The Study Of The Tertiary Lymphoid Structures And Its Gene Signature In The TME Of Ovarian Cancer

BackgroundOvarian cancer(OC)is a secret killer of women’s health.Ovarian cancer has the lowest ovarian cancer rate.As the standard treatment for OC,comprehensive staging surgery supplemented by platinum-based chemotherapy has achieved certain clinical remission,but it still faces a series of intractable problems such as chemotherapy resistance,tumor recurrence and metastasis.In recent years,immunotherapy based on immune checkpoint inhibitors has brought revolutionary improvement for the precision therapy of various cancers.However,current clinical trials have shown that ovarian cancer has a limited response to immunotherapy.Although it has not achieved satisfactory expected results,clinical practice has provided a new research perspective and direction for the development of OC therapy.Such as tumor microenvironment(TME).The immunosuppressive properties of TME are involved in the occurrence of tumor immune escape,thus limiting the effect of immunotherapy.Therefore,systematic evaluation of the characteristics and function of immune cells in TME can explore new targets and markers,so as to stratified the treatment population and screen potential benefit groups.It was found that Tumor Infiltrating Lymphocyte(TIL)in the tumor microenvironment could effectively aggregate to form ecstatic Lymphoid Structures,namely Tertiary Lymphoid Structures(TLS).Published studies have shown that TLS is associated with better outcomes in cancer patients.However,there is insufficient research evidence to support the importance of TLS in the prognosis and treatment of OC.Purposes1.To verify the existence of TLS in OC from the organizational level,and analyze the structural performance of TLS.2.To explore the relationship between TLS and prognosis of OC patients.3.To establish the characteristics of TLS related genes in OC,and explore their immune landscape,prognostic significance and value in predicting immunotherapy.Methods1.Sample inclusion and data preprocessing(1)Experimental samples:60 patients who underwent surgical treatment in Qilu Hospital of Shandong University from 2014 to 2017 and were diagnosed as OC by postoperative pathology were included in this study.Clinical information of patients was collected and tissue sections were prepared.(2)Sample public database:Download The data and clinical information of OC patients by RNA sequencing(RNA-Seq)from the Human Cancer Genome Atlas Program(TCGA)database,and after normalization and screening in the R language software,362 patients with OC were included;In the GEO Series GSE140082 of the Gene Expression Omnibus(GEO)database,380 OC patients were screened and included.(3)Immunotherapy cohort:The IMvigor210 cohort consisted of patients with advanced urothelial carcinoma receiving atrilizumab treatment.348 patients were enrolled after screening,and RNA-seq data and clinical information of patients were downloaded using R language software.2.Analysis of histological features and prognostic significance of TLS:HE staining was performed on slices of 60 OC samples to preliminatively confirm the presence of TLS.At the same time,immunohistochemical staining was performed to label CD20+B cells,CD3+T cells and CD8+T cells to analyze the tissue characteristics of TLS,the composition and spatial location of TIL,and the relationship between TLS and clinicopathologic features was analyzed by chi-square test.COX regression analysis and Kaplan-Meier analysis were conducted to explore the relationship between TLS and Progression Free Survival(PFS)and Overall Survival(OS)in OC patients.3.Bioinformatics analysis of TLS gene characteristics:In this study,single-factor COX regression analysis was performed on TLS background gene set in the TCGA-OC training set,and genes associated with prognosis were screened(P<0.05),which were defined as TLS gene characteristics in OC,and the independent prognostic significance of these gene characteristics was verified by COX regression.CIBERSORT,ESTIMATE and TIDE algorithms were used to analyze the relationship between the gene characteristics and tumor immune invasion.The significance of this gene profile in predicting immunotherapy was initially determined using the IPS(Immunophenoscore)scoring system and validated in the IMvigor210 cohort.Finally,the relationship between this gene signature and existing immunotherapy markers(tumor mutation load and immune checkpoint genes)was explored.Results1.Analysis of histological features and prognostic significance of TLS(1)TLS exists in OC and has two phenotypes:mature type and immature type.In this study,HE staining and immunohistochemical staining were combined to define TLS as lymphocyte aggregates.The positive expression rate of TLS in OC tissues was 51.67%(31/60)and the negative expression rate was 48.33%(29/60).There was no significant difference in clinicopathological parameters between groups with and without TLS(P>0.05).TLS can be divided into immature type and mature type.Immature type of TLS is the accumulation of lymphocytes in HE staining showed irregular patchy distribution,and CD20+B cells and CD3+T cells were scattered and few in number.In mature TLS,HE staining showed clusters of densely distributed lymphocytes.CD20+B cells clustered to form circular or oval lymphoid follicular structures,and CD3+T cells formed bands around CD20+B cells.OC patients with TLS were divided into mature TLS(19/31,61.29%)and immature TLS(12/31,38.71%).The location of TLS in tumor interstitium of OC includes two types:tumor center and tumor margin,among which TLS in tumor margin is mostly mature type,while TLS in tumor center is mostly immature type.