Correlation Between Clinical Features And HPV Specific Immune Response In Advanced Cervical Squamous Cell Carcinoma

Objective: 1)To analyze the clinical and pathological characteristics of patients with advanced cervical squamous cell carcinoma,to compare the clinical and pathological differences between HPV positive and negative patients,and to explore the relationship between HPV infection and lymph node metastasis and prognostic characteristics.2)To analyze the correlation between the frequency and intensity of HPV E1 and E2 protein antigen polypeptide specific CTL immune response and clinical features and prognosis.3)To compare the expression of CTLA-4,PD-1 and Tim-3 on CD4+T cells and CD8+T cells in peripheral blood and cancer tissues,and to analyze the co-expression of PD-1 and Tim-3.To investigate the changes of CTLA-4,PD-1 and Tim-3 expression in peripheral blood of patients with advanced cervical cancer during radiotherapy.Methods: 1)One hundred and ninety-four patients with advanced cervical squamous cell carcinoma who were hospitalized in the affiliated Cancer Hospital of Xinjiang Medical University from January 2014 to December 2017 were collected.The clinical and pathological data and follow-up of the patients were collected.2)In the first part of the study,66 patients with advanced cervical squamous cell carcinoma infected with HPV16 and 60 HPV16 negative healthy controls were selected.Peripheral venous blood of 15 mL was collected from the subjects.Specific immune response level of peripheral T cells in peripheral blood mononuclear cells(PBMC)of patients and control subjects to E1 and E2 protein antigen peptide of HPV expression were measured by using ELISPOT assay.3)Sixty-three patients with advanced cervical squamous cell carcinoma infected with HPV16 were included in the study.Peripheral venous blood 15 mL was collected before treatment,during treatment and at the end of treatment.The cervical cancer tissues of patients before treatment were collected.The expression of CTLA-4,PD-1 and Tim-3 on CD4+ and CD8+T cells in peripheral blood and cancer tissues was detected by multicolor flow cytometry.Results: 1)HPV was detected in 155 of 194 patients with advanced cervical squamous cell carcinoma,HPV was negative in 39 patients,lymph node metastasis was found in 85 patients,and no lymph node metastasis was found in 109 patients.There was significant difference in SCC-Ag and lymph node metastasis between HPV positive and negative patients(P<0.05).The expression of SCC-Ag in HPV positive group was higher than that in HVP negative group,and the diameter of lymph node in HPV positive group was higher than that in HPV negative group.The positive rate of HPV was 87.71% in the first station,78.57% in the second station,100% in the third station,and the positive rate of HPV was the highest in the third station.There was significant difference in PFS and OS between HPV positive and negative patients.There was significant difference in PFS and OS between HPV positive patients with lymph node metastasis and patients without lymph node metastasis(P<0.05).2)The frequency of CTL immunoreactivity of HPV16 E1 and E2 antigen peptides in patients with cervical squamous cell carcinoma was significantly higher than that in healthy controls(P<0.05).The CTL immunoreactive intensity of HPV16 E1 antigen peptides in patients with cervical squamous cell carcinoma was higher than that in healthy controls.There was significant difference in the frequency of HPV E1 specific CTL reaction among tumor stage,TSGF and lymph node metastasis(P≤0.05).Univariate analysis showed that age was the influencing factor of PFS,HPV16 E1 reaction was the influencing factor of PFS and OS.Multivariate analysis showed that age and lymph node metastasis were the important factors affecting PFS,and HPV16 E1 reaction was the important influencing factor of PFS and OS.According to the survival curve,PFS and OS in HPV16 E1 positive group were better than those in HPV E1 negative group.3)The expression of CD8+CTLA-4+T cells were highest in peripheral blood of cervical cancer,and the expression of CD8+PD-1+T cells were highest in cervical cancer tissues.The expression of PD-1 and Tim-3 on CD4+ and CD8+T cells in cervical carcinoma were higher than that in peripheral blood.The expression of CTLA-4 in CD8+T cells in cervical peripheral blood was significantly higher than that in cancer tissues(P<0.05).The expression of CD8+CTLA-4+T cells in cervical peripheral blood were significantly higher than that in cancer tissues(P<0.05).Correlation analysis showed that the expression of PD-1 and Tim-3 on CD4+,CD8+T cells in peripheral blood increased with the increase of expression in tissues.There was significant difference in the pathological grade of CD4+CTLA-4+T cells in peripheral blood of patients(P<0.05).There were significant differences in BMI,FIGO stage and lymph node metastasis of CD4+Tim-3+T cells in peripheral blood(P<0.05).There was significant differences in pathological grade of CD8+CTLA-4+T cells in peripheral blood(P<0.05).There was significant difference in tumor size grade of CD8+PD-1+T cells in peripheral blood of the patients(P<0.05).There were significant difference in BMI,FIGO stage grade of CD8+Tim-3+T cells in peripheral blood of the patients(P<0.05).There were significant difference in BMI,FIGO stage grade of CD4+Tim-3+T cells in cancer tissue of the patients(P < 0.05).There was significant difference in tumor size stage grade of CD8+PD-1+T cells in cancer tissue of the patients(P<0.05).There was significant difference in pathological and tumor size stage grade of CD8+Tim-3+T cells in cancer tissue of the patients(P<0.05).The expression of PD-1+Tim-3-was the highest in CD4+ and CD8+T cells in peripheral blood and tissues of the patients.It was found that the expression of PD-1+Tim-3+,PD-1+Tim-3-and PD-1-Tim-3+T cells in tissues was higher than that in peripheral blood.There was significant difference in the expression of CTLA-4,PD-1 and Tim-3 immune checkpoint molecules on CD4+T cells between before and at the end of treatment(P<0.05).Conclusion: 1)HPV positive patients with advanced cervical squamous cell carcinoma have higher SCC-Ag index,more prone to lymph node metastasis and poor prognosis.Therefore,it is necessary to improve the anti-tumor ability of patients with cervical cancer infected with HPV.2)HPV16 E1 and E2 specific T cell immune responses were detected in peripheral blood of patients with advanced cervical squamous cell carcinoma,which were higher than those of healthy controls.The intensity of HPV E1 specific T cell immune response was correlated with FIGO stage,TSGF and lymph node metastasis.The PFS and OS of patients with HPV16 E1 specific T cell negative reaction were worse,suggesting that HPV16 E1 protein can induce T cell immune response in patients with cervical squamous cell carcinoma,which may be a new immune target for the treatment of cervical cancer.3)The expression of CTLA-4,PD-1 and Tim-3 molecules in CD4+,CD8+T cells was detected in peripheral blood and cancer tissues of patients with cervical squamous cell carcinoma.The expression of PD-1 and Tim-3 in CD4+ and CD8+T cells was higher than that in peripheral blood,and the expression of PD-1 and Tim-3 in CD4+,CD8+T cells in peripheral blood increased with the increase of cancer tissue.The immune checkpoint molecules expressed by CD4+ and CD8+T cells were associated with BMI,pathological grade,clinical stage,tumor size and lymph node metastasis.The co-expression of PD-1 and Tim-3 in CD4+,CD8+T cells was found in peripheral blood and cancer tissues of the patients.Radiotherapy can cause changes in the immune system of patients,and the expression of immune checkpoint molecules in CD4+T cells increases gradually during radiotherapy.This study provides guidance for the use of combined immunocheckpoint inhibitors for cervical cancer.