The Expression And Significance Of Tertiary Lymphoid Structure In The Colorectal Tumor Microenvironment

Objective:Tertiary lymphoid structures(TLS),which are immune cell aggregates found in tumor sites within the last few years,have been shown to provide a critical functional environment for lymphocyte and humoral immunity,promoting and sustaining anti-tumor immune responses.The tumor stroma has a potent immunosuppressive function that can exclude tumor-infiltrating lymphocytes from the tumor bed,leading to a “cold” phenotype.Despite TLS and tumor stroma percentage(TSP)being significantly correlated with patient prognosis for various solid tumors,the exact roles and interrelationships of TLS and TSP remain unknown.Additionally,the potential of TLS in the colorectal tumor immune microenvironment(TIME)and the intrinsic link between TLS and neoadjuvant chemoradiotherapy(CRT)are unclear.This study focuses on TLS and aims to explore the potential role of TLS in the tumor immune microenvironment in CRC.We also examine the intrinsic link between TLS and neoadjuvant CRT.Methods:1.We included 114 and 60 nm CRC patients as the training and external validation sets,respectively,to evaluate the correlation between TLS,TSP,clinicopathological features,and their prognostic value using HE and IHC methods.Finally,we construct a nomogram including TLS,TSP,and tumor-node-metastasis(TNM)staging to predict the probability of 2-year and 5-year recurrence-free survival(RFS)in nm CRC patients.2.We included 114 patients with orthotopically resected locally advanced rectal cancer(LARC)to evaluate the effect of TLS on tumor-infiltrating lymphocytes(TILs)using H&E and IHC methods.3.We assessed the association between TLS-score and immune cells and immunerelated pathways(using ss GSEA)in rectal cancer patients from the TCGA-READ(N=58)databases.Then,we performed gene expression profile analysis on the high TLS-score and low TLS-score groups to determine the differential genes.The cluster Profiler tool was used to perform GO enrichment analysis on the genes with significant differences,focusing on the critical role of TLS-score in shaping the tumor immune microenvironment.4.We included the neoadjuvant therapy cohort(N=93),GSE119409(N=61),and GSE150082(N=39)datasets to assess the effect of TLS or TLS-score in biopsy tissue on the response to neoadjuvant therapy using H&E,IHC,and bioinformatics methods.5.We included patients from the neoadjuvant therapy cohort(N=95,incompletepathological remission)and surgery cohort(N=114,LARC)to analyze the effect of neoadjuvant therapy on TLS formation and prognostic value using H&E and IHC methods.Results:1.Peritumoral TLS(P-TLS),intratumoral TLS(In-TLS),and high TSP(H-TSP,>50%)were present in 99.1%,26.3%,and 41.2% of patients,respectively.H-TSP tumor tends to have lower P-TLS density(P =0.0205).The low P-TLS density(< 0.098/mm2)was significantly associated with reduced RFS(HR=6.597 95% CI: 2.882-15.103,P <0.001)and reduced overall survival(OS)(HR=6.628 95% CI: 2.893-15.183,P < 0.001)of nm CRC patients.In-TLS was not of significance in evaluating the clinical outcomes of nm CRC patients.H-TSP was significantly associated with reduced RFS(HR=0.126 95% CI: 0.048-0.333,P <0.001)and reduced OS(HR=0.125 95% CI: 0.047-0.332,P <0.001)of nm CRC patients.The 5-year RFS of the high P-TLS,low-TLS,H-TSP,and L-TSP groups were 89.7%,47.2%,53.2%,and 92.5%,respectively.The P-TLS density,TSP and TNM stage were independent prognosis factors of nm CRC patients.The Nomogram,including the PTLS density,TSP and TNM stage,outperformed the TNM stage.2.High TLS density is significantly associated not only with prolonged RFS in LARC patients(HR=0.3493 95% CI: 0.1814-0.6275,p=0.0016),but also with increased infiltration of adaptive immune cells(CD4+ T cells,CD8+ T cells,CD20+ B cells,CD45RO+ lymphocytes cells).Additionally,bioinformatic analyses demonstrate that high TLS-score is closely associated with increased infiltration of immune cell infiltration and activation of immune-related pathways.3.Compared to TLS(-)patients in pre-treatment biopsy tissues,TLS(+)patients not only had a higher response rate(63.6% vs 35.2%,p=0.0258)and pathological complete response(p CR)rate(45.5% vs 16.9%,p=0.0096),but also had longer RFS(HR=0.3574,95% CI: 0.1489-0.8578,p=0.0213),which was confirmed in GSE119409 and GSE150082.4.Neoadjuvant chemoradiotherapy can significantly impair the TLS density and predictive value.We calibrated TLS using tumor burden and construct TLS/tumor ratio.We observed higher infiltration of CD4+ T cells,CD8+ T cells,and CD45RO+ lymphocytes in tumors with a high TLS/tumor ratio.Finally,a high TLS/tumor ratio was an independent protective factor for RFS in neoadjuvant CRT cohort patients.Conclusion:1.P-TLS is present in almost all nm CRC patients and is associated with favorable prognosis,while low P-TLS density is associated with an immunosuppressive tumor stroma.2.High TLS density is not only associated with longer RFS in LARC patients,but also with more infiltration of adaptive immune cells.3.High TLS-score is associated with T cell inflamed tumor immune microenvironment.4.TLS or TLS-score in biopsy tissue can predict response to neoadjuvant chemoradiotherapy in rectal cancer patients.5.Neoadjuvant chemoradiotherapy can significantly impair TLS density and prognostic value,which may be related to decreased tumor burden.6.High TLS/tumor ratio is not only associated with more infiltration of adaptive immune cells,but also an independent protective factor for RFS in neoadjuvant-treated patients.