Prognostic value and clinicopathological features of PD-1/PD-L1 expression with mismatch repair status and desmoplastic stroma in Chinese patients with pancreatic cancer

Pancreatic cancer (PC) is a highly lethal cancer. Thus, the immune molecular markers which help to select PC patients are especially important. In this study, we aimed at systematically analyzing the expression of MLH1, MSH2, PD-L1 and PD-1, investigate their clinical significance and prognostic value. We found that high expression of PD-L1 on cancer cell membranes correlated with lymph node metastasis (P = 0.033) and strongly correlated with poor-differentiation (P = 0.008); high expression of PD-1 on cell membranes of T-cells correlated with well-differentiation (P = 0.018) and strongly correlated with advanced T stage (P = 0.004); high PD-1 expression was associated with a significantly superior OS and was an independent prognostic factor (P = 0.031). Then we found an inverse correlation between MSH2 expression and PD-L1 expression (Spearman correlation coefficient r = −0.295, P = 0.004). In subgroup analyses, we observed that PD-1 expression level was associated with OS only at low PD-L1 expression subgroup (P = 0.021). Finally, when we stratified the cases into four subgroups based on PD-1 expression and stroma density, we found that patients with high PD-1 expression and dense stroma had a better OS, while patients with low PD-1 expression and moderate stroma showed a worst outcome. Our result may provide more effective molecular markers for immunotherapeutic strategies of PC patients in clinical practice.


INTRODUCTION
Pancreatic cancer (PC) is a highly lethal cancer, with a 5-year survival of approximately 5% [1][2].The potentially resectable of PC is less than 20%, for the diagnosis of pancreatic cancer is often difficult, and most patients are diagnosed at an advanced stage.However, even in rescetable cases, the median survival time is only 15-18 months.Recently, surgery, radiotherapy and chemotherapy for pancreatic cancer have been improved, but the effect was limited.Thus, it is very important to find more effective molecular markers for the treatment and prognosis of PC patients in clinical practice.
Program death ligand 1 (PD-L1) is a member of the B7 family of immune-regulatory cell-surface proteins.As a receptor of PD-L1, programmed death-1 (PD-1) is typically expressed on activated T cells.PD-1/PD-L1 axis plays an important role in the negative regulation of cell-mediated immune responses as an immune checkpoint.The expression of PD-1/PD-L1 is up-regulated in many tumors and their microenvironment [3][4][5].PC is characterised by an abundant desmoplastic stroma which Research Paper www.impactjournals.com/oncotargetcontains dense activated stellate cells.PC is considered as a non-immunogenic tumor with few effector T cells infiltrating into the tumor tissue.The stroma of PC is poor-vascularized and often prevents T-cell from infiltrating [6][7][8][9].Although the antibodies of PD-1/ PD-L1 have shown improving outcome in patients with melanoma, renal cell carcinoma, non-small lung cancer, the results of treating pancreatic cancer with single-agent immune checkpoint inhibitors have been disappointing [10][11][12][13].However, anti-tumor activity was preliminarily reported for some PC patients at the 2014 ASCO annual meeting.Thus, the immune molecular markers which help to select PC patients are especially important [14].
High PD-L1 expression was firstly suggested to predict the response to anti-PD-1/PD-L1 antibody therapies.Then, pembrolizumab has been reported to be highly effective in gastrointestinal cancers in patients with mismatch repair deficiencies [15][16].Deficiencies of mismatch repair enzymes (MLH1, MSH2, MSH6, and PMS2) were originally identified by Dr. Henry Lynch [17].Meanwhile, mismatch repair deficiency (MMR-D) cancer appears to be more antigenic than mismatch repair proficient (MMR-P) cancer and has a special susceptibility to immunotherapy.Since somatic mutations have the potential to create immunogenic neoantigens, patients with mismatch repair defects, which promote somatic mutations, may have increased intratumoral effector T cell responses to the neoantigens [15][16].Thus, effective immune-based therapeutic strategies are highly demanded for the majority of malignancies that are naturally immune quiescent.
Although many studies showed that the expression of PD-1 and PD-L1 was correlated with the clinical outcome in several malignancies including PC [18][19], some studies did not find any prognostic impact of PD-1/PD-L1 [20].Also, the factors which affected the expression of PD-1/ PD-L1 in PC were not fully illustrated.Moreover, the role of mismatch repair enzymes in this disease and correlation with PD-1/PD-L1 expression remains largely unknown.In this study, we firstly aim to evaluate the correlation of clinical/pathological characteristics and the expression of PD-1/PD-L1, MLH1, MSH2 respectively and demonstrated the prognostic role of them in a tissue microarray (TMA) including 94 well-documented, clinically annotated PC specimens.Then, we evaluated the correlation of PD-1, PD-L1, MLH1, MSH2 and desmoplastic stroma density and investigated their clinical significance in PC progression.

