Expression And Significance Of PD-1、PD-L1 And CMTM6 In Oral Squamous Cell Carcinoma

BackgroundOral squamous cell carcinoma（OSCC）,as the most common type of disease in oral and maxillofacial malignancies,is extremely harmful to patients’quality of life due to the invasion and metastasis.At present,although there are surgery,radiation therapy,chemotherapy and other methods for OSCC in clinical practice,the efficacy of patients with advanced cancer is still not satisfactory.In recent years,it was reported that immunotherapy brings hope to patients with OSCC and effectively improved the prognosis of patients.It was found that the inhibition of T cell function in tumor microenvironment plays an important role in tumor immune escape,and especially programmed death 1（PD-1）and its ligand（programmed cell death-ligand1,PD-L1）,as a pair of negative costimulatory molecules,are the important immunoregulators that affect T cell function.Clinically,the treatment of monoclonal antibody against PD-1/PD-L1 at the immunoassay points has made great breakthroughs in various tumor treatments,especially in the treatment of melanoma,lung cancer,kidney cancer and bladder cancer.However,with the large load,microenvironment changes in the patient’s body and immune tolerance in advanced tumors,single immunotherapy was insensitive,becausing of the body’s inability to effectively activate the immune response.Therefore,it is necessary to use combined immunotherapy to control disease and improve efficacy.The latest study reported that chemokine-like factor superfamily 6（CMTM6）,which co-localized on the plasma membrane and circulated nucleus with PD-L1,could enhance the expression of PD-L1 and strengthen the ability of tumors to suppress immune responses,in addition,the expression of PD-L1 and the ability of tumor-specific T cell activity would reduce lack of the elimination of CMTM6.CMTM6 is expected to become a new target for tumor combined immunotherapy,opening up the new avenues for immunotherapy.Until recently,there are relatively few reports about the expression of PD-1,PD-L1and CMTM6 in OSCC.Moreover,the correlation between CMTM6 and PD-1/PD-L1in OSCC has not been elucidated systematically.This paper aims to provide insight into the expression and biological significance of PD-1,PD-L1 and CMTM6 in OSCC by quantitative real time polymerase chain reaction（qRT-PCR）and Immunohistochemistry（IHC）,and further explore its pathogenic mechanism to provide a theoretical basis for the treatment of OSCC.ObjectiveIn this study,we detected the expression of PD-1,PD-L1 and CMTM6 from both protein and mRNA level in OSCC and normal oral mucosa tissues,and analyzed the relationship between three factors and clinical case parameters of patients with OSCC.The correlation between CMTM6andPD-1/PD-L1was analyzed and the pathogenic mechanism of OSCC was explored further,providing a theoretical basis for immunotherapy of OSCC.MethodsIn this experiment,66 cases of Formalin-Fixed and Paraffin-Embedded（FFPE）（44 cases of OSCC and 22 cases of normal oral mucosa tissue）were collected from the Department of Pathology inZhengzhou University Affiliated Tumor Hospital.The methods of quantitative real time polymerase chain reaction（qRT-PCR）and Immunohistochemistry（IHC）were used to detect the expression of PD-1,PD-L1 and CMTM6 from both mRNA and protein levels in OSCC and normaloral mucosa tissues.2^-△Ct method and semi-quantitative method were used to calculate the mRNA expression and the protein expression,respectively.The experimental results were analyzed by SPSS19.0 software.The Wilcoxon rank sum test was used to compare the quantitative data.The comparison between the groups of qualitative data was performed byχ2 test or Fisher exact probability method,and Spearman rank correlation was used for correlation analysis.P<0.05 was considered statistically significant.Results1.The expression of PD-1 mRNA in oral squamous cell carcinoma and normal oral mucosa tissues was 0.0008601±0.0000020,0.0000199±0.0000000.The expression of PD-L1 mRNA in oral squamous cell carcinoma and normal oral mucosa tissues was 0.0099626±0.0003928,0.0008427±0.0000024.The expression of CMTM6 mRNA in oral squamous cell carcinoma and normal oral mucosa tissues was0.0180891±0.00100000,0.0063536±0.00021324.There was a significantly statistical difference in three groups（P=0.035,P=0.041,P=0.033<0.05）.2.The expression of PD-1,PD-L1 and CMTM6 protein in oral squamous cell carcinoma:The expression of PD-1 protein was detected in the cytoplasm and membrane of lymphocytes.PD-L1 and CMTM6 proteins were mainly expressed on the cytoplasm and cell membrane of tumor cells,with the brownish yellow and brown.The positive rates of PD-1,PD-L1 and CMTM6 in tumor tissues were55%,66%,25%,and the positive rates in normal oral mucosa tissues were 23%,25%,0,respectively.The differences were statistically significant in three groups（P=0.014,P=0.002,P=0.010<0.05）.3.The correlation between PD-1,PD-L1 and CMTM6 protein and clinicopathological parameters in OSCC.The positive expression rates of PD-L1protein in stage I^II and III^IV stage with OSCC patients were 53.57%and 87.50%,respectively.There was a significantly statistical difference（P=0.022<0.05）.The positive expression rates of PD-L1 protein in lymph node metastasis（87.50%）with OSCC patients was higher than that in without lymph node metastasis group（53.57%）,and the difference was statistically significant（P=0.022<0.05）.However,there was no any correlation between the positive expression rates of PD-1 or CMTM6 and clinicopathological characteristics,including sex,age,smoking,drinking,tumor size,pathological grading,lymph node metastasis and clinical stage in OSCC（all P>0.05）.4.The correlation of CMTM6andPD-1/PD-L1 in OSCC showed that the expression rates of CMTM6 protein was positively correlated withPD-L1 protein（rs=0.510,P=0.000<0.05）and there was a positive correlation between the expression of PD-L1 mRNA and CMTM6 mRNA（rs=0.474,P=0.003<0.05）.There was no significant correlation between PD-1 and CMTM6（P>0.05）.5.The correlation between PD-1,PD-L1 and CMTM6 mRNA and PD-1,PD-L1and CMTM6 protein expression was analyzed in OSCC.The results of Speaerman test showed that the expression of PD-1 mRNA and protein,PD-L1 mRNA and protein,CMTM6 mRNA and protein were all positively correlated,respectively（r_s=0.756,P=0.030<0.05;rs=0.841,P=0.000<0.05;rs=00629,P=0.028<0.05）.Conclusions1.PD-1,PD-L1 and CMTM6 are involved in the immune escape mechanism of OSCC,which is closely related to the occurrence of oral squamous cell carcinoma.2.The expression level of PD-L1 is related to the severity and prognosis of patients with OSCC.3.CMTM6 might has a synergistic effect with PD-1/PD-L1 in regulating the immune response of tumor microenvironment,and CMTM6 may be activated or enhance the PD-1/PD-L1 signaling pathway and play a role in immunosuppression.4.CMTM6 is expected to be a new checkpoint for OSCC,and combined with PD-1/PD-L1 immunotherapy maybe improve clinical efficacy.