA2 in HCC.
Previous studies reported discrepant results among different cancer types on the correlations between immune cell infiltration and HHLA2 expression. In ovarian cancer, HHLA2 expression was independently correlated with high CD8 + TILs count [20]. However, no significant correlation was found between the presence of TILs and HHLA2 expression for osteosarcoma [30]. Furthermore, a negative association was observed between HHLA2 over-expression and cytotoxic T cells [19]. And some researchers had revealed that HHLA2 inhibited the proliferation and cytokine production of both human CD4 + T cells and CD8 + T cells, and functions as a T-cell co-inhibitory molecule [15, 16]. Thus, it indicates that high level of HHLA2 potentially plays an important role in tumor progression through immune suppression. In this study, we observed that HHLA2 over-expression was associated with low density of stromal TILs in HCC, which was in line with the result of many study and indicated that HHLA2 may inhibited the proliferation of T cells.
In our study, PD-L1 positive rate on TC and IC in 547 HCC cases was 39.7% and 53.4%, respectively. This result was a little higher than those currently reported in the literature [11–13]. Different PD-L1 immunohistochemistry assays, various scoring systems, and evaluation of different tumor compartments may be the reasons for this observed diversity. In our study, positive PD-L1 either on TC or on IC was found to have a significant correlation with unfavorable OS, and these were consistent with other HCC studies [31, 32]. Meanwhile, we identified that PD-L1 ( TC and IC) positive cases had prominent T cell infiltration, which was also consistent with some previous studies [19, 31], but contradicted with the report from Schalper et al. concerning lung cancer [33]. PD-L1 expression, based on the molecular basis, is speculated to have a negative impact on survival, whereas its positive correlation with T cell infiltration mainly exert an opposing impact and consequently leads to the uncertainty of prognostic significance [34]. The infiltration of T cells, especially CD8 + T cells, was a known factor that indicated favorable survival and this finding was also confirmed in our cohort (Table S8). Schalper et al. suggest the discrepant prognostic significances of PD-L1 may not be a limitation for its possible role as a therapeutic target and a predictive biomarker for treatment response [33]. In summary, our study suggests that PD-L1 expression was considerable in HCC and was associated with higher densities of stromal TILs and poor prognosis.
Although the expression of both HHLA2 and PD-L1 is induced by related immunoregulatory factors, the limited overlap between the expression of PD-L1 and HHLA2 indicates their potential non-redundant biological functions or distinct spatial and/or temporal contributions to immune evasion. A previous report indicated that HHLA2 and PD-L1 co-expression predicts poor prognosis in patients with clear cell renal cell carcinoma [18]. Similarly, in our results, we observed that the percentage of HHLA2 (+)/ PD-L1 TC (-) and HHLA2 (-)/ PD-L1 TC (+) cells were 18.8% (103/547) and 26.7% (146/547) respectively. Immune checkpoint inhibitor (ICI) therapy targeting anti-PD-1 or its ligand (anti-PD-L1) is the backbone of numerous combination regimens aimed at improving the objective response and survival of patients with HCC [35]. Similar to PD-L1 [33], the discrepant prognostic significances of HHLA2 may also not be a limitation for its possible role as a therapeutic target and a predictive biomarker for treatment response. In our study, the expression of HHLA2 was correlated with an inhibitory TME (Tumor microenvironment) featured by decreased TILs, which indicated the possible role of HHLA2 as an immunotherapeutic target.
Despite the large number of cases in our cohort and the satisfactory results obtained in our research, there were also some limitations. Firstly, this was a single-center in china and further multicenter verification is needed to determine whether the results could apply to other populations. Secondly, this is a retrospective study, HHLA2 expression and biological function in HCC are warranted to further elucidate. Thirdly, we used immune-histochemistry to examine the relationship among HHLA2, PD-L1, and TILs, different research approaches and different statistical analysis may contribute to different results, therefore, standard experimental procedures as well as statistical analysis, multicenter verification is necessary before HHLA2 may be used in clinical study. Lastly, the use of TMA sections in this study may weaken tumor representation or heterogeneity of the markers.