Correlations between MLH1/MSH2/PD-1/PD-L1 and clinicopathological characteristics
The relationships of the expression levels of MLH1, MSH2, PD-1 and PD-L1 with clinicopathological features of PC were evaluated.For MLH1 and MSH2, as summarized in Table 2, we did not observe any correlation between their expression levels and clinicopathological characteristics (Table 2).High expression of PD-L1 on cancer cell membranes correlated with lymph node metastasis (P = 0.033) and strongly correlated with poor-differentiation (P = 0.008), but did not correlate with patient's gender, age, tumor location, T stage, distant metastasis, vascular invasion and nervous invasion (P > 0.05) (Table 3).Meanwhile, high expression of PD-1 on cell membranes of TILs correlated with well-differentiation (P = 0.018) and strongly correlated with advanced T stage (P = 0.004), but did not correlate with patient's gender, age, tumor location, N stage, distant metastasis, vascular invasion and nervous invasion (P > 0.05) (Table 3).The relationships of the stroma density with clinicopathological features of PC were evaluated by haematoxylin-eosin staining.As summarized in Table 3, no correlation was observed between stroma density and patients' clinicopathologic features.

Correlations of MMR gene expression, stroma density and PD-1/PD-L1 expression in PC tissues
As shown in Table 5, an inverse correlation was identified between low expression of MSH2 and high expression of PD-L1 (Spearman correlation coefficient r = −0.295,P = 0.004, Table 5).No correlations were found between PD-L1 expression and MLH1expression (P > 0.05, Table 5) and between PD-L1 expression and stroma density (P > 0.05, Table 5).Then, we used serial sections from the same PC tissue to depict the co-distribution of MSH2 and PD-L1 in PC tissue.As seen in Figure 3E, low MSH2 expression strongly correlated with high PD-L1 expression.
For PD-1, as summarized in Table 6, we did not observe any correlation between PD-1 expression level and the expression of MMR gene (P > 0.05, Table 6).Also, PD-1 expression level was not associated with PD-L1 (P > 0.05, Table 6).Then, we further made subgroup analyses to evaluate the prognostic impact of PD-1 expression stratified by PD-L1.Kaplan-Meier analysis and log-rank test result  showed that PD-1 expression level was associated with OS only at low PD-L1 subgroup (Figure 3A, 2B, P = 0.021).Moreover, high expression of PD-1 on cell membranes of TILs strongly correlated with moderate stroma (P < 0.001, Table 6).Previous studies indicated that a better survival was seen in surgically resected PC patients with higher levels of TILs within the tumor microenvironment and dense stroma made PC tissue poor-vascularized and can prevent T-cell from infiltrating (Supplementary Figure S3).To further investigate the association of OS with PD-1 expression and stroma density, we stratified the cases into four subgroups based on PD-1 expression and stroma density (Figure 3C).Kaplan-Meier analysis showed that the OS of four subgroups had significant differences (log-rank test: P = 0.006).Interesting, patients with high PD-1 expression and dense stroma had a better OS (median OS > 24 months), while patients with low PD-1 expression and moderate stroma showed a worst outcome (median OS = 7 months).Likewise, when the first three subgroups were combined into a single subgroup named "all others", a similar result was found (log-rank test P = 0.006, Figure 3D).

