Abstract
Background/Aim: The development and application of cancer immunotherapy to pancreatic cancer has not progressed because its efficacy has not been proven in clinical trials. In this study, we aimed to explore potential targets of immune checkpoint inhibitor therapy for pancreatic cancer treatment. Materials and Methods: We collected resected specimens from 40 patients with pancreatic cancer who underwent resection at our Institution without any preoperative treatment. We evaluated the expression of molecules in the programmed death receptor-1 (PD-1), T cell immunoglobulin mucin-3 (Tim-3)/Galectin-9, and CD155/T cell immunoreceptor with Ig and ITIM domains (TIGIT) pathways using immunohistochemical staining. The correlation between the expression pattern of these molecules and patient prognosis were assessed using Kaplan-Meier analysis. Results: An increased number of CD8+ T cells in pancreatic cancer tissue was significantly associated with a better patient prognosis. Additionally, patients with a higher ratio of PD-1 expression to CD8+ T cells had a worse prognosis. We observed no correlation between the Tim-3/Galectin-9 and CD155/TIGIT pathways and patient prognosis. Conclusion: Modifications in the immune environment to increase T cell infiltration into tumors could result in the PD-1 pathway becoming a potential target to treat pancreatic cancer using immune checkpoint inhibition.
Pancreatic cancer has an extremely poor prognosis. At the time of diagnosis, approximately 80-85% of patients progress to either an unresectable or metastatic state, which accounts for a 5-year survival rate of less than 10% (1). Even in the small subset of patients who have the opportunity to undergo surgery (10%), the prognosis remains poor, with only 20% of patients surviving after 5 years (2). The effectiveness of chemotherapy and radiotherapy is limited; therefore, the development of new treatment methods is urgently needed.
Recently, the efficacy of cancer immunotherapy has been shown in gastrointestinal cancers, and immunotherapy using immune checkpoint inhibitors (ICIs) has been funded by the Japanese National Health Insurance System for gastric, esophageal, and liver cancers. Since the use of ICIs as a standalone treatment has been ineffective in clinical trials, the development of pancreatic cancer immunotherapy has been slow; in a phase I study of gemcitabine (GEM) and tremelimumab in combination with chemotherapy, none of the patients responded to the treatment (3). The highly immunosuppressive tumor microenvironment of pancreatic cancer is thought to be the reason for this failure of immunotherapy, thus making an immunological approach difficult (4). Although there are potential anti-tumor effects of immune checkpoint inhibition in pancreatic cancer (5, 6), few studies have evaluated the immune microenvironment in clinical specimens. In this study, we aimed to evaluate the microenvironment of pancreatic cancer by assessing the number of cytotoxic T lymphocytes (CTLs) and the expression of immune checkpoint molecules in tumorinfiltrating lymphocytes (TILs) and the ligands of these molecules in tumor cells; these molecules are involved in pathways that could be potential targets to treat pancreatic cancer with ICI therapy.
Materials and Methods
This study was approved by the Ethical Review Committee of the Kagoshima University (number: 200288). Written informed consent was obtained from each participant of the study after a full explanation of the purpose and nature of all procedures used. This study was conducted in accordance with the Declaration of Helsinki.
Sample collection. Forty patients with pancreatic cancer who had undergone resection without any preoperative treatment at the Department of Digestive Surgery, Kagoshima University Hospital between 2004 and 2013 were included in the study. The TNM staging system (UICC 8th edition) (7) was used for tumor staging. Immunohistochemical staining was performed on resected specimens to evaluate the number of CTLs (CD8+ T cells) and the expression of immune checkpoint molecules in TILs and the ligands of these molecules in tumor cells. The immune checkpoint molecules examined were programmed death receptor-1 (PD-1), T cell immunoglobulin mucin-3 (Tim-3), and T cell immunoreceptor with Ig and ITIM domains (TIGIT). The ligands assessed were programmed cell death 1 ligand 1 (PD-L1), programmed cell death 1 ligand 2 (PD-L2), Galectin-9, and poliovirus receptor (CD155). Immunohistochemical staining was performed by preparing paraffin-embedded sections of 3 μm thickness from resected specimens following the procedure recommended by Vector Laboratories (Burlingame, CA, USA). The primary antibodies used are listed in Table I. Normal serum blocking and secondary antibody conjugation were performed using ImmPRESS Universal Reagent Anti-Mouse/Rabbit Ig (MP-7500, VEC, Burlingame, CA, USA). Antibody dilution was performed using REAL™ Antibody Diluent (S202230, Agilent, Palo Alto, CA, USA). Liquid DAB+ Substrate Chromogen System (K3468, Agilent) was used for color development with the 3,3’-diaminobenzidine substrate.
