The Effect Of Ectopic Lymphoid Tissue On The Prognosis Of Endometrial Cancer And Its Related Mechanism

Research backgroundEndometrial cancer(EC)ranks second among gynecologic malignant tumor in China.According to statistics of the National Cancer Center,the number of EC in China was 63,400 and 21,800 deaths in 2015[1].According to etiology,EC can be divided into two types.Among them,type ? is estrogen-dependent well differentiated endometrioid cancer,which mostly comes from benign endometrial lesions and atypical endometrial hyperplasia(AEH).It accounts for about 80%of EC,with a good prognosis.Type ? is non-estrogen-dependent EC,also known as EC with poor histology.It mainly includes serous carcinoma,clear cell carcinoma,carcinosarcoma,undifferentiated carcinoma,and poorly differentiated endometrioid cancer.It accounts for about 10-20%of endometrioid cancer,and the prognosis is poor,the precursor lesions of which are unknown[2].Most patients with EC are diagnosed in the early stage.About 68%of the lesions are confined to the uterus,20%have local or lymph node metastasis,and 8%have distant metastasis.According to statistics,the five-year survival rate of patients in FIGO stage ? is 80-90%,that of stage ? is 70-80%,and that of patients with stage ?C-? is only about 20%.The standard treatment for EC is a comprehensive staging operation including total uterus+bilateral adnexectomy.For patients with high prognostic factors,postoperative adjuvant radiotherapy or chemotherapy is used.How to improve the prognosis of advanced patients is the current difficulty in the treatment of EC[3].In recent years,tumor immunotherapy has developed rapidly.In 2013,cancer immunotherapy was ranked first in Top 10 global breakthroughs by scientific journals.Nature and Science have successively published tumor immunotherapy special issues.EC has shown good therapeutic response to immune targeted therapy,and the exploration of related targeted biomarkers has become a hot research field[4].The host's immune system is the main environment for antitumor immunotherapy.The accumulation of tumor immune cells for resisting tumors is an important part of tumor genetics[5].The synergistic effect of tumor cells and immune cells constitutes the tumor microenvironment(TME),which further controls the occurrence and metastasis of tumors.On the one hand,the immune system recognizes and kills tumor cells through the normal immune system.On the other hand,tumor cells escape the body's immune surveillance and form immunosuppression by multiple ways.Immune cells provide a microenvironment for developing inflammation and tumor metastasis[6-8].Therefore,the basic strategy of tumor immunotherapy is always focused on how to avoid impaired immune surveillance and how to prevent damage to the immune system.T cell-mediated cellular immunity is the main immune response.Anti-PD-1 and anti-PD-L1 are used as immune checkpoint inhibitors to regulate the function of T cells.PD-L1 on the tumor was prevented from binding to PD-1 of effector T cells,thus relieving the inhibitory effect of tumor cells on T cells and upregulating the killing function of T cells[9-11].Results of the clinical trial showed that Pembrolizumab(anti-PD-1)was used to treat advanced or recurrent EC in patients with MSI-H and DMMR,with a 57%response rate,which indicated that a promising survival rate for advanced EC[12]Ectopic lymphoid tissue,also known as tertiary lymphoid structure(TLS),is an ectopic lymphoid organ formed with chronic inflammation in non-lymphoid tissues,mainly composed of B lymphocytes[13].Its typical structural feature is the presence of a large number of aggregated CD20+B cells clusters in the inflammatory tissue[14].The studies have found that this structure also exists in a variety of solid tumors,such as breast cancer,lung cancer,and colorectal cancer,and is related to prognostic benefits,which has attracted widespread attention in the field of tumors[15-17].The TLS serves as an effective site for tumor reactions in TME[18].The up-regulation of TLS may cause a significant anti-tumor response,which is related to the beneficial survival.It is reported that the anti-tumor response of TLS may be related to B cells and its pathways.As an important part of the human immune response,B cells are responsible for the production and release of antibodies against specific antigens,and participate in antibody-mediated immune responses.As an enrichment area of B cells in tumor tissue,TLS triggers an inflammatory response through B cells or other TILs,triggering effective