| Objective: 1)The specific T cell immune response stimulated by HPV16 E6 and E7 antigenic peptides in peripheral blood of patients with cervical squamous cell carcinoma was detected by flow cytometry,and the expression patterns of IFN-γ,IL-2,TNF-α and IL-13 secreted by CD4+T cell subsets and CD8+T cell subsets were compared,and the T cell function was evaluated.2)To analyze the expression of PD-1 on T cells in peripheral blood and cancer tissues of patients with cervical squamous cell carcinoma,and to confirm the correlation between PD-1/PD-L1 and clinical features and prognosis combined with the expression of PD-L1 in tumor tissues.3)Combined with the first two parts,the second-generation sequencing technology was used to analyze the mutation differential genes in different groups,including whether to produce specific immune response and different PD-L1 status.Then,we analyze the correlation between TMB level and mutation types and clinical characteristics,and to predict new antigens in patients with possible immune drug resistance,so as to provide theoretical basis for predicting immunotherapy benefits and individualized or combined immunotherapy in patients with cervical squamous cell carcinoma.Methods: 1)87 patients with cervical cancer treated in the third Clinical Medical College of Xinjiang Medical University from September 2017 to November 2020 were enrolled in this group.The peripheral blood of the patients before treatment was extracted and PBMCs was isolated for intracellular cytokine staining induced by HPV16 E6 and E7 antigen peptides in vitro.Then,the clinicopathological features of the patients were collected.2)95 patients with advanced cervical cancer who were treated in the third Clinical Medical College of Xinjiang Medical University from September 2017 to October 2019 were enrolled in the group.The peripheral blood and tissue of cervical cancer were collected before treatment,and the PBMCs in peripheral blood and TILs in tissue were isolated at the same time.In the later stage of the study,the pathological paraffin sections of the patients were arranged and prepared.Flow cytometry was used to detect the expression of PD-1 on CD4+ and CD8+T cells in peripheral blood and cancer tissues.Detection of PD-L1 expression in cancer tissues by immunohistochemical staining.Then the prognosis was analyzed combined with follow-up.3)A total of 45 patients who were enrolled in the previous group were selected for NGS sequencing.According to the results of the first two parts,the subgroup analysis was conducted to explore the correlation between mutant differential genes and TMB and clinical features.Results: 1)In HPV16 E6 stimulated group,the expression of IFN-γ,IL-2 and TNF-α in CD4+T cell subset in peripheral blood of cervical squamous cell carcinoma patients was higher than unstimulated group,and the expression of IL-2 and IL-13 in CD8+T cell subset was higher than unstimulated group,the differences were statistically significant,respectively(P<0.05).In HPV16 E7 stimulated group,the expression of IL-2 and TNF-α in CD4+T cell subset in peripheral blood of cervical squamous cell carcinoma patients was higher than unstimulated group,and the expression of IL-2 and IL-13 in CD8+T cell subset was higher than unstimulated group,the differences were statistically significant,respectively(P<0.05).In the CD4+T cell specific immune response positive group,more than half of the patients had multiple cytokine secretion and the co-expression frequency of double cytokines was better than that in the immune response negative group.Besides,i MFI analysis showed that the more kinds of co-expressed cytokines,the stronger intensity of specific immune response.In addition,the specific immune response of HPV16 E6 was related to tumor size and pelvic lymph nodes,the differences were statistically significant,respectively(P<0.05).2)The PD-1 expression of CD4+ and CD8+T cells in 95 patients with cervical squamous cell carcinoma was significantly higher than that in peripheral blood(P<0.05).Combined with the clinical features,it was found that the expression of CD4+PD-1+ in cancer tissue was related to the tumor diameter,and the expression of PD-L1 was related to the stage of the patient.the difference was statistically significant(P<0.05).In addition,the expression