List of antibodies used in this study.
Analysis of immunohistochemistry images. Using the image processing software ImageJ Fiji (NIH/http://imagej.nih.gov/ij/download.html, NIH/http://fiji.sc/Fiji), the number of T cells was measured in tumors at hotspots with 5 fields of view at 400× magnification. The expression of ligands for immune checkpoint molecules was evaluated by randomly selecting 5 fields of view and measuring the number of cells using the same method as for T cells. The correlation between the expression of these molecules and patient prognosis, such as overall survival (OS) and recurrence-free survival (RFS) from surgery, was assessed to identify potential therapeutic targets.
Statistical analysis. Receiver operating characteristic (ROC) curves were generated by the number of positive cells that expressed the examined molecules to evaluate the prognosis of the two groups classified according to a prognostic factor described in the TNM classification. The ROC curve analysis was used to calculate the area under the curve (AUC), and the optimal cutoff value for each parameter was determined using the Youden Index. An AUC value greater than 0.5 was considered significant. Survival curves were generated using the Kaplan-Meier method, and significance tests were performed using the log-rank test and Wilcoxon signed-rank test. Statistical significance was set at p<0.05, and all statistical analyses were performed using JMP pro14 software (SAS, Institute Inc, Cary, NC, USA).
Results
Clinical and pathological characteristics of patients. The clinical and pathological characteristics of the 40 patients with pancreatic cancer are presented in Table II. There were 19 (47.5%) males and 21 (52.5%) females, with a median age of 69 years (range=42-81 years). The tumors were localized in the pancreatic head, body, and tail in 24 (60%), 11 (27.5%), and 5 patients (12.5%), respectively. The tumors were classified as stage I, II, III, and IV in 11 (27.5%), 13 (32.5%), 14 (35%), and 2 patients (5%), respectively. The histopathological diagnoses were adenocarcinoma and adenosquamous carcinoma in 38 (95%) and 2 patients (5%), respectively, and 29 patients (73%) had lymph node metastases. Postoperative adjuvant therapy was performed in 32 patients, and the regimens used were GEM, S1, and a combination of GEM and S1 in 18, 13, and 1 patient, respectively. During the median surveillance period of 21 months (range=3-141 months), 37 patients (92.5%) died, and 3 patients (7.5%) survived. As the time of recurrence was unknown in 1 patient who died, this patient was excluded from the RFS assessment.
Characteristics of the 40 patients with pancreatic cancer in this study.
CD8+ T cell infiltration in tumor and prognosis. Immunohistochemical staining showed that all tumors were infiltrated with CD8+ T cells, but the number of cells differed among patients (Figure 1A). When the cutoff value for the CD8+ cell count was set at 236.6, the AUC of the ROC curve was 0.745, and 23 patients had high CD8 expression levels (CD8-High) and 17 had low CD8 expression levels (CD8-Low). OS was significantly prolonged in the CD8-High group (log-rank test p=0.031, Wilcoxon test p=0.018) (Figure 1B), and RFS was also prolonged in the CD8-High group (log-rank test p=0.088, Wilcoxon test p=0.032) (Figure 1C).
Correlation between the number of CD8+ cells and prognosis of patients with pancreatic cancer. (A) Immunohistochemical staining with anti-CD8 antibodies in pancreatic cancer tissue. (B) Kaplan-Meier analysis of overall survival and the number of CD8+ cells. (C) Kaplan–Meier analysis of recurrence-free survival and the number of CD8+ cells.
Analyses of the PD-1 pathway. PD-1 was expressed on the cell membranes of TILs in pancreatic cancer tumors (Figure 2A). When the cutoff value for the number of PD-1+ cells was set at 20.6, the AUC of the ROC curve was 0.617. Moreover, when the cutoff value for the ratio of PD-1+ cells to CD8+ T cells (PD-1/CD8) was set at 0.121, the AUC of the ROC curve was 0.729. Although there was no correlation between the number of PD-1+ cells and OS of patients (Figure 3A), OS was prolonged in the group with a lower PD-1/CD8 ratio (log-rank test p=0.055, Wilcoxon test p=0.031) (Figure 3B). PD-L1 and PD-L2 were not expressed in the pancreatic cancer tissue (Figure 2B).
