Tertiary Lymphoid Structures And Their Marker CXCL13 Indicate Prognosis And Predict Response To ICI In Patients With Lung Adenocarcinoma

BackgroundThe incidence of lung cancer has been increasing year by year in recent years and has become the disease with the highest number of deaths among cancer patients,with lung adenocarcinoma being the most prevalent lung cancer subtype.Tertiary lymphoid structure(TLS)has been reported to show an association with reduced risk of recurrence and survival of cancer patients in many cancers,such as breast,colorectal and intrahepatic cholangiocarcinoma,however,the clinical importance of tertiary lymphoid structure and its marker CXCL13 in lung adenocarcinoma has not been fully investigated,and therefore the study of tertiary lymphoid structure in lung adenocarcinoma is of great value.MethodsIn this study,a total of 364 patients with lung adenocarcinoma from January 2015 to December 2015 and 121 patients from January 2020 to December 2020 were screened,and clinical traits of corresponding patients were collected(this protocol was approved by the Ethics Committee of the Affiliated Hospital of nantong university).The presence status of tertiary lymphoid structures within HE(hematoxylin-eosin staining)sections of paraffin tissue specimens of lung adenocarcinoma was first assessed microscopically,and the density,distribution and differentiation of TLSs in lung adenocarcinoma were evaluated by hematoxylin-eosin staining and immunohistochemistry,and then the relationship between tertiary lymphoid structures and clinicopathological features was analyzed in 364 patients with lung adenocarcinoma.Then,the expression characteristics of CXCL13,a TLS related marker,were studied by online database and IHC(immunohistochemical staining),and the correlation between CXCL13 and the formation of TLS and tumor immunoinfilt--rating cells was analyzed.The correlation between CXCL13 and PD-L1,a commonly used clinical immune checkpoint indicator,was verified by immunohi--stochemical staining.Finally,the biological function enrichment analysis of CXCL13 was conducted to explore the functional pathways involved in CXCL13 and further clarify its role in the formation of TLS.Results1.The presence status of TLS in lung adenocarcinoma tissues was confirmed:hematoxylin and eosin staining and immunohistochemical staining of 364 lung adenocarcinoma samples showed that TLS was mainly manifested as clusters of CD3-positive T cells surrounding CD20-positive B cells,which were present in both intratumoral stroma(i TLS)and peritumoral tissue(p TLS),and that B cells occupied the the major component of TLS.2.Different levels of TLS maturation were distinguished: in mature TLS,CD21-positive follicular dendritic cells were present in a B-cell-dominated follicular structure and many T cells clustered around and inside the follicles.In contrast,T and B cells were scattered in immature TLSs without forming follicular structures,and almost no CD21 FDCs were observed.CD3+ T cells,CD4+ T cells,CD8+ T cells and CD20+ B cells were found in both mature and immature TLSs.In addition,most of the positive TLSs were mature TLSs according to the experimental results.3.The relationship between TLSs and clinicopathological features was analyzed: the presence of TLSs was positively correlated with tumor size,absence of lymph node metastasis and VPI(pleural invasion).In addition,the presence of TLSs indicated a better prognosis for patients with lung adenocarcinoma.High-density TLSs showed a trend of small tumor size,good tumor differentiation,low TMN stage,absence of lymph node metastasis,and VPI.High-density TLSs had better prognosis than low-density TLSs and non-TLSs.4.CXCL13 gene expression in lung adenocarcinoma tumor tissues was significantly correlated with the presence of TLS,lymphocyte infiltration,and good prognosis of LUAD: We found that CXCL13 was highly expressed in TLS by IHC staining and the high expression of CXCL13 was significantly increased in TLS-positive samples,and TIMER database analysis confirmed the widespread expression of CXCL13 in the immune The TIMER database analysis confirmed the widespread expression of CXCL13 in the microenvironment and that CXCL13 expression was associated with B cells(r =0.468,P = 9.18e-28),CD4+ T cells(r = 0.335,P = 4.05e-14),CD8+ T cells(r = 0.344,P= 5.14e-15),dendritic cells(r = 0.249,P = 2.61e-08),and neutrophils(r = 0.249,P =2.83e-08)were positively correlated.In the KM analysis,patients with high CXCL13 expression had a significantly better prognosis(P = 0.048).It was also demonstrated again using the UALCAN database analysis that CXCL13 expression was significantly higher in lung adenocarcinoma tissues than in adjacent normal tissues,and that CXCL13 was more common in female lung adenocarcinoma patients.5.CXCL13 expression was strongly correlated with immune checkpoint-related targets: The correlation between CXCL13 and immune checkpoint-related targets in lung adenocarcinoma was analyzed by the TIMER algorithm,and CXCL13 expression levels were correlated with PD-L1/CD274(cor = 0.348,P = 3.84e-16),PD1/PDCD1(cor =0.666,P = 2.24e-67),CTLA4(cor = 0.67,P = 0e+00),LAG3(cor = 0.603,P = 0e+00),TIGIT(cor = 0.732,P = 0e+00),TIM-3/HAVCR2(cor = 0.404,P = 0e+00),VISTA/C10ORF54(cor = 0.366,P = 8.38e-18)were positively correlated.Finally,immunohistochemical staining proved that CXCL13 was significantly correlated with the expression of PD-L1,a commonly used clinical immune checkpoint indicator.6.Biological function enrichment analysis showed that CXCL13 was related to a variety of immune functional pathways,especially the activation of T cells and B cells.ConclusionTertiary lymphoid structures and their marker CXCL13 indicate prognosis and predict response to ICI in patients with lung adenocarcinoma.