DISCUSSION
PD-1/PD-L1 axis, as an immune checkpoint, has been widely studied in many different types of malignant.The expression of PD-1/PD-L1 was often used as a biomarker of immune checkpoint inhibitors [21][22].Moreover, PD-1/PD-L1 antibody has been shown to be highly effective in gastrointestinal cancers as a single agent in patients with mismatch repair deficiencies.Deficient MMR results in the incorporation of mismatched   nucleotides, with MMR-deficient colorectal cancer cells exhibiting 10-100 times the number of somatic mutations as those with proficient MMR [23][24].Thus, high somatic mutation loads, which have the potential to create "non-self" immunogenic neoantigens, correlated with increased intratumoral effector T cell responses to the neoantigens Supplementary Tables S1, S2 [25].Meanwhile, some studies indicated that MMR-deficient status was associated with higher expression of PD-L1 in gastric and colorectal cancer [26][27][28].However, the relationship between MMR gene and PD-1/PD-L1 in pancreatic cancer remains largely unknown.Moreover, the correlations between PD-1/PD-L1 and prognosis are variants among different tumor types.Although Angela Diana et al. reported the expression of PD-1/PD-L1 and their prognostic value in pancreatic cancer [18].However, the correlations of MMR-D/MMR-P and PD-1/PD-L1 and their clinical significant in pancreatic cancer were not fully understood, especially in East Asia.
Our study aimed at systematically analyzing the expression of MLH1, MSH2, PD-L1 and PD-1 in pancreatic cancer cells and tumor infiltrating cells, investigate their clinical significance and prognostic value.We found that high expression of PD-L1 on cancer cell membranes correlated with lymph node metastasis and strongly correlated with poor-differentiation, high expression of PD-1 on cell membranes of TILs correlated with well-differentiation and strongly correlated with advanced T stage, high PD-1 expression was associated with a significantly superior OS and was an independent prognostic factor.Then we found an inverse correlation between MSH2 expression and PD-L1 expression.In subgroup analyses, we observed that PD-1 expression level was associated with OS only at low PD-L1 expression subgroup.Finally, when we stratified the cases into four subgroups based on PD-1 expression and stroma density, we found that patients with high PD-1 expression and dense stroma had a better OS, while patients with low PD-1 expression and moderate stroma showed a worst outcome.
In our study, high expression of PD-1 on cell membranes of TILs strongly correlated with advanced T stage.Our observations are in line with most of previous studies in pancreatic cancer [18].Also, our study reported that high expression of PD-L1 correlated with lymph node metastasis and strongly correlated with poor-differentiation.In previous study, Guo Y et al. made a meta-analysis to evaluate the prognostic and clinicopathological value of PD-L1 in breast cancer [29].The result showed that positive/higher PD-L1 expression was significantly associated with positive lymph node metastasis and poor nuclear grade.[30].The underline mechanisms remain unclear.Thus, part of the patients with metastatic lymph nodes, poorer nuclear grade may benefit from anti-PD-L1 therapy, which would improve their prognosis.PD-L1 expression was reported to be over expressed in MMR-deficient cancers, usually because MMR-deficient cancer creates high accumulated mutations which lead many tumor-specific neoantigens and is associated with high immune activities presumably aroused by mutated neoantigens.However, in present study, MSH2 was showed an inverse association with PD-L1 expression but not MLH1.These differences could be attributed to the several factors, such as the heterogeneity in population 1.917 0.969-3.7930.062 HR: hazard ratio, 95% CI: 95% confidence interval.Significant values (P < 0.05) have been marked with bold.and different tumor type but also analysis method.In our study, high expression of PD-1 on cell membranes of TILs correlated with well-differentiation and a favorable clinical outcome in PC.Our results are in line with some studies in pancreatic cancer [18], but the mechanisms are currently unclear.Ashton et al. reported that antigenspecific immune activation following T-cell receptor stimulation leads to PD-1 upregulation on TILs [31].Badoual et al. found higher expression of the immune activation markers HLA-DR and CD38 in PD-1+ TILs [32], so the presence of PD-1+ TILs could reflect an endogenous antitumor immune response that occurred upon activation of TILs.It could be an argument for this hypothesis that PD-1 expression level was associated with OS only at low PD-L1 expression subgroup.PC is characterised by an abundant desmoplastic stroma which is poor-vascularized and often prevents T-cell from infiltrating.The mechanisms include activated pancreatic stellate cells that drive apoptosis and sequestration of TILs, and the physical barrier imposed by dense collagen [33].Analysis of PD-1+ TILs expression showed significantly higher infiltration in patients with moderate stroma density compared to patients with dense stroma and vice versa in our study.Moreover, patients with both low PD-1 expression and moderate stroma had a worse OS and vice versa.Intriguingly, Wang et al. reported stroma density constituted an independent prognostic marker in PC patients treated with adjuvant chemotherapy [8].Some studies have reported higher TILs motility and migration in tumor areas with loose collagen [34].Therefore, we speculated that low PD-1+ TILs in moderate stroma may reflect the lower TILs motility and migration and poor immune response of some patients.Obviously, these patients may have a worse outcome.

Similar result was reported by Abbas M et al. in clear cell renal cell carcinoma
To our knowledge, the present study is the first study concerning PD-1/ PD-L1 and MMR statues analyses in East Asia population, which specifically investigated their clinical significant in PC.However, we would like to acknowledge the limitations of the study.Although we have analyzed a large cohort, the present study is a retrospective analysis and there is a potential for selection bias.And we did not include therapy responses in our studies which may affect the result of multivariate analysis.
In summary, we evaluated the correlation between clinicopathological characteristics and MMR, PD-1/PD-L1 expression, and found the prognosis value of PD-1 expression.In subgroup analyses, we observed that PD-1 expression level was associated with OS only at low PD-L1 expression subgroup.When we stratified the cases into four subgroups based on PD-1 expression and stroma density, we found that patients with high PD-1 expression and dense stroma had a better OS, while patients with low PD-1 expression and moderate stroma showed a worst outcome.Our result may provide more effective molecular markers for immunotherapeutic strategies of PC patients in clinical practice.