Immunohistochemical staining depicting the expression of immune checkpoint-related molecules in pancreatic cancer tissue. (A) Expression of PD-1 in tumor infiltrating lymphocytes (TILs). (B) No expression of PD-L1 and PD-L2 in tumor cells. (C) Expression of Tim-3 in TILs. (D) Expression of Galectin-9 in tumor cells. (E) Expression of TIGIT in TILs. (F) Expression of CD155 in tumor cells.
Kaplan-Meier analyses of the correlation between the expression of immune checkpoint-related molecules and patient prognosis. (A) Analysis of PD-1 expression and overall survival (OS). (B) Analysis of PD-1/CD8 ratio and OS. (C) Analysis of Tim-3 expression and OS. (D) Analysis of Galectin-9 expression and OS. (E) Analysis of Galectin-9 expression and recurrence-free survival (RFS). (F) Analysis of TIGIT/CD8 ratio and OS. (G) Analysis of CD155 expression and OS. (H) Analysis of CD155 expression and RFS.
Analysis of Tim-3/Galectin-9 pathway. A small number of Tim-3+ cells was found among TILs (Figure 2C), and the number of Tim-3+ cells did not affect the prognosis between the two groups that were separated at a cutoff value of 5.6 (AUC=0.587) (Figure 3C). Galectin-9, a ligand for Tim-3, was expressed on the membrane of tumor cells (Figure 2D). At the cutoff value of 221.4 (AUC=0.685), the high Galectin-9 expression group showed significant prolongation of OS (log-rank test p=0.007, Wilcoxon test p=0.008) and RFS (log-rank test p=0.028, Wilcoxon test p=0.062) (Figure 3D, E). The evaluation of the Tim-3/CD8 ratio was excluded because of the low AUC value of 0.492 (p<0.50).
Analyses of the CD155/TIGIT pathway. TIGIT was expressed on the cell membranes of TILs in pancreatic cancer tumors (Figure 2E). When the cutoff value of the ratio of TIGIT+ cells to CD8+ T cells (TIGIT/CD8) was set at 0.236, the AUC of the ROC curve was 0.599. There was no correlation between the TIGIT/CD8 ratio and patient prognosis (Figure 3F). CD155, the ligand for TIGIT, was expressed on the membrane of tumor cells (Figure 2F), and when the cutoff value for the number of CD155+ cells was set at 491.4, the AUC of the ROC curve was 0.534. There was no significant correlation between the number of CD155+ cells and OS or RFS (Figure 3G, H). The evaluation of TIGIT by itself was excluded because of the low AUC value of 0.482 (p<0.50).
Discussion
PD-L1 overexpression has been associated with a poor prognosis in many types of cancer (8-11); therefore, immunotherapy targeting the PD-1 pathway is considered to be effective in such cancers. Furthermore, a correlation between PD-L1 expression and therapeutic efficacy has been shown (12). In this study, we examined ligands of immune checkpoint molecules in pancreatic cancer tissue and found no expression of PD-L1 or PD-L2. This result suggests that the use of anti-PD-1/PD-L1 antibodies, the most prevalent ICI, would be an ineffective pancreatic cancer treatment. Investigation of PD-1 expression in TILs showed that patients with a low PD-1/CD8 ratio tended to have a better prognosis, and a high PD-1/CD8 ratio indicated an immune environment that suppressed anti-tumor immunity; however, without the expression of the ligands PD-L1 and PD-L2, the PD-1 pathway is not a suitable therapeutic target. Nevertheless, the expression of PD-L1 can be induced in tumors via interferon-gamma, which is produced by T cells in tumors (13, 14). As pancreatic cancer has fewer TILs than other types of cancer, increased T cell infiltration may promote PD-L1 expression and effectively inhibit the PD-1 pathway. The expression of PD-L1 in tumors has been used as a marker for prognosis and prediction of therapeutic efficacy, but the issue of altering PD-L1 expression has also been highlighted (15). Peripheral and gastric invasive PD-1 expression has been reported as a potential prognostic marker in patients with gastric adenocarcinoma (14). This study suggests that the PD-1/CD8 ratio in TILs may be used as a predictive marker of the PD-1 pathway.
