Prognostic and Clinicopathological Significance of PD-L1 in Patients with Cholangiocarcinoma: A Meta-Analysis

Background In recent years, there is growing literature on the prognostic significance of programmed death-ligand 1 (PD-L1) in cholangiocarcinoma (CCA); however, data have been conflicting. Therefore, the objective of this study was to assess the correlation between PD-L1 and prognosis in CCA through meta-analysis. Methods Published studies were retrieved from the Web of Science, PubMed, Embase, and Cochrane Library up to April 17, 2020. The relationships between PD-L1 expression and survival outcomes were assessed using hazard ratios (HRs) and 95% confidence intervals (CIs). Results Eighteen studies consisting of 2012 patients were included. Overexpression of PD-L1 was significantly associated with worse overall survival (OS) (HR = 1.58, 95%CI = 1.30 − 1.92, p < 0.001) but not with poor disease-free survival (DFS) (HR = 1.03, 95%CI = 0.68 − 1.55, p = 0.895) in CCA. Moreover, PD-L1 was associated with low differentiation (OR = 1.43, 95%CI = 1.09 − 1.87, p = 0.010) and higher pN stage (OR = 1.45, 95%CI = 1.10 − 1.92, p = 0.009) but not with sex, TNM stage, vascular invasion, perineural invasion, age, or tumor size. Conclusion High PD-L1 expression was associated with worse OS, poor differentiation, and higher pN stage in patients with CCA. PD-L1 could be a potential prognostic marker in CCA.


Introduction
Cholangiocarcinoma (CCA) is the second most frequent type of primary liver cancer, with aggressive nature and a high mortality rate, accounting for 20% of liver-related deaths [1]. The incidence of CCA is increasing during the past decades in Western countries, and the 5-year survival rate is approximately 10% [2,3]. Surgical resection is the definitive treatment option for CCA; however, recurrence remains high and maintains a poor prognosis [4,5]. Emerging treatment options, including targeted therapies and immunotherapy with checkpoint inhibitors, are in clinical trials and provide personalized therapeutic strategies for patients with CCA [5]. Efficient prognostic biomarkers are still lacking for CCA; therefore, a reliable prognostic marker is needed for optimal therapeutic strategy selection [6].

Materials and Methods
This meta-analysis was conducted based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement [29]. Ethical approval and patient consent were not performed because all data collected were from previously published studies.
2.1. Literature Search. PubMed, Web of Science, Cochrane Library, and Embase were reviewed till April 17, 2020. The search terms used were "PD-L1" or "programmed death ligand 1" or "PDL1" or "B7-H1" or "CD274", and "bile duct neoplasms" or "cholangiocarcinoma" or "bile duct cancer". The reference lists in relevant studies were also examined for potential inclusions.

Inclusion and Exclusion
Criteria. The criteria for inclusion were (1) patients histologically diagnosed with CCA; (2) PD-L1 expression detected by immunohistochemistry (IHC); (3) studies reporting the relationship between PD-L1 and survival outcomes including overall survival (OS) and disease-free survival (DFS); (4) sufficient data available for the calculation of hazard ratios (HRs), odds ratios (ORs), and 95% confidence intervals (CIs); and (5) studies published in English.
The exclusion criteria were (1) conference abstracts, case reports, reviews, or letters; (2) studies with insufficient data for analysis; (3) animal studies; and (4) studies recruited overlapping patients.

Data Extraction.
Two independent investigators (Q.X. and L.W.) collected data from the included studies and any discrepancies were settled by discussion with a senior investigator (S.Z.). The following baseline information was extracted: author, year, study country, study design, sample size, treatment method, follow-up, survival outcomes, posi-tive rate of PD-L1 expression, and detection method. Detailed information on PD-L1 antibodies used for IHC (specie, clone, dilution, source, and cutoff value) was also extracted. The HR and 95% CIs of OS and DFS were obtained directly if reported or were calculated by Tierney's method [30].
2.4. Quality Assessment. The Newcastle-Ottawa Scale (NOS) was applied to evaluate the quality of eligible studies [31]. The NOS evaluated each study in three aspects. The score ranges from 0-9, and studies with NOS scores of ≥6 are considered high-quality studies.

Discussion
CCA is an aggressive cancer, and most patients present at an advanced stage at the time of diagnosis [32,33]. The current meta-analysis containing 18 studies with 2012 patients showed that high PD-L1 expression was a significant prognostic factor for low OS (HR = 1:58). Particularly, the mortality risk of patients with CCA with high PD-L1 expression increased by 58% compared with that of patients with low PD-L1 expression. PD-L1 expression was not significantly correlated with DFS. In addition, we found that PD-L1 was positively associated with poor differentiation and higher pN stage in CCA. Generally, these results demonstrated that PD-L1 overexpression was associated with invasive clinical features and suggested poorer prognosis of CCA. The tumor microenvironment in CCA consists of cancer cells, stromal cells, and various immune cells including CCA cells, cancer-associated fibroblasts, tumor-associated macrophages, tumor-infiltrating lymphocytes, and CD8+ cytotoxic T lymphocytes [34]. PD-1 is expressed on B cells, activated CD4+ and CD8+ T cells, and dendritic cells [35]. PD-L1 is a ligand of PD-1 and is expressed on different cell types [36]. Targeting PD-1/PD-L1 is a new strategy for cancer immunotherapy [37]. Recent studies showed that nivolumab (a PD-1 inhibitor) showed considerable safety in patients with metastatic CCA [15]. PD-L1 is mainly expressed by intertumoral immune cells in CCA [38]. Thus, the overexpression of PD-L1 may lead to immune tolerance in the tumor environment and result in tumor progression. This could be a possible mechanism for the correlation between PD-L1 elevation and poor differentiation in CCA.
Recent studies have demonstrated that PD-L1 overexpression is associated with unfavorable prognosis in various types of cancer [39,40]. A recent meta-analysis showed that high expression of PD-L1 was significantly associated with a poor OS (HR = 1:22, 95%CI = 1:01 − 1:48, p = 0:04) in colorectal cancer [41]. Another metaanalysis including 13 studies also demonstrated that tumor cell PD-L1 expression was correlated with poor OS (HR = 2:128, 95%CI : 1:341 -3:378, p = 0:001) in patients with diffuse large B-cell lymphoma [42]. These findings              Notably, several limitations of this study should be acknowledged. First, the cutoff values of PD-L1 varied in the included studies (Table 2), which may introduce heterogeneity. Further investigations used uniform antibody and a cut-off value of PD-L1 are needed. Second, only one included study was a prospective trial, and the remaining were retrospective studies. Therefore, high-quality prospective studies are still needed. Third, some HRs and 95% CIs were calculated according to survival curves, which may not be as precise as the original data. Fourth, the sample was relatively small. Only 2012 patients were enrolled and most patients were of Asian ethnicity. More studies recruiting patients of diverse ethnicities were needed to verify the results of this meta-analysis. Because of these limitations, well-designed large cohort studies or randomized controlled trials may be recommended to confirm our findings.

Conclusions
Our study indicates that PD-L1 was associated with worse OS, poor differentiation, and higher pN stage in patients with CCA. PD-L1 could be a potential prognostic marker for CCA.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request. 10 Disease Markers