Abstract
Clinical significance of 2,3-dioxygenase (IDO) has been studied in types of tumors, but the role that IDO played in gastric adenocarcinoma (GAC) is still unclear. Here, we aim to investigate the prognostic value of IDO expression in patients with GAC. We examined intratumoral IDO expression in retrospectively enrolled 357 patients with GAC undergoing gastrectomy at Zhongshan Hospital of Fudan University in 2008 by immunohistochemical staining. The Kaplan-Meier method and Cox regression models were used to evaluate the prognostic value of IDO expression and its association with clinical pathological factors. We generated a predictive nomogram by integrating IDO expression with the TNM staging system for overall survival of GAC patients. High expression of intratumoral IDO predicted a dismal outcome. Intratumoral IDO expression gave a further discrimination for the prognosis of GAC patients. By Cox multivariate analysis, IDO expression was defined as an independent prognosticator. The generated nomogram performed well in predicting the 3- and 5-year overall survival of GAC patients. Conclusively, IDO is a potential prognostic biomarker for overall survival of patients with GAC after gastrectomy.
Similar content being viewed by others
Introduction
Despite the steadily decreasing incidence and mortality rates in the majority of the world, gastric adenocarcinoma (GAC) remains the fourth most common malignancy and the third leading cause of cancer death worldwide. Moreover, incidence rates are much higher in East Asia, particular in Korea, Japan and China, than that in North America and most parts of Africa1. Due to the mild and atypical symptoms at the early stage, over 80% of the patients were clinically diagnosed at an advanced stage, which generally indicated a dismal outcome2,3.
Currently, UICC/AJCC TNM system is the widely used risk stratification system for patients with GAC. The underlying molecular and cellular processes during the carcinogenesis of GAC are ignored in this system while broad evidences emerge to state that the disease is heterogeneous with unpredictable nature history4. Thus, illumination of the involved molecules and the underlying mechanisms in the development and progression of GAC might contribute to disease prognosis and provide novel therapeutic targets for the treatment.
Indoleamine 2,3-dioxygenase (IDO) is responsible for the first enzymatic step of tryptophan catabolism by the kynurenine pathway performed as the rate-limiting enzyme5. Increasing evidences implicated that IDO, as well as tryptophan catabolism, generated immune tolerance to foreign antigens in tissue microenvironment. Within a tryptophan-depleted microenvironment, the expression of IDO shields the tumor from an immune response against it either by a direct suppression of T cell function and proliferation or indirect suppression of anti-tumor immune responses by accumulation of IDO catabolites into the environment6. Moreover, IDO expression was also reported to be associated with M2-polarization of tumor-associated macrophages and impaired function of nature killer cells7,8,9.
In non-small cell lung cancer10, colorectal cancer11,12, ovarian cancer13, endometrial cancer14, breast cancer15, as well as in childhood acute myeloid leukemia16, aberrant expression of IDO had been determined as a prognosticator for poor prognosis. These suggested a therapeutic potential for IDO inhibitors. 1-methyl tryptophan (1MT) is a specific inhibitor of IDO17. Several studies have offered evidence that IDO inhibition with 1MT could exert antitumor effects18. In H. pylori-infected human gastric mucosa, an enhanced expression of IDO was detected19. However, divergent results have been reported about IDO expression in GAC and its correlation with tumor stage20,21. The expression of IDO in GAC and its clinical significance have not been fully clarified and need further study.
In this study, we sought to investigate IDO expression in patients with GAC and explore its correlation with the clinical pathological characteristics and clinical outcome. Moreover, a nomogram integrated IDO expression and depth of tumor invasion, lymph node metastasis status was constructed to present a quantitative assessment for the 3- and 5-year overall survival in GAC patients after surgery.
Results
IDO expression in gastric adenocarcinoma
The positive staining of IDO was observed in the cytoplasm and/or on the membrane of neoplastic epithelial cells and mononuclear cells in the stroma (Fig. 1a,b). The integrated optical density (IOD) of the immunostaining in each specimen varied greatly in the cancer tissue. The measured IOD of the staining in tumor tissue was 352.25 ± 291.58 (median, 284.00; range from 1.62 to 587.78). With the X-tile software, the cut-off point was 282, which was determined using the method of minimum p value. Thus, 175 patients (49%) were separated into the IDO low expression subgroup and 182 patients (51%) were separated into the IDO high expression subgroup.
Correlations between IDO Expression and clinical pathological features
The relationship between clinical pathological characteristics and IDO expression is shown in Table 1. Intratumoral IDO expression was significantly associated with the depth of tumor invasion (p = 0.045) and lymph node metastasis (p < 0.001).For the other clinical pathological characteristics, no significant association was found.
