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Gallbladder cancer (GBC) is a rare biliary tract malignancy seen in most developed countries, widespread with extensive geographic and ethnic variance [1]. Annually, GBC affects less than two out of 100,000 individuals but is more commonly observed in India, Chile, Japan, and Korea than in Western countries [2,3]. Most patients present with an advanced stage at diagnosis and the 5-year survival rate is < 10% [4]. In Korea, the overall incidence of GBC from 2009 to 2013 was 2.96 of 100,000 people among males and 2.79 of 100,000 people among females [5]. The 5-year survival rate is 30% and the median survival is 10.7 months [6]. Ulsan, where the hospital in this study is located, showed the highest incidence during 2009 to 2013 (4.31/100,000 in men and 4.09/100,000 in women) as compared with the national incidence [5,6].
In recent years, research on various tumor entities has increasingly focused on immunomodulatory drugs than directly cytotoxic cancer therapies. Genomic sequencing studies have identified a host of genetic aberrations that are potentially targetable in GBC [7,8]. In particular, the immunomodulatory therapy approach targeting the interaction between programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) has become increasingly significant. The aberrant expression of PD-L1 allows for tumor cells to escape the host immune system and continue to proliferate. Previous research has demonstrated the association of PD-L1 with tumor aggressiveness and poor prognosis in gastric, esophageal, and hepatocellular carcinoma as well as colonic and lung cancers [9,10]. It is expected that the therapeutic agents known as immune checkpoint inhibitors will be a key emerging strategy in treating the subgroup of advanced GBC.
Throughout the published literature, scant information is available on the use of PD-L1 as a prognostic marker in GBC. Existing research by Neyaz et al. [11] and Lin et al. [12] has reported inconsistent and contradictory results. Furthermore, although the possibility of immunotherapy has been studied, relevant information in this area is also very limited so far [13,14]. This study aimed to investigate the expression of PD-L1 and determine the potential association with prognostic impact in GBC. We also reviewed associations with clinicopathological parameters and survival.
MATERIALS AND METHODS
Specimens and patient selection
Formalin-fixed, paraffin-embedded (FFPE) primary GBC tissues were derived from 101 patients at Ulsan University Hospital (UUH) between January 2013 and December 2018. Clinical data were recorded from the UUH electronic medical records, including age, sex, size, location, risk factors (e.g., gallstone, cholecystitis, diabetes mellitus, hypertension), margin of tumor resection, histologic grade, TNM stage, lymph node involvement, lymphovascular invasion or perineural invasion by tumor, adjuvant chemotherapy, and follow-up time in months. Follow-up was completed on April 8, 2019. Overall survival (OS) was the interval either between the initial diagnosis and death or between the initial diagnosis and the last observation among surviving patients, respectively. Progression-free survival (PFS) was the interval between the initial diagnosis and progressive changes in the typical imaging appearance on computed tomography and/or magnetic resonance imaging. No patient underwent chemotherapy before surgery. The pathologic diagnosis was confirmed according to the eighth edition of the American Joint Committee on Cancer staging system [15] and the World Health Organization classification systems.
Automated immunohistochemistry
A representative paraffin block from each specimen was chosen for immunohistochemical analysis. We immunohistochemically analyzed PD-L1 expression on 3- to 5-μm tissue sections of FFPE specimens. The primary PD-L1 antibody (rabbit monoclonal antibody clone SP263; Roche Holding AG, Basel, Switzerland) was used in all cases in a concentration of approximately 1.61 μg/mL. A negative control for all cases was also developed using the same antibody to control for potential false-positive staining. Placental tissues served as positive controls. Immunohistochemistry assays were performed on a VENTANA BenchMark ULTRA instrument (Roche Holding AG) according to the manufacturer’s instructions.
Evaluation of immunohistochemistry
The PD-L1 expression proportion score was assessed as the percentage of positive membranous expression on tumor cells, whereas cytoplasmic expression was regarded as negative. Tumor cells with any membranous staining intensity were judged to be positive. Various PD-L1 antibodies and cutoff levels were used in different studies. We assessed PD-L1 expression using the cutoff levels of 1%, 10%, and 50% (Fig. 1).
Statistical analysis
SPSS ver. 24.0 (IBM Corp., Armonk, NY, USA) was used to conduct statistical analyses. To determine the association between two or more variables and PD-L1 expression, Pearson’s chi-square test or Fisher exact test where appropriate were applied, with statistical significance at p < .05. The univariable analysis of OS and PFS was completed using the Kaplan-Meier method and log-rank test.