The use of pembrolizumab, an anti-PD-1 antibody, was approved in Japan for unresectable solid tumors with high-frequency microsatellite instability (MSI-high) in 2018. However, in the KEYNOTE-158 clinical trial of pembrolizumab in MSI-high solid tumors, pembrolizumab was less effective in pancreatic cancer than in other types of cancers (16). These tumors typically have a strong tumor immune response due to the large number of neoantigens, and there are usually more TILs in these tumors. This limited therapeutic effect in pancreatic cancer, even in MSI-high solid tumors, may be due to the suppressive immune environment of the cancer that prevents TIL infiltration.
In the tumor microenvironment of pancreatic cancer, the numerous immunosuppressive cells, such as regulatory T cells, myeloid-derived suppressor cells, and tumor-associated macrophages, as well as the large proportion of stromal cells, in the tumor are thought to prevent the infiltration of T cells (17). Therefore, few CD8+ cells (CTLs) are considered to be present among the TILs in pancreatic cancer. However, in this study, the survival of patients with pancreatic cancer was significantly prolonged in the group with a large number of CTLs, similar to that of patients with other cancer types. This suggests that there may be anti-tumor immunity in patients with pancreatic cancer in which there are many infiltrated CTLs.
While clinical trials of ICI therapy in combination with chemotherapy and radiation therapy have been performed to improve the immune environment in pancreatic cancer, treatments have not been effective. For immunotherapy to be effective in treating pancreatic cancer, it is important to make the tumor microenvironment conducive to the infiltration of T cells. The use of low-dose vascular endothelial growth factor inhibitors improves anti-tumor immunity via vascular normalization, thereby allowing the infiltration of numerous T cells into the tumor (18). Immunotherapy combined with low-dose vascular endothelial growth factor inhibitors may be effective in the treatment of pancreatic cancer. Moreover, the quality of neoantigens in pancreatic cancer is associated with T cell infiltration into the tumor; thus, this attribute may be useful in selecting patients who could benefit from immunotherapy (19).
We also evaluated the Tim-3/Galectin-9 and CD155/TIGIT pathways as novel immunotherapy targets to treat pancreatic cancer. However, no prognostic disadvantage was observed in the groups with a high expression of immune checkpoint-related molecules in either pathway; thus, we could not prove that these pathways are effective targets for pancreatic cancer treatment.
High expression of Tim-3 has been reported to be significantly higher in pancreatic cancer tissues than in healthy pancreas, suggesting that Tim-3 may be involved in immune invasion, evasion, and metastasis in pancreatic cancer (20). While antigen-specific tumor-reactive CD8+ T cells can be identified by PD-1 expression (21), PD-1+Tim-3+ cells are considered to be terminally exhausted T cells (22). The presence of PD-1+Tim-3+ T cells in the tumor microenvironment indicates that the blockade of both Tim-3 and PD-1 may be effective in cancer patients (23). However, in this study, a significantly prolonged prognosis was observed in the group with higher expression of Galectin-9, a ligand of Tim-3 that regulates several intracellular processes including Tim-3-mediated T cell death (24, 25). In addition, Galectin-9 has been reported to inhibit tumor growth by inducing apoptosis of cancer cells (26, 27). The results of this study suggest that Galectin-9 may act as a tumor suppressor rather than a tumor immunosuppressor in pancreatic cancer.
The limitations of the current study include the relatively small sample size and the regimen of adjuvant therapy not being standardized among patients. Additionally, this study only evaluated immunohistochemical staining, and the assessment of mRNA might have been necessary as ligand expression in tumor tissue can fluctuate.
Conclusion
A low ratio of PD-1 expression in TILs to the number of CD8+ cells in the tumor is correlated with a better prognosis for patients with pancreatic cancer. Therefore, the PD-1 pathway may be a potential target to treat pancreatic cancer with ICI if the immune environment is modified to increase T cell infiltration into the tumor.
Acknowledgements
This work was supported by JSPS KAKENHI (Grant Number JP20H03753).
Footnotes
Authors’ Contributions
C.N., T.I. and H.S. carried out the experiment. C.N. wrote the manuscript with support from K.T., Y.S. and D.M. C.N., Y.M., H.K. AND T.I. were responsible for collecting and preparing the sample. K.Y., Y.H., A.N. and T.A. helped supervise the project. C.N. and K.T. conceived the original idea. T.O. supervised the project.
Conflicts of Interest
The Authors declare that there are no conflicts of interest.
- Received April 9, 2022.
- Revision received May 14, 2022.
- Accepted May 23, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