Prognostic evaluation of IDO expression in gastric adenocarcinoma
By separating the patients into two subgroup as mentioned above, we applied Kaplan-Meier analysis to determine the overall survival. Log rank test was used to give the p values. As shown in Fig. 2a, high expression of IDO was associated with poor overall survival (p < 0.001). The median survival time for IDO low expression group was 51.4 months while that for IDO high expression group was 30.7 months.
The overall survival of the two subgroups was significantly different in different tumor invasion depth (Fig. 2b), lymph node metastasis status (Fig. 2c), TNM stage (Fig. 2d), Lauren’ classification (Fig. 2e) and histology (Fig. 2f), with all the p values less than 0.001. Furthermore, statistically significant difference in overall survival was found between the two subgroups in patients without adjuvant chemotherapy (p = 0.001, Fig. 3a), as well as in patients with adjuvant chemotherapy (p = 0.005, Fig. 3b). However, in Fig. 2d, for example, the overall survival for the patients of TNM I stage did not differ significantly in the IDO low and high expression subgroups although the p value for the whole model was less than 0.001.
Thus, we calculated the hazard ratios of IDO high expression in GAC in different tumor invasion depth, lymph node metastasis status, TNM stage, Lauren’ classification, histology and adjuvant chemotherapy status respectively to give a further analysis. As shown in Table 2, we found that in T3 and T4, N3, TNM III, Lauren’s intestinal type, patients of IDO high expression group had a significantly increased risk. Meanwhile, patients in IDO high expression group suffered a higher risk of death regardless of adjuvant chemotherapy and histological type.
In the univariate Cox regression analysis of overall survival, IDO expression was defined as a prognostic factor (p < 0.001). Multivariate analysis revealed that intratumoral IDO was a risk factor for death, independent of depth of tumor invasion, lymph node metastasis, Lauren’s classification and adjuvant chemotherapy (Fig. 4a).
Predictive Nomogram for overall survival of patients with gastric adenocarcinoma
A predictive nomogram was constructed to give a quantitative model to stratify GAC patients into different risks. Independent factors for overall survival selected by multivariate analysis, excluding adjuvant chemotherapy, were integrated in the nomogram. Since T1 and T2, T3 and T4, N0 and N1 had a similar hazard ratio for overall survival (Fig. 4a), we combined T1 with T2, T3 with T4, N0 with N1 in the nomogram. In the nomogram, a higher total point predicts a worse prognosis. The total point was raised by adding the score of tumor depth (0 for T1 + T2, 94 for T3 + T4), lymph node metastasis (0 for N0 + N1, 51 for N2, 100 for N3) and IDO expression (0 for low and 52 for high) for each patients correspondingly (Fig. 4b). Calibration curves for nomogram predicted 3- and 5-year overall survival were built to give the internal validation, which performed well with the ideal model (Fig. 4c,d). The Harrell’s c-index for the generated nomogram was 0.702 (95% CI, 0.663-0.741), higher than that for TNM stage (0.684; 95% CI, 0.646–0.722), indicating that the nomogram gave a better prediction for overall survival of the patients.
Discussion
Studies on IDO expression in various tumors and antigen presenting cells have confirmed that IDO played an immunosuppressive role in carcinogenesis and served as a biomarker for poor prognosis in some tumors10,11,12,13,14,15. However, few studies focused on the expression of IDO and its clinical significance in GAC. Here, we demonstrated the prognostic value of IDO expression in GAC and gave a simple risk classification for the patients according to IDO expression. Cox multivariate analysis revealed IDO expression as an independent prognostic factor for overall survival of the patients. The nomogram model based on the integration of depth of tumor invasion, lymph node metastasis and IDO expression seemed to perform better than TNM staging system in predicting overall survival of the patients.
The immune system gains increasing interests for its crucial role in carcinogenesis of a bunch of tumors, not only for its suppression of tumor growth by ruining cancer cells or inhibiting their invasion, but also for the promotion of tumor progression either by selecting tumor cells that are more accommodative to survive in an immunocompetent host or by establishing requisites in tumor microenvironment that facilitate tumor invasion22. Lymphocytes, especially T cells, are very sensitive to particular amino acids in the local environment23. Both tryptophan depletion mediated by IDO in a cellular microenvironment and tryptophan catabolites contributed to establish a regulatory environment affecting CD8+ as well as CD4+ T cell function and not only was tryptophan catabolism an effector pathway of immunologic tolerance, but it also led to GCN2-dependent formation and activity of Treg cells24 and myeloid derived suppressor cells (MDSCs)25. Further, some studies stated that aberrant expression of IDO was implicated in the polarization of IL-10-secreting M2 immunosuppressive macrophages (CD14+/CD206+)26. Thus, it is conceivable that aberrant IDO expression in GAC created an immunosuppressive microenvironment that favored the infiltration and invasion of the primary tumor, which could give a possible explanation to the finding in the study that IDO expression in GAC was associated with depth of tumor invasion and lymph node metastasis (Table 1).