sites in downstream pathways,thus exerts effective anti-tumor biological effects[19,20].However,in the current field of gynecological oncology[21],no literature reported TLS in EC.TLS is an effective site of B cell pathways,and its related molecular pathways have certain biomarkers.The TLS and related B cell pathways may become new antitumor targets after the T cell therapy revolution.In this study,the EC patient database of Peking Union Medical College Hospital(PUMCH)and Kyoto University Hospital(KHU)were respectively used to observe the expression of TLS in EC.The structure of TLS was explored and verified by immunohistochemistry(IHC)and immunofluorescence(IF)methods.After that,we used DNA panel and RNA-seq skills to explore B cell and related pathways.The relationship between TLS and ECGA molecular class classification was futher researched.Research method1.The research on composition of TLS in EC and its relationship with prognosisThe clinical data of EC patients in the Department of Obstetrics and Gynecology of KHU were retrospectively collected.The expression of TLS structure in tumor tissues were observed by HE staining and IHC.We explored the relationship between TLS and the prognosis of EC patients.2.The research on TLS expression verification and protein pathway research in EC(1)The clinical information and pathological characteristics of EC patients from 2010 to 2014 in single operation team in the Department of Obstetrics and Gynecology of PUMCH were retrospectively collected,who were diagnosed by biopsy pathology and underwent hysterectomy.Patients with any of the following characteristics were excluded:receiving neoadjuvant chemotherapy or neoadjuvant radiotherapy;and rare pathological types other than endometrioid adenocarcinoma,uterine serous carcinoma(USC),or mixed type(endometrioid and serous adenocarcinoma).The expression of CD20+B cells and structure of TLS were also verified by HE staining and IHC.(2)Exploring the expression of CXCL13/CXCR5,as TLS-related B cell molecular pathways by IF,and observing its distribution and localization.(3)Exploring the prognosis of EC patients with TLS via univariate and multivariate analysis.3.The research on molecular pathway of TLS in EC and biological information analysis(1)10 fresh tumor specimens of EC patients who underwent surgery from November 2020 to February 2021at the team of Pro.Pan in the Department of Obstetrics and Gynecology of PUMCH were collected.The differential genes expression analysis between TLS and non-TLS were analyzed through DNA panel and RNA-seq.Then we used GO and KEGG analysis to explore its downstream enrichment pathways,and explored its TME through GSVA analysis.And the relationship between TLS and TCGA molecular classification was further explored.(2)To Use the TCGA database of EC to research TLS-related genes in EC,and analyze differences in gene expression and its relationship with prognosis.Research result1.The research on composition of TLS in EC and its relationship with prognosis(1)In this chapter,104 patients with EC were enrolled as the experimental group,and 10 normal endometrial tissues and 10 AEH tissues were used as the control group.By observing HE staining and CD20 under microscope,it was confirmed that CD20+B cells in clusters were positively expressed in tumor tissues,but were not expressed in the control group.This indicates that TLS is present in EC,but not in the control group.(2)TLS consisted of clusters rich in CD20+B cells,CD8+T cells,CD4+T cells,and CD38+PCs,with or without CD23+GCs.According to the morphological structure of TLS,TLS can be divided into aggregate TLS and decentralized TLS.According to the location,TLS can be divided into infiltration in the internal stroma and the marginal stroma of tumor cells.According to the expression of CD23+GC,TLS is divided into mature stage TLS(with positive CD23+GC)and naive stage TLS(without positive CD23+GC).(3)Among the 104 patients with EC,81(77.9%)had TLS,and 23(22.1%)had no TLS.The PFS in EC patients with TLS is better than patients without TLS(P<0.001),regardless of pathological differentiation.The results of multivariate analysis showed that the absence of TLS was an independent risk predictor of recurrence in patients with endometrial cancer(HR=0.154,95%CI 0.044-0.536,P=0.003).2.The research on TLS expression verification and protein pathway research in EC(1)In this chapter,93 patients with EC in PUMCH were enrolled.By observing HE staining and IHC staining,CD20+B cells were positively expressed in EC.This also indicates that TLS exists in EC,and its morphology and maturity are similar to those in previous studies.