(2)TIL significantly increased in OC tissues with TLS,and the number of CD20+B lymphocytes was correlated with the number of CD3+T and CD8+T lymphocytes.TIL distribution was different between groups with and without TLS,and the number of CD20+B cells,CD3+T cells and CD8+T cells in the group with TLS was significantly higher than that in the group without TLS(P<0.001).In the TLS positive group,the proportion of CD20+B cells,CD3+T cells and CD8+T cells in the high expression group was significantly higher than that in the low expression group(P<0.001).Meanwhile,the number of CD3+T cells and CD8+T cells was significantly increased in the group with high CD20+B cell expression(P<0.001),and the number of CD8+T cells in the group with high CD3+T cell expression was significantly higher than that in the group with low CD3+T cell expression(P<0.001).(3)The existence of TLS can affect OS and PFS of OC patients.After Log-Rank test,multivariate COX regression analysis showed that the absence of TLS was an independent risk factor for OS and PFS(OS:HR=3.34,95%CI=1.67-6.69;P<0.001;PFS:HR=2.77,95%CI=1.48-5.18,P<0.001).Kaplan-Meier survival curve showed that the TLS group had better OS(P<0.05)and PFS(P<0.05).2.Bioinformatics analysis of TLS gene signature(1)The TLS gene signature associated with OC could be used as independent prognostic factors.In this study,after univariate COX regression analysis of 21 TLS background genes in the TCGA-OC training set,9 TLS genes associated with prognosis were screened out(P<0.05),which were defined as TLS gene signature associated with prognosis in OC.In TCGA-OC and GSE-OC,the OS and PFS of TLS gene in high expression group were significantly higher than those in low expression group(P<0.05).Single and multifactor COX analysis of TCGA-OC showed that TLS gene characteristics were significantly correlated with OS(P<0.05).Considering the lack of statistical significance of tumor stage in the TCGA-OC data for the prognosis of OC patients(P>0.05),the results of multi-factor COX analysis of the GSE-OC data as validation showed that TLS gene characteristics were still significantly correlated with OS(P<0.05).Therefore,TLS gene signature is an independent prognostic predictor for OC patients.(2)The expression of TLS gene signature was correlated with tumor immunoinfiltration.CIBERSORT analysis showed that the number of CD8+T cells,memory CD4+T cells and M1 macrophages in the high TLS group was significantly higher than that in the low TLS group(P<0.05).The ESTIMATE showed that the percentages of immune score,matrix score and estimate score in the high TLS group were significantly higher than those in the low TLS group(P<0.05),indicating a higher degree of immune infiltration in the high TLS group.The TIDE score in low TLS group was significantly higher than that in high TLS group(P<0.05),indicating the existence of tumor immune escape in low TLS group.(3)TLS gene signature can be used as a marker to predict the benefit of immunotherapy.The IPS-related scores of the high TLS group were higher than those of the low TLS group(P<0.05),indicating that the high expression TLS group may have a better response to immunotherapy.After applying this TLS gene signature to the IMvigor210 cohort,patients were assigned to the high(N=174)and low(N=174)TLS groups based on the median TLS score of the sample calculated by the ssGSEA algorithm.Kaplan-Meier survival analysis showed that OS in the high TLS group was significantly higher than that in the low TLS group(P<0.05),TLS scores of immunoinflammatory phenotypes were significantly higher than those of specific immune desert and immune exclusion phenotypes(P<0.001),and after receiving PD-L1 treatment,TLS scores in complete response and partial response were significantly higher than those in stable and progressive disease groups(P<0.05),confirming that TLS gene signature can be used as a marker to predict the benefit of immunotherapy.(4)TLS gene signature can be used in combination with existing immunotherapy markers.The mutation load calculation results of TCGA-OC showed that the proportion of high TMB in the high TLS expression group was significantly higher than that in the low TLS expression group(52%vs 48%),although there was no statistically significant difference in the number of TMB between the two groups(P=0.23).Compared with the low TLS group,The expression of programmed death 1(PD-L1)and cytotoxic T-lymphocyte associated protein 4(CTLA-4)in high TLS group was significantly increased(P<0.05).Conclusions1.TLS exists in OC and is a highly ordered lymphoid tissue structure in OC.2.Expression of TLS is correlated with the prognosis of OC patients.3.TLS gene signature can be used as prognostic markers and immunotherapy efficacy markers.Innovation and limitationInnovation1.The existence,structure and spatial location of TLS in OC tissues were proved by HE staining and immunohistochemical staining.2.The relationship between TLS and TIL and its influence on the prognosis of OC were explored.3.In the gene expression profile of public data,the gene signature of TLS associated with OC were determined,the identification procedure of TLS was simplified,and the predictive value of this gene characteristics on the prognosis and immunotherapy of OC was further explored.Limitation1.The sample size of the experiment is relatively small and it is a single-center study,so the sample size should be further expanded and the multi-center study should be carried out.2.Studies on the pathways and mechanisms related to TLS gene characteristics are lacking.3.The TLS gene signature established were derived from the OC public database,which lacked the verification of gene sequencing samples and immunotherapy cohort of OC patients from the research center.