A Study On GITR Expression In Common Malignancies And Its Relationship With Clinicalpathological Features

BackgroundGITR (glucocorticoid-induced tumor necrosis factor receptor family-related protein) is commonly expressed on the surface on regulatory T cells (Treg) and activated CD4(+) and CD8(+) T cells, providing co-stimulatory signals for T cell activation. It is very important for immunomodulation. As applied in animal tumor models, including lymphoma, melanoma, colon cancer, fibrosarcoma, anti-GITR has been demonstrated to be able to stimulate immune system to suppress tumor more efficiently. These findings are highly-valued for tumor immunotherapy. However, GITR’s expression in tumor bearing bodies especially in tumor regional lymph nodes and tumor tissues has not been investigated systematically, and its correlations with tumor clinicalpathological features and prognosis have neither been revealed.PurposeTo identify the GITR expression in common tumor regional lymph node and tumor tissues, and their relationship with clinicalpathological characters. The possible roles of GITR positive cells in tumor and its implication for immunotherapy will also be discussed, in the hope to provide evidences for clinical application of anti-GITR.Methods1. All the cases of common tumors were retrieved from the files of the Department of Pathology Cancer Hospital, Fudan University between January2007and December2010. In the first part research, we collected129cases of common tumor regional lymph nodes in2010, including gastric cancer (45cases), breast cancer (18cases), ovarian cancer(11cases), prostate cancer(10cases), colon cancer (13cases), lung cancer(12cases), melanoma(10cases) and renal cancer(10cases). In the second part, we collected102cases of common tumor tissues in2010, including gastric cancer (40cases), breast cancer(8cases), ovarian cancer(11cases), prostate cancer (6cases), colon cancer(9cases), lung cancer(12cases), melanoma (6cases) and renal cancer (10cases), and another139cases of colorectal cancer between January2007and December2008. All the histological slides were reviewed for confirmation of the pathologic diagnosis by professional pathologists. And the clinicalpathological characters were collected, including gender, age, tumor size, pathologic diagnosis, differentiation of tumor tissue, number of regional lymph nodes with tumor metastasis, number of all the regional lymph nodes, presence of metastasis in vessels, presence of neural invasion and clinical stages of each case.2. Using immunohistochemistry, GITR protein was detected by EnVision two steps in formalin-fixed, paraffin-embedded tissues as needed. The139cases of colorectal cancer tissues were detected additionally for CD8expression also by EnVision two steps. And then the slides were observed under microscope, Olympus BX51and recorded in pictures by the matched camera DP25for further calculating. GITR protein detected slides were observed and taken pictures to record the5highest GITR expression fields under200X microscope magnification, the average GITR positive cells of which equaled the score of this slide for GITR expression. CD8protein detected slides were taken the10highest CD8expression fields under100X microscope magnification. Image pro-plus was used for calculating CD8positive cells in each picture, after which the number was divided by100. Summation of the10pictures’numbers within each case was recorded as the score of CD8expression for this case.3. Statistical analyses were done by SPSS19, using t-test, Pearson correlation and Kaplan-Meier Survival Analysis. Significance was determined when p<0.05.Results1GITR expression was observed in all kinds of tumor’s regional lymph nodes, but the expression level varied between tumor kinds and cases of one kind of tumor. Confined by cases in this research, regional lymph nodes from lung cancer expressed highest GITR score (97.76), but those from breast cancer expressed the lowest score (31.78). Several correlations were found between GITR scores with clinicalpathological features. In gastric cancer, tumor regional lymph nodes with presence of neural invasion had higher GITR scores than those tumors without presence of neural invasion (p=0.049); Poor differentiation of tumor tissue was correlated with lower GITR score in regional lymph nodes (p=0.0482). In breast cancer, cases with more total retrieved lymph nodes had higher GITR scores in tumor regional lymph nodes (p=0.0502). In lung cancer and ovarian cancer, those lymph nodes with metastasis in them had more GITR positive cells than those without metastasis (p<0.05). In melanoma, younger age was associated with better GITR expression score in tumor regional lymph nodes (p=0.0385). Except all the results presented above, we didn’t find any more correlations between clinicalpathological characters and GITR expression in tumor regional lymph nodes.2GITR expression was also observed in all kind of tumor tissues. There was no relationship between GITR expression scores in tumor tissues and clinicalpathological features, except that in the139cases of colorectal cancer, more total lymph nodes retrieving was connected with more GITR positive cells in tumor tissues. In the additional139cases of colorectal cancer, more GITR positive cells in tumor tissues was associated with more CD8positive cells(p=0.000) and was also a factor linked with better prognosis:30patients with recurrence or tumor metastasis survived without disease after first operation for between2.5months and47.5months, and higher GITR scores correlated with longer disease free survival(p=0.004);16cases in death group had lower GITR scores than those46cases in living group(p=0.024), and by Kaplan-Meier Survival Analysis, patients with lower GITR expression scores than the mean level of139cases had poorer outcome(p=0.01).Conclusions1GITR expression can be observed in tumor tissues and tumor regional lymph nodes from8kinds of common malignancies, including gastric cancer, colorectal cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, melanoma, renal cancer, which provides solid evidence for future application of anti-GITR immunotherapy.2GITR expression in tumor regional lymph nodes correlated with some clinicalpathological features of tumor, such as tumor invasion (presence of metastasis or neural invasion), age, which show that GITR expression is related with T cell immune response. However, to explore deeper about this correlation, more cases would be need in further study.3In colorectal cancer, higher GITR expression was correlated with higher CD8expression. More GITR positive cells infiltrated in tumor sites of colorectal cancer are related with longer disease free survival before metastasis or recurrence and better chance to survive, so it can be considered as another cancer prognostic factor in the family of immune system.