RESULTS
Clinicopathological characteristics
The study group included 101 primary GBC cases, with a female predominance (56.4%). The mean age of the included patients was 68.0 years (range, 40 to 90 years) and 97 patients (96.0%) were aged older than 45 years. Fifty-two patients (51.5%) were diagnosed via simple cholecystectomy specimens and 10 of these underwent further surgery after diagnosis. Risk factors included gallstone (28.7%), cholecystitis (91.0%), hypertension (28.7%), and diabetes (32.7%). The majority of cases showed adenocarcinoma not otherwise specified (n = 82, 81.2%), and the most common type was well-differentiated (n = 45, 44.6%). Although subtype-specific components accompanying adenocarcinoma were present, no cases were diagnosed as either undifferentiated carcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, or neuroendocrine carcinoma. This cohort included mostly patients with early stages of disease; 84 patients (83.2%) presented with pT1 or pT2 category. Among 72 patients eligible for the evaluation of pathologic stage status, 37 (51.4%) presented with stage I or stage II disease. The clinicopathological characteristics of our GBC patients are shown in Table 1.
Correlation of clinicopathological parameters with PD-L1 expression
PD-L1 expression in tumor cells was observed in 19 patients (18.8%) with a cutoff level of 1%, 14 patients (13.8%) with a cutoff level of 10%, and eight patients (7.9%) with a cutoff level of 50%. The finding of any positive PD-L1 expression was significantly correlated with poorer and other differentiation (1% cutoff: p = .001; 10% cutoff: p < .001; 50% cutoff: p < .001) and the presence of lymphovascular invasion (1% cutoff, p = .015; 10% cutoff, p = .001; 50% cutoff, p = .005). Positive PD-L1 expression with cutoff levels of 10% and 50% was associated with the presence of perineural invasion (10% cutoff, p = .032; 50% cutoff, p = .023), higher T category (10% cutoff, p = .012; 50% cutoff, p = .026), and higher pathologic stage (10% cutoff, p = .045; 50% cutoff, p = .010). In addition, positive PD-L1 expression with 1% and 10% cutoff levels was correlated with larger tumor size (1% cutoff, p = .040; 10% cutoff, p = .007). No significant differences were observed with regard to sex; age; tumor location; margin of tumor resection; adjuvant chemotherapy; or primary risk factors for GBC such as gallstones, cholecystitis, diabetes, and hypertension. The associations between PD-L1 expression in tumor cells and clinicopathological characteristics of GBC patients are shown in Table 2.
Survival analysis
At the time of analysis, the median OS was 14 months (range, 0 to 71 months). Thirty-three patients (32.6%) died during the follow-up period. Meanwhile, a total of 24 patients showed disease progression, and 19 of these patients died. Survival analysis using Kaplan-Meier analysis was performed to evaluate the prognostic impact of PD-L1 expression and other parameters. OS was significantly associated with histologic grade (p = .003), T category (p < .001), N category (p < .001), pathologic stage (p < .001), lymphovascular invasion (p < .001), perineural invasion (p < .001), growth pattern (p = .019), and resection margin (p = .006). Worse mean survival was observed in histologic grade progressing from well-differentiated to poorly differentiated, undifferentiated, or other. The patients with higher T categories, nodal metastasis, higher pathologic stages, presence of lymphovascular invasion, and presence of perineural invasion showed poorer OS, whereas those with a polypoid growth pattern and complete resection showed better OS. These parameters were more significantly associated with PFS. Significant differences in both OS and PFS according to PD-L1 expression were seen only at the 50% cutoff statistically (1% cutoff: p = .14; 10% cutoff: p =.259; 50% cutoff: p = .049 for OS and 1% cutoff: p = .095; 10% cutoff: p = .178; 50% cutoff: p = .028 for PFS) (Fig. 2). We observed a high expression of PD-L1 correlated with poor prognostic significance of both survival types, especially PFS. Old age (≥ 68 years) was correlated with poor OS and larger tumor size (≥ 2.7 cm) was correlated with poor PFS, respectively. No significant associations with sex, histologic type, adjuvant chemotherapy, gallstone status, cholecystitis, diabetes, or hypertension were evident. Correlations between OS or PFS and clinicopathological parameters are shown in Table 3.
DISCUSSION
Immune checkpoint inhibitors targeting the PD-1–PD-L1 pathway have exhibited potent efficacy in some cancers such as triple-negative breast cancer, renal cell carcinoma, and non-small cell lung cancer [16-20]. Clinical benefits were strongly correlated with high PD-L1 expression and certain drugs have been approved for use in conjunction [20,21]. While PD-L1 expression significantly correlates with poor prognosis in gastric cancer, hepatocellular carcinoma, and esophageal cancer, both better and worse results have been observed in lung cancer, colorectal cancer, and melanoma [22]. PD-L1 expression has been suggested as an important prognostic factor, but few studies have evaluated the expression levels of PD-L1 in GBC patients and there are no consistent results regarding its value as a predictor.