Notably, since Lauren introduced in 1965 GAC occurred in two subtypes, intestinal type and diffuse type, debates have continued whether Lauren’s classification could give a risk stratification in patients with GAC. Our study found that while there was no difference between overall survival rates of IDO high and low expression patients in Lauren’s diffuse type, the overall survival rates significantly differed in Lauren’s intestinal type. That raised the hypothesis that tumors of the two subtypes could totally be two different kind of diseases, although they both occurred in the stomach and were so called “gastric adenocarcinoma”.
In this study, we demonstrated the prognostic value of IDO expression in GAC. According to intratumoral IDO expression, patients could be separated into two subgroups regardless of adjuvant chemotherapy. IDO expression proved to be an independent adverse prognosticator for overall survival in patients with GAC. Stratification analyses revealed IDO expression could give some additional prognostic information in tumors of different stages and of different histology types. Further, we constructed a nomogram by integrating IDO expression, depth of tumor invasion and lymph node metastasis status to give a prediction for the 3- and 5-year overall survival of the patients. Calibration plots and c-indices indicated the generated nomogram performed better than TNM staging system in discriminating the patients with different clinical outcomes.
However, owing to the limitation of retrospective study, we only found a close relationship between IDO expression and tumor stage. The causal relationship between higher IDO expression and progression of GAC remains elusive. The exact role of IDO in the progression of GAC would be investigated in our future work. Moreover, the number of patients enrolled in the study is relatively small. A large, multi-center, prospective data is needed to validate these results.
In conclusion, we have identified aberrant expression of IDO in GAC as an independent prognosticator, which could be incorporated with depth of tumor invasion and lymph node metastasis status to generate a nomogram to give a better stratification for patients with different prognosis.
Methods
Patients
The study enrolled 357 GAC patients from Zhongshan Hospital of Fudan University (Shanghai, China). All the patients included were neoadjuvant therapy naïve and received the semi-elective gastrectomy performed by the same surgical team in 2008 after sufficient preoperative evaluation. All the patients received a radical (R0) resection with a D2 lymphadenectomy. We retrospectively collected the clinical pathological and baseline demographic characteristics of the patients, including age, gender, tumor size, tumor differentiation, Lauren’s classification and tumor stage. We were grateful to the two independent gastroenterology pathologists from Department of Pathology, Zhongshan Hospital for their reassessment for tumor stage according to the 7th Edition of the UICC/AJCC TNM Staging System. Patients with adjuvant chemotherapy received at least one cycle of 5-fluorouracil based chemotherapy. All the patients were followed up until April 2014 with a median follow-up time of 41 months. Overall survival was defined as the time from the date of surgery to the date of death or last visit. All methods were approved by the research medical ethics committee of Zhongshan Hospital, Fudan University and were carried out in accordance with the approved guidelines. Written informed consent on the use of clinical specimens from each patient was achieved.
Tissue microarray, Immunohistochemical Staining and Evaluation
The immunohistochemical protocols were as previously described27 and the tissue microarray was established with formalin-fixed paraffin-embedded surgical specimens. Anti-Indoleamine 2,3-dioxygenase antibody (0.5 mg/ml, ab55305, Abcam, Cambridge, MA, USA) was used as the primary antibody in the immunohistochemical analysis. The density of positive staining was measured by the computerized image system composed of an Olympus CCD camera connected to a Nikon eclipse Ti-s microscope. The stained sections were scanned at ×200 magnification and three independent microscopic fields with the strongest staining were captured by NIS-Element F3.2 software to ensure representativeness and homogeneity. Each photo used an identical setting.
The density of immunostaining was measured by Image-Pro Plus version 6.0 software (Media Cybernetics Inc., Bethesda, MD, USA).Integrated optical density (IOD) of all the positive staining in the sections was measured to give a quantitative assessment for the staining. The mean IOD of the three captured microscopic fields was regarded as the density of IDO expression in the represented tissue. Two observers who were blinded to the patient outcomes and clinical pathological characteristics gave the evaluation of immunostaining. The cut-off points for the definition of high/low expression subgroups were determined by X-tile software28.
