Expression And Significance Of PD-1 And TIM-3 In Epithelial Ovarian Cancer

Ovarian cancer is the fifth most common cancer that causes death in women.Due to the late onset of symptoms,most patients were diagnosed as advanced(III-IV),and the 5-year relative survival rate was only 29 percent.Ascites usually accumulate in the abdominal cavity in patients with advanced diseases,promoting the spread of tumor cells in the abdominal cavity.Standard treatments include surgical resection of the tumor to reduce the size of the tumor,followed by platinum-based chemotherapy.Although this method can improve the sensitivity of tumor cells to radiotherapy,long-term chemotherapy will cause immunosuppression,increase peripheral tolerance,leading to the acquisition or production of drug resistance.Therefore,there is an urgent need to develop new treatments.Tumor immunotherapy is one of the development directions of tumor therapy.it refers to the method of controlling or eliminating tumor cells by activating or enhancing the anti-tumor immune response of the immune system.the purpose of immunotherapy is to develop combined methods.At the same time to enhance immunity and prevent local immunosuppression.The progress of immunotherapy provides a broad prospect for the treatment of ovarian tumors.Of which.Immune checkpoint is an inhibitory signaling pathway existing in the immune system.the intensity and persistence of immune response in peripheral tissue are regulated to prevent tissue damage and play a role in maintaining the tolerance of autoantigen.In recent years,it has been found that immune checkpointsplay an important role in tumor immune escape.Tumor immune escape refers to the phenomenon that tumor cells grow and metastasize through various mechanisms to escape the recognition and attack of the immune system.it is an important strategy for tumor survival and development.Programmed death ligand receptor-1(PD-1),T cell immunoglobulin mucin-3(TIM-3),indoleamine2,3-dioxygenase-1(IDO1),Lymphocyte activation gene-3(LAG-3)and cytotoxic T lymphocyte associated antigen 4(CTLA-4),They're called immune checkpoints,It can inhibit the activation of effector T lymphocytes and eventually lead to tumor immune escape.Human PD-1 is a type I transmembrane protein encoded by PDCD1 gene located on chromosome 2q37,which is composed of 288amino acid residues and belongs to immunoglobulin CD28 family.PD-1 protein is a type I transmembrane protein with molecular weight of 55-60 kDa.It is composed of extracellular IGV-like region,hydrophobic transmembrane region and intracellular region.There are two independent phosphorylation sites in the C-terminal and N-terminal amino acid residues of PD-1:immune receptor tyrosine inhibitory motif(ITIM)and immune receptor tyrosine switching motif(ITSM).PD-L1(B7-H1 or CD274)is the first PD-1ligand discovered,belonging to the B7 family,located on the human chromosome9p24.2.Its amino acid structure is similar to that of PD-1.When PD-1 binds to PD-L1,the ITSM region is phosphorylated,activating a series of intracellular signaling pathways,inhibiting the activation of immune cells,thereby reducing the secretion of antibodies and cytokines by immune cells,and even exhausting immune cells.To achieve effective immunosuppression.TIM-3,also known as HAVCR2,belongs to the TIM gene family.In humans,the TIM family,including TIM-1,TIM-3 and TIM-4,is located on chromosome5q33.2.They have similar structures:variable immunoglobulin domain(IGV),glycosylated mucin domain of different lengths of extracellular domain and single transmembrane region.in addition to TIM-4,all TIM molecules also contain a C-terminal cytoplasmic tail,GAL-9,a conserved tyrosine-based signal motif,is the first ligand to be discovered and is a sugar-binding protein.It can specifically recognize the structure of N-sugar chain in the variable region of TIM-3immunoglobulin.TIM-3 binds to Gal-9 to promote the influx of calcium ions into the intracellular region of Th1 cells,thus inducing apoptosis.At present,there are few joint studies on PD-1 and TIM-3 in epithelial ovarian cancer.Some studies have shown that PD-1 and TIM-3,are co-expressed on(TILs)of infiltrating lymphocytes in many other malignant tumors,such as gastric cancer,colorectal cancer,thyroid carcinoma and so on.Combined blocking of TIM-3 and PD-1 often produces better anti-tumor effect than blocking PD-1 alone.ObjectiveThe purpose of this study was to detect the positive expression rate of PD-1 and TIM-3 and the expression of mRNA in different ovarian tissues by immunohistochemistry and qRT-PCR method.So as to provide ideas and opportunities for PD-1 and TIM-3 as a combined target for anticancer therapy.Materials and methods1.Subjects:From September 2016 to March 2018,100 patients with ovariectomy were enrolled in the third affiliated Hospital of Zhengzhou University.,the age was(13-69)years old,with a median age 48 years old.Among them,30 cases of normal epithelial ovarian tissue(normal group),the specimens came from cervical cancer or other benign diseases for adnexal resection,postoperative pathology confirmed as normal ovarian tissue;There were 30 cases of benign epithelial ovarian tumors(benign group)and 40 cases of epithelial ovarian cancer(malignant group).All cases did not receive radiotherapy,chemotherapy,drugs and other treatment before operation,except for other organs of metastatic cancer,postoperative tissue specimens were confirmed by pathology.All the organizations were collected and approved by the Ethics Committee of the third affiliated Hospital of Zhengzhou University,and the informed consent of the patients was obtained.2.Methods:(1)the positive expression rates of PD-1 and TIM-3 protein in normal ovarian tissues,benign epithelial ovarian tumors and epithelial ovarian cancer tissues were detected by immunohistochemical SP method.