The Genome Variations And Immune Landscape Of Cervical Cancer

Part 1 A single-cell atlas of the immune ecosystem of cervical cancerObjective:To explore the differences of tumor immune microenvironment in different pathological types of cervical cancer and to find potential immunotherapeutic targets.Methods:78 cervical squamous carcinoma(SC)and 31 adenocarcinoma(AC)patients were enrolled in this study.Fresh tumors and adjacent tissues were digested into single cell suspension for CyTOF test together with paired PBMC samples.The phenotypes of immune cells were described by means of clustering,and the frequency of different immune cell subsets in different pathological types of cervical cancer were compared.Multiplex immunohistochemical staining(m IHC)was used to reveal the infiltration and spatial distribution of immune cells.Results:Cervical cancer patients had higher CD4~+effector memory T cells and lower CD4~+na(?)ve T cells in PBMC than healthy donors.Compared with adjacent tissues,the infiltration of CD4~+/CD8~+central memory T cells and CD8~+effector memory T cells were higher in tumor,while the infiltration of CD4~+/CD8~+terminal differentiated effector memory T cells were lower in tumor.T cells in tumor tissues exhibited a more exhausted phenotype than PBMC and adjacent tissues,and CD39~+T cells were only infiltrated in tumors.CD4~+/CD8~+CD39~-tumor resident memory cells were positively correlated with later FIGO stage,lymph node metastasis and vascular invasion.High expression of CD8~+CD39~+were correlated with better survival in cervical cancer patients.Cervical adenocarcinoma patients had lower frequency of CD8~+effector memory T cells in PBMC and lower CD4~+effector memory T cells as well as higher CD4~+central memory T cells in tumor tissue,compared with cervical squamous carcinoma.Generally,cervical adenocarcinoma had lower infiltration of CD45~+immune cells,especially lower infiltration of CD3~+T cells and NKp46~+NK cells,while the infiltration of tumor associated neutrophils,CD206~+macrophages and CD57~+NK cells were higher than that of squamous carcinoma.Conclusion:The characteristics of immune cells in the peripheral blood and tissues of patients with cervical cancer were quite different.The infiltration of immune cells in cervical adenocarcinoma was lower than that in squamous carcinoma,with lower infiltration of T cells and higher inhibitory immune cells.Inhibitory immune cells may be a potential immunotherapy target.Part 2 The genetic and immune microenvironmental characteristics of recurrent cervical cancerObjective: This study aimed to explore the intrinsic driving factor of cervical cancer recurrence,illuminate the correlation between genomic variation and immune cell infiltration,identify key factors affecting cervical cancer recurrence,and construct a predicting model to predict the recurrence of cervical cancer.Methods: 1.77 recurrent and 31 non-recurrent cervical cancer patients were enrolled in this study.DNAs were extracted from FFPE samples of primary tumors and paired adjacent normal tissues,and then sequenced by whole exome sequencing.Driver gene mutations and TMB(tumor mutation burden)were compared between these two groups.The correlation between genomic variation and immune cell infiltration were explored.2.The density and distribution of different immune cells in tumors from recurrent and nonrecurrent cervical cancer patients were examined by m IHC(multiplex immunohistochemistry assay)and then compared between the two groups.The relationships between different immune factors and the prognosis of cervical cancer patients were determined thereafter.3.Risk factors of cervical cancer recurrence were identified by survival analysis,and a model was established to predict the recurrence of cervical cancer patients.Results: WES analysis demonstrated that the C > T mutation was the main mutation type of cervical cancers in our cohort.EP300 mutation frequency was higher in recurrent patients than nonrecurrent patients(24% vs 4%,P < 0.05),whereas FAT1 mutation frequency was higher in non-recurrent patients(18% vs 4%,P < 0.05).The TMB of non-recurrent patients was higher than that of recurrent patients(8.42 vs 5.81 muts/Mb,P < 0.05).High tumor mutation burdens were positively correlated with the mutation of KMT2C/KMT2D/EP300/ARID1A/FAT1/NOTCH1/CASP8/NFE2L2.m IHC assays showed that PD-L1 expression was higher in recurrent patients than non-recurrent patients.Patients with higher TMB had higher infiltration of CD4~+/CD56~+/Treg cells.Driver gene mutations were correlated with the infiltration of immune cells,for example,patients with EP300 mutation had higher infiltration of CD20~+/CD4~+/CD8~+/CD8~+PD-1~+/Treg cells.High TMB,higher infiltration of CD56~+ NK cells and CD8~+ T cells were correlated with better survival.Patients with high TMB and high CD8~+ T cells had better disease-free survival.EP300 mutant combined with low CD8~+ T cells infiltration were predictors of cervical cancer recurrence and low overall survival rate.Conclusion: The gene mutation and tumor immune microenvironment are markedly different between recurrent and non-recurrent cervical cancer patients.The recurrence of cervical cancers is likely the results of the combinatorial effects of intrinsic driver gene mutation and extrinsic immune microenvironment.Targeting driver genes and immune cells simultaneously may provide a new avenue for the treatment of patients with recurrent cervical cancer.