of TILs CD4+PD-1+ and CD8+PD-1+ in PD-L1 positive group was significantly higher than that in PD-L1 negative group(P<0.05).The results of survival analysis showed that the prognosis of patients with cervical squamous cell carcinoma with positive expression of PD-L1 was poor.Subgroup analysis showed that the prognosis of the group with high expression of CD8+PD-1+T cells in TILs was worse,the difference was statistically significant(P<0.05).COX regression analysis showed that simultaneous radiotherapy and chemotherapy(HR=0.161 95%CI=0.046-0.560P=0.004)and positive PD-L1(HR=3.364 95%CI=1.180-9.589 P=0.023)were independent prognostic factors in patients with cervical squamous cell carcinoma.3)Comparing 14 ICS test positive patients with 12 negative patients,somatic SNV found that 27% of the patients had FAT1 gene mutation.The mutation rates in the positive group and the negative group were 42.86% and 8.33%,respectively,the difference was statistically significant(P=0.044).The expression of CNV differential genes occurred in the negative group of specific immune reactions.Compared with the positive group,the incidence of CNV of gene MYC,DDR1 and MDC1 was 25%(3/12),8.3%(1/12),and8.3%(1/12),and the differences were statistically significant,respectively(P<0.05).Comparing 22 PD-L1 positive patients with 19 negative patients,somatic SNV found that44%(18/41)had PI3 KCA gene mutation,and 22%(9/41)patients had FAT1 gene mutation.The mutation of KMT2 D gene was found in 12%(5/41)of patients,and the mutation of DNMT3 B gene was found in 10%(4/41)of patients,and the differences were statistically significant,respectively(P<0.05).The differential expression of CNV genes all occurred in PD-L1 positive group.Compared with the negative group,the incidence of CNV of gene CD274,JAK2 and PDCD1LG2 was 13.6%(3/22),9.1%(2/22)and 9.1%(2/22),respectively(P<0.05).The value of TMB in HPV positive patients was higher than that in HPV negative patients,and the difference was statistically significant(P=0.032).The value of TMB in FAT1 mutation group was higher than that in non-mutation group,and the difference was statistically significant(P=0.018).The second-generation sequencing revealed the mutation of the drug resistance gene in 4patients with immunotherapy,and the new antigen was predicted and analyzed based on the results of HLA and somatic SNV.HLA typing HLA-A*02: 01 and HLA-A*30: 01 correspond to more new tumor antigens.Conclusion: 1)The CD4+T cell subsets in E6 and E7 stimulated groups of 87 patients with cervical squamous cell carcinoma more reflected the function of Th1 helper cells and secreted more IL-2,IFN-γ and TNF-αcytokines.In the CD4+T cell specific immune response positive group,more than half of the patients had multiple cytokine secretion and the co-expression frequency of double cytokines was better than that in the immune response negative group.Besides,i MFI analysis showed that the more kinds of co-expressed cytokines,the stronger intensity of specific immune response.In addition,the specific immune response of HPV16 E6 was related to tumor size and pelvic lymph nodes.2)The PD-1 expression of CD4+ and CD8+T cells in TILs was higher than that in PBMCs in 95 patients with cervical squamous cell carcinoma.In addition,the expression of TILs CD4+PD-1+ and CD8+PD-1+ in PD-L1 positive group was higher than that in PD-L1 negative group.Survival analysis showed that the prognosis of patients with cervical squamous cell carcinoma with PD-L1 positive expression was poor,and the prognosis of patients with high expression of CD8+PD-1+T cells in TILs was even worse.COX regression analysis showed that simultaneous radiotherapy and chemotherapy and positive PD-L1 were independent prognostic factors in patients with cervical squamous cell carcinoma.3)There was mutant differential gene FAT1,both in HPV antigen specific immune reaction positive group and PD-L1 positive group.Meanwhile,the TMB value in FAT1 mutation group was higher than that in non-mutation group.In addition,the expression of TMB in HPV positive group was higher than that in HPV negative group.HLA typing of HLA-A*02: 01 and HLA-A*30: 01 in 4 patients with drug resistance gene mutations in immunotherapy corresponded to more new tumor antigens. |
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