(2)qRT-PCR method was used to detect the expression of PD-1 and TIM-3mRNA in normal ovarian tissues,benign epithelial ovarian tumors and epithelial ovarian cancer tissues.3.Statistical Methods:SPSS21.0 was used for statistical analysis.The measurement data of normal distribution are expressed as x±s,the measurement data of skewness distribution are represented by median,and the counting data are expressed as percentages(%).R×C?~2 test and Fisher exact probability method were used to analyze the positive expression rate of PD-1 and TIM-3 protein in three kinds of ovarian epithelial tissues and the relationship between their expression and different clinicopathological parameters.The expression of PD-1 and TIM-3 mRNA in ovarian tissues of normal group,benign group and malignant group were compared by one-way ANOVA,and Dunnett'T3 method was used for comparison between the two groups.The correlation between PD-1 and TIM-3 protein expression was analyzed by Spearman correlation analysis.the test level was?=0.05.Results1.Expression of PD-1 and TIM-3 protein in three groups of Ovarian tissues.The positive expression of PD-1 protein and TIM-3 protein were located in cell membrane and cytoplasm,and distributed in brownish yellow granules under microscope.The positive expression rates of PD-1 protein in normal group,benign group and malignant group were 6.7%(2/30),36.7%(11/30)and 75.0%(30/40),respectively.The positive expression rate of malignant group was significantly higher than that of normal group and benign group(P<0.001),and the positive expression rate of benign group was higher than that of normal group.The difference was statistically significant(P<0.001).The positive expression rates of TIM-3 protein in normal group,benign group and malignant group were 10%(3/30),43.3%(13/30)and 85.0(34/40),respectively.The positive expression rate of TIM-3 protein in malignant group was significantly higher than that in normal group and benign group.The difference was statistically significant(P<0.001).The positive expression rate in benign group was higher than that in normal group.The difference was statistically significant(P<0.001).2.Relationship between positive expression of PD-1 and TIM-3 protein and clinicopathological features of epithelial ovarian cancerThe positive expression rates of PD-1 protein in FIGO stage I-II and III-IV stage epithelial ovarian cancer were 50%(7/14)and 88.5%(23/26),respectively.with the increase of surgical and pathological stages,The positive expression rate of PD-1protein was significantly higher than that of control group,and there was significant difference between the two groups(P<0.05).The positive expression rate of PD-1protein was 88.9%(16/18)in epithelial ovarian carcinoma with lymph node metastasis confirmed by pathology after operation.The positive expression rate of PD-1 protein in epithelial ovarian carcinoma without lymph node metastasis was50.0%(6/12),the difference was statistically significant(P<0.05).However,the expression of PD-1 protein was not correlated with age,histological classification and degree of tissue differentiation(P>0.05).The positive expression rates of TIM-3 protein in well-differentiated epithelial ovarian carcinoma,moderately differentiated ovarian cancer and poorly differentiated epithelial ovarian carcinoma were 40.0%(2/5),83.3%(10/12)and 95.7%(22/23),respectively,the difference was statistically significant(P<0.05).The positive expression rate of TIM-3 protein was 100.0%(18/18)in epithelial ovarian carcinoma with lymph node metastasis confirmed by pathology after operation.The positive expression rate of TIM-3 protein in epithelial ovarian carcinoma without lymph node metastasis was 66.7%(8/12),the difference was statistically significant(P<0.05).However,the expression of TIM-3 protein was not correlated with age,histological classification and FIGO stage(P>0.05).3.Expression of PD-1 and TIM-3 mRNA in ovarian tissues of three groupsThe relative expression of PD-1 mRNA in normal ovarian tissues,benign epithelial ovarian tumors and epithelial ovarian tissues was 0.037±0.019,0.149±0.039 and 0.678±0.275,respectively.The relative expression of PD-1 mRNA in malignant group was significantly higher than that in benign group and normal group(P<0.05).The relative expression of PD-1 mRNA in benign group was higher than that in normal group,and the difference was statistically significant(P<0.05).The relative expression of TIM-3 mRNA in normal ovarian tissues,benign epithelial ovarian tumors and epithelial ovarian tissues was 0.037±0.020,0.179±0.076 and 0.665±0.271,respectively.The relative expression of TIM-3 mRNA in malignant group was significantly higher than that in benign group and normal group(P<0.05).The relative expression of PD-1 mRNA in benign group was higher than that in normal group,and the difference was statistically significant(P<0.05).4.Correlation between the expression of PD-1 and TIM-3 in epithelial ovarian cancer.Spearman correlation analysis showed that there was a positive correlation between the expression of PD-1 protein and TIM-3 protein in epithelial ovarian tissues(r=0.566,P<0.001).Conclusions1.The positive expression of PD-1 and TIM-3 protein and the expression level of mRNA in epithelial ovarian carcinoma were significantly higher than those in normal ovarian epithelial tissue and benign epithelial ovarian tumor tissue.2.The expression level of PD-1 protein can predict the clinicopathological stage and lymph node metastasis in epithelial ovarian cancer,and the expression level of TIM-3 protein can predict the degree of tissue differentiation and lymph node metastasis.3.The expression of PD-1 and TIM-3 is positively correlated in epithelial ovarian cancer,which may be involved in the occurrence and development of epithelial ovarian cancer.