Patients and treatment
This study was approved by the Ethics and Research Committees of Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, and was conducted in accordance with the Declaration of Helsinki Principles.
The TMA used for this study includes 94 unselected, non-consecutive, primary, and sporadic PC treated between March 2012 and August 2014 in Pancreatic Cancer Center, Shanghai Jiao Tong University School of Medicine.Formalin-fixed, paraffin-embedded tissue blocks from resected PC were made.Tissue cylinders with a 2.0 mm diameter were punched from representative tissue areas.The histological types were confirmed by experienced pathologists.The TMAs contained well-documented clinicopathological information, including patients' age, sex, location, tumor differentiation, T stage, lymph node metastasis, distant metastasis, nervous invasion, vascular invasion and follow-up data (ended in March, 2016).In total, 94 patients were included, 63 males and 31 females, with a median age of 62 years old (ranging from 31 to 78 years old).We got follow-up data of 77 patients in this cohort.The overall survival time ranged from 1.75 to 30.00 months, with a median of 14 months.Detailed information can be found in Table 7.

Immunohistochemical staining
Immunohistochemistry was performed based on the standard streptavidin-peroxidase (S-P) method (Zymed, San Francisco, CA).Briefly, the tissue microarrays were dewaxed and dehydrated in a xylene and alcohol bath solution.Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0.After that, endogenous peroxidase activity was then blocked with 0.3% hydrogen peroxide for 10 mins.The slides were cooled to room temperature and blocked by incubating with normal goat serum at room temperature for 1h and were subsequently incubated overnight at 4°C with primary antibodies as follows: PD-1 (dilution1:50, Abcam), PD-L1 (dilution  S2).The sections were incubated with biotinylated secondary antibodies (Zymed, San Francisco, CA) for 30 min at room temperature, followed by incubation with streptavidin horseradish peroxidase complex.Finally, sections were visualized by 3, 3′-diaminobenzidine staining.Meanwhile, slides were stained with haematoxylin and eosin (H&E) as described before.
Samples with a final staining score of < 6 were classified as low expression, while those with score of ≥ 6 were considered to be high expression.The extent of TILs was assessed in HE stained TMA.The expression of PD-1 in TILs by IHC method was evaluated by measuring cell density as previously reported.Scoring was as follows: (1) absent cells; (2) < 25% cell density; (3) 25-50% cell density; (4) >50% cell density.Samples with a score of 1 or 2 were considered negative (low expression) and those with a score of 3 or 4 were considered positive (high expression).Stromal density based on HE staining, and classified as moderate or dense as previously described.The stromal density was evaluated on H&E-stained TMAs.Quality was defined as moderate or strong on the basis of its morphologic appearance.Moderate stroma had a paucicellular matrix of loosely packed connective tissue fibres with occasional oedematous appearance.Cases with dense stroma showed a densely packed network of fibres with intense staining.Thus, dense stroma presents mature collagen fibres packed into multilayers with intense staining and others were considered to be "moderate" [7][8]35].

Statistical analysis
All statistical analyses were carried out by the SPSS 16.0 software.The χ 2 test and Fisher's exact test were used to analyze the correlations between categorical variables.Overall survival (OS) was defined as the interval from date of diagnosis until death from any cause.Data were censored for living patients and patients lost between follow-ups.The OS was estimated using the Kaplan-Meier method and compared using the log-rank test.Significant variables were further analyzed by multivariate analysis to test for independent prognosis.Bivariate correlations between variable factors were calculated by Spearman rank correlation coefficients.P-values < 0.05 were considered statistically significant.

Figure 3 :
Figure 3: Correlations of MMR gene expression, stroma density and PD-1/PD-L1 expression in PC tissues.(A) OS according to PD-1 expression in high PD-L1 expression subgroup.(B) OS according to PD-1 expression in low PD-L1 expression subgroup.(C) OS in four subgroups straitified by PD-1 expression and stroma density.(D) Low PD-1 expression and moderate stroma showed a worse outcome than the combination of other three group named "all others".(E) Serial sections were used from the same PC tissue to depict an inverse correlation between MSH2 expression and PD-L1 expression.Top left and top right: low MSH2 expression vs. high PD-L1 expression; Lower left and lower right: high MSH2 expression vs. low PD-L1 expression.