Statistical Analysis
SPSS 19.0 (SPSS Inc., IL, Chicago) and R software version 3.0.2 with the “rms” package (R Foundation for Statistical Computing, Vienna, Austria) were used to perform the analyses. Pearson χ2 test or Fisher’s exact test was used to compare categorical variables. Continuous variables were analyzed by Student’s t test. Kaplan-Meier analysis was used to determine the overall survival. Log-rank test was used to compare the overall survival of the patients between subgroups. Cox proportional hazards model was used to perform multivariate analysis. Nomogram was generated by R software with “rms” package. Calibration plots for 3- and 5-year overall survival were constructed to examine the performance characteristics of the generated nomogram. The prognostic accuracy was measured by calculating the Harrell’s concordance indices (c-indices). All statistical analyses were two-sided and p < 0.05 was regarded as statistically significant.
Additional Information
How to cite this article: Liu, H. et al. Increased expression of IDO associates with poor postoperative clinical outcome of patients with gastric adenocarcinoma. Sci. Rep. 6, 21319; doi: 10.1038/srep21319 (2016).
References
Torre, L. A. et al. Global Cancer Statistics, 2012. CA Cancer J Clin. 65, 87–108 (2015).
Mayer, R. J., Venook, A. P. & Schilsky, R. L. Progress Against GI Cancer During the American Society of Clinical Oncology’s First 50 Years. J Clin Oncol. 32, 1521–1530 (2014).
Siegel, R., Ma, J., Zou, Z. & Jemal, A. Cancer Statistics, 2014. CA Cancer J Clin. 64, 9–29 (2014).
Bass, A. J. et al. Comprehensive Molecular Characterization of Gastric Adenocarcinoma. Nature. 513, 202–209 (2014).
Terentis, A. C. The Heme Environment of Recombinant Human Indoleamine 2,3-Dioxygenase. Structural Properties and Substrate-Ligand Interactions. J Biol Chem. 277, 15788–15794 (2002).
Katz, J. B., Muller, A. J. & Prendergast, G. C. Indoleamine 2,3-Dioxygenase in T-cell Tolerance and Tumoral Immune Escape. Immunol Rev. 222, 206–221 (2008).
Munn, D. H. et al. Inhibition of T Cell Proliferation by Macrophage Tryptophan Catabolism. J Exp Med. 189, 1363–1372 (1999).
Seymour, R. L., Ganapathy, V., Mellor, A. L. & Munn, D. H. A High-Affinity, Tryptophan-Selective Amino Acid Transport System in Human Macrophages. J Leukocyte Biol. 80, 1320–1327 (2006).
Chiesa, M. D. et al. The Tryptophan Catabolite L-kynurenine Inhibits the Surface Expression of NKp46- and NKG2D-activating Receptors and Regulates NK-cell Function. Blood. 108, 4118–4125 (2006).
Astigiano, S. et al. Eosinophil Granulocytes Account for Indoleamine 2,3-Dioxygenase-Mediated Immune Escape in Human Non-Small Cell Lung Cancer. Neoplasia. 7, 390–396 (2005).
Ferdinande, L. et al. Clinicopathological Significance of Indoleamine 2,3-Dioxygenase 1 Expression in Colorectal Cancer. Brit J Cancer. 106, 141–147 (2011).
Brandacher, G. Prognostic Value of Indoleamine 2,3-Dioxygenase Expression in Colorectal Cancer: Effect On Tumor-Infiltrating T Cells. Clin Cancer Res. 12, 1144–1151 (2006).
Okamoto, A. Indoleamine 2,3-Dioxygenase Serves as a Marker of Poor Prognosis in Gene Expression Profiles of Serous Ovarian Cancer Cells. Clin Cancer Res. 11, 6030–6039 (2005).
Ino, K. et al. Indoleamine 2,3-Dioxygenase is a Novel Prognostic Indicator for Endometrial Cancer. Brit J Cancer. 95, 1555–1561 (2006).
Travers, M. T., Gow, I. F., Barber, M. C., Thomson, J. & Shennan, D. B. Indoleamine 2,3-Dioxygenase Activity and L-Tryptophan Transport in Human Breast Cancer Cells. BBA-Biomembranes. 1661, 106–112 (2004).
Folgiero, V. et al. Indoleamine 2,3-Dioxygenase 1 (IDO1) Activity in Leukemia Blasts Correlates with Poor Outcome in Childhood Acute Myeloid Leukemia. Oncotarget. 8, 2052–2064 (2014).
Cady, S. G. & Sono, M. 1-Methyl-DL-tryptophan, beta-(3-benzofuranyl)-DL-alanine (The Oxygen Analog of Tryptophan) and Beta-[3-Benzo(B)thienyl]-DL-alanine (The Sulfur Analog of Tryptophan) are Competitive Inhibitors for Indoleamine 2,3-Dioxygenase. Arch Biochem Biophys. 291, 326–333 (1991).