(2)The relationship between CD20+B cells and other lymphoid infiltrating cells(TILs)in TLS was further verified,as well as the relationship between different TILs and prognosis.The density of CD20+B cells increased as the number of TLSs increased(P<0.001).The density of CD20+B cells were positively correlated with CD8+T cells(P<0.001,r=0.357),CD4+T cells(P<0.001,r=0.48).The high co-expression of CD20+B cells and CD8+T cells had better PFS than patients in other groups(P=0.028).(3)CXCR5 is the expression receptor of CXCL13,and there is a positive correlation between these two molecular expressions(r=0.344,P<0.001).The CXCL13/CXCR5 axis,as a kind of B cell pathway,is specifically expressed in TLS(P<0.001).(4)Among the 93 patients with EC,35(37.6%)had TLS,and 58(62.4%)had no TLS.The PFS(P=0.032)and OS(P=0.046)in EC patients with TLS were significantly better than those without TLS.The multivariate analysis result showed that Absence of TLS was independently associated with tumor progression(HR=0.125,95%CI 0.024-0.667,P=0.015).3.The research on molecular pathway of TLS in EC and biological information analysis(1)In this chapter,10 tumor specimens were collected.There significant statistical difference between TLS expression and TCGA molecular classification of EC(P=0.019).There are TLS in POLE mutation(20%),MSI-H(10%)and CN-high(30%)patients,while no TLS exist in CN-L patients(40%).One of the patients with POLE mutation was highly positive for TLS expression.(2)The differential gene expression analysis was performed on the TLS group and the non-TLS group.Compared with non-TLS group,the up-regulated gene in TLS group included CCDC106,KRT128P,etc.The down-regulated gene in TLS group included LAMP5-AS1,ANKRD34C,OAT,etc.This part of the differential gene expression may be related to the histone HE-K9 methylation metabolic pathway.(3)The tumor immune microenvironment was further explored by GSVA analysis.The results showed that TLS tumor tissue is richer in activated B cell,activated CD4+T cell,activated CD8+T cell,Tfh cell and other TILs.This is consistent with the results of IHC in the second chapter.(4)Based on the bioinformatics database of EC,T lymphocytes(CD8,CTLA4,FOXP3,TCF7),B lymphocytes(CD23),plasma cells,chemokine family genes may be related to different prognostic of EC patients.Research conclusion1.This study respectively used Chinese and Japanese EC patient's databases to confirm that CD20+B lymphocytes aggregate in EC tumor tissues and form a TLS structure,which is the first report in the world.TLS exists in EC,and the expression rate is about 40-70%.2.TLS consisted of clusters rich in CD20+B cells,CD8+T cells,CD4+T cells,and CD38+PCs,with or without CD23+GCs.According to the morphological structure of TLS,TLS can be divided into aggregate TLS and decentralized TLS.According to the location,TLS can be divided into infiltration in the internal stroma and the marginal stroma of tumor cells.According to the expression of CD23+GC,TLS is divided into mature stage TLS(with positive CD23+GC)and naive stage TLS(without positive CD23+GC).This is consistent with the TLS reported in other solid tumors in the literature.The density of CD20+B cells increased as the number of TLSs increased.There is positive correlation between CD20+B cells and other TILs.The CXCL13/CXCR5 axis,as a kind of B cell pathway,is specifically expressed in TLS.3.The PFS in EC patients with TLS is better than patients without TLS,regardless of pathological differentiation.The results of multivariate analysis showed that the absence of TLS was an independent risk predictor of recurrence in patients with endometrial cancer.3.Compared with non-TLS group,the up-regulated gene in TLS group included CCDC106,KRT128P,etc.The down-regulated gene in TLS group included LAMP5-AS1,ANKRD34C,OAT,etc.This part of the differential gene expression may be related to the histone HE-K9 methylation metabolic pathway.There significant statistical difference between TLS expression and TCGA molecular classification of EC.The EC patients with POLE mutation was highly positive for TLS expression.Based on bioinformatics database of EC,and the preliminary IHC experiment was further verified.How to induce formation of the TLS and how to inhibit tumorigenesis with the TLS will become research directions in the future.The TLS and B cell-related pathways need to be further explored as important immune signals and new immunotherapy targets after the T cell therapy revolution.