Various studies suggest that PD-L1 expression is associated with poor prognostic factors or survival in different tumor types. These studies observed that tumors with poor differentiation, vascular invasion, nodal metastasis, higher stage, adenocarcinoma histology, and lower survival rate were correlated with higher PD-L1 expression. Table 4 summarizes recent studies covering the prognostic value of PD-L1 [11,12,23-32].
In patients with advanced cholangiocarcinoma or gallbladder adenocarcinoma, results from phase I KEYNOTE-028 and phase II KEYNOTE-158 research indicated that pembrolizumab, a humanized monoclonal antibody against PD-1, constitutes a possible treatment option regardless of PD-L1 expression [14]. The PD-L1 antibody (22C3) and a 1% cutoff level were used in these trials. Elsewhere, Ha et al. [33] found a high level of soluble PD-L1 in the serum represents a negative prognostic factor in advanced cholangiocarcinoma and GBC patients who received palliative chemotherapy. Recently, two other studies evaluated the predictive value of PD-L1 expression using immunohistochemistry in GBC tissues. Neyaz et al. [11] examined the relationship between PD-L1 expression in tumor cells and tumor-infiltrating lymphocytes (TILs) at cutoff levels of 1%, 10%, and 50% and clinicopathological characteristics or OS. Their study ultimately showed significant correlations existed in terms of histologic type, histologic grade, TIL density, and stage of disease at all cutoff levels but did not find any significant correlations in conjunction with OS. Lin et al. [12] evaluated the expression of PD-L1, PD-L2, and the density of CD8+ TIL in association with OS, PFS, and risk factors in gallbladder adenocarcinoma by analyzing PD-L1 expression at a 5% cutoff level and performing four subgroup analyses according to PD-L1 expression and CD8+ TILs. According to the results, there were no correlations observed with PD-L1 expression in tumor cells alone except for regarding CD8+ TIL density and worse OS. Instead, the study demonstrated the coevaluation of CD8 TIL and PD-L1 had the significant prognostic value, and patients with high TILs and/or PD-L1 positivity had the worst PFS and OS.
Based on the above studies, PD-L1 expression in tumor cells as a predictive marker is controversial in GBC. In this study, we evaluated the expression of PD-L1 in 101 GBC cases and investigated the relationship between PD-L1 expression and various clinicopathological parameters or survival. A standard positive cutoff level or biomarker for PD-L1 has not been established [34-36]. Different antibodies (e.g., SP263, SP142, 22C3, 22-8, and E1L3N clones) and cutoff levels (e.g., 1%, 5%, 25%, and 50%) are used in various studies; we used the monoclonal antibody SP263 and the 1%, 10%, and 50% cutoff levels in our investigation. Our study showed a strong positive correlation in poor histologic grade and lymphovascular invasion at any cutoff level of PD-L1 expression. Also, other unfavorable parameters such as perineural invasion, higher T category, and higher pathologic stage of disease showed a significant correlation with PD-L1 expression at the 10% and 50% cutoff levels. Our final aim was to evaluate the prognostic impact of the clinicopathological parameters in survival. In this study, the association between PD-L1 expression at the 50% cutoff level and OS or PFS achieved statistical significance. Other parameters including the presence of lymphovascular invasion and perineural invasion; incomplete resection; higher histologic grade; higher T category, N category, and pathologic stage; and nonpolypoid growth pattern were also significantly associated with poor OS and PFS.
In summary, although opposite results have been reported regarding the use of PD-L1 expression as a predictive parameter in GBC, our results supported the negative clinical impact of PD-L1 expression as described by Lin et al. [12]. We found that GBC cases with high PD-L1 expression were significantly associated with poor clinicopathological parameters and survival at the 50% cutoff level. Interestingly, although a significant association with PD-L1 expression was found in the two studies using E1L3N and SP263, SP263 did not display any such significance in the previous study by Neyaz et al. [11]. We have to consider the following reasons for discrepancies in PD-L1 expression: dissimilar cutoff levels and anti-PD-L1 antibodies, heterogeneity of tumor, interobserver and intra-observer variability, and the influence of relationships with other indicators such as PD-L1 expression in TILs. Future research with larger study populations focused on elucidating detailed evaluation criteria and identifying the benefit of PD-L1–inhibiting immunomodulating therapies should be conducted.
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