Muller, A. J., DuHadaway, J. B., Donover, P. S., Sutanto-Ward, E. & Prendergast, G. C. Inhibition of Indoleamine 2,3-Dioxygenase, an Immunoregulatory Target of the Cancer Suppression Gene Bin1, Potentiates Cancer Chemotherapy. Nat Med. 11, 312–319 (2005).
Larussa, T. et al. Enhanced Expression of Indoleamine 2,3-Dioxygenase in Helicobacter Pylori-Infected Human Gastric Mucosa Modulates Th1/Th2 Pathway and Interleukin 17 Production. Helicobacter. 1, 41–48 (2015).
Kim, J. W. et al. Prognostic Implications of Immunosuppressive Protein Expression in Tumors as Well as Immune Cell Infiltration within the Tumor Microenvironment in Gastric Cancer. Gastric Cancer, 10.1007/s10120-014-0440-5 (2014).
Zhang, R. P. et al. The Correlation Between the Subsets of Tumor Infiltrating Memory T Cells and the Expression of Indoleamine 2,3-Dioxygenase in Gastric Cancer. Digest Dis Sci. 58, 3494–3502 (2013).
Schreiber, R. D., Old, L. J. & Smyth, M. J. Cancer Immunoediting: Integrating Immunity’s Roles in Cancer Suppression and Promotion. Science. 331, 1565–1570 (2011).
Edinger, A. L. & Thompson, C. B. Antigen-Presenting Cells Control T Cell Proliferation by Regulating Amino Acid Availability. P Nalt Acad Sci USA 99, 1107–1109 (2002).
Fallarino, F. et al. The Combined Effects of Tryptophan Starvation and Tryptophan Catabolites Down-Regulate T Cell Receptor Zeta-Chain and Induce a Regulatory Phenotype in Naive T Cells. J Immunol. 176, 6752–6761 (2006).
Smith, C. et al. IDO is a Nodal Pathogenic Driver of Lung Cancer and Metastasis Development. Cancer Discov. 2, 722–735 (2012).
Francois, M., Romieu-Mourez, Li, M. & Galipeau, J. Human MSC Suppression Correlates with Cytokine Induction of Indoleamine 2,3-Dioxygenase and Bystander M2 Macrophage Differentiation. Mol Ther. 1, 187–195 (2012).
Zhang, H. et al. Infiltration of Diametrically Polarized Macrophages Predicts Overall Survival of Patients with Gastric Cancer After Surgical Resection. Gastric Cancer. 18, 740–50 (2014).
Camp, R. L., Dolled-Filhart, M. & Rimm, D. L. X-Tile: A New Bio-Informatics Tool for Biomarker Assessment and Outcome-Based Cut-Point Optimization. Clin Cancer Res. 10, 7252–7259 (2004).
Acknowledgements
This study was funded by grants from National Natural Science Foundation of China (31100629, 31270863, 31300671, 31470794, 81471621, 81472227), Program for New Century Excellent Talents in University (NCET-13-0146) and Shanghai Rising-Star Program (13QA1400300). All these study sponsors have no roles in the study design, in the collection, analysis and interpretation of data.
Author information
Authors and Affiliations
Contributions
H.L., Z.S. and J.X. carried out conception and design; Z.W., H.Z. and X.W. carried out analysis and interpretation of data; H.L., Z.W. and Z.S. carried out statistical analysis; H.L., H.Z. and X.W. prepared all figures and tables. H.L., Y.S. and J.X. carried out drafting of manuscript; Y.S., J.Q. and X.Q. carried out technical and material support. All authors reviewed the manuscript.
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Rights and permissions
This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
About this article
Cite this article
Liu, H., Shen, Z., Wang, Z. et al. Increased expression of IDO associates with poor postoperative clinical outcome of patients with gastric adenocarcinoma. Sci Rep 6, 21319 (2016). https://doi.org/10.1038/srep21319
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/srep21319
This article is cited by
-
Immunometabolism: a new dimension in immunotherapy resistance
Frontiers of Medicine (2023)
-
Microenvironmental influences on T cell immunity in cancer and inflammation
Cellular & Molecular Immunology (2022)
-
Up-regulation of DGAT1 in cancer tissues and tumor-infiltrating macrophages influenced survival of patients with gastric cancer
BMC Cancer (2021)
-
The cancer metabolic reprogramming and immune response
Molecular Cancer (2021)
-
The prognostic value of IDO expression in solid tumors: a systematic review and meta-analysis
BMC Cancer (2020)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.