Table15_Gene set-based identification of two immune subtypes of diffuse large B cell lymphoma for guiding immune checkpoint blocking therapy.XLSX

Background: Diffuse large B cell lymphoma (DLBCL) is the most common lymphoma in adults. Tumour microenvironment is closely related to tumour prognosis and immune checkpoint blocking therapy (ICBT). This study aimed to investigate the immunological and prognostic characteristics of the tumour microenvironment (TME), as well as the regulatory mechanisms.Methods: Gene expression profiles and clinical data of patients with DLBCL were obtained from GEO database. ESTIMATE, CIBERSORT, and ssGSEA analyses were used to explore microenvironment characteristics and regulatory mechanism of the immune subtypes, which were identified by consistent clustering. The differences in enriched pathways were showed by GSEA. Hub genes associated with CD8+ T cells, which were identified by WCGNA, were exhibited biological functions through GO and KEGG. Immune-related gene scores (IRGSs) based on hub genes were used to evaluate the prediction of immune subtypes and ICBT, and retrospective analysis was used for validation. Finally, prognostic genes were screened to construct risk models.Results: Consensus clustering divided patients with DLBCL into two subtypes with significant heterogeneities in prognosis and immune microenvironment. Low immune infiltration was associated with poor prognosis. Subtype C1 with high immune infiltration was enriched in multiple immune pathways. We observed that two common mutated genes (B2M and EZH2) in DLBCL were closely related to MHC-I and microenvironment. Our further analysis manifested that MYD88L265P may be the main cause of TLR signalling pathway activation in subtype C1. Hub genes (SH2D1A, CD8A, GBP2, ITK, CD3D, RORA, IL1R2, CD28, CD247, CD3G, PRKCQ, CXCR6, and CD3E) in relation with CD8+ T cells were used to establish IRGS, which was proved an accurate predictor of immune subtypes, and patients in high-IRGS group were more likely to benefit from ICBT. Retrospective analysis showed that absolute lymphocyte count (ALC) was higher in the group that responded to the PD-1 inhibitor. Finally, the risk model was constructed based on two genes (CD3G and CD3D), and the low-risk group showed better prognosis.Conclusion: DLBCL immune classifications with highly heterogeneity are a powerful predictor of prognosis and ICBT. The IRGS is proved to be a reliable tool to distinguish immune subtypes as a substitute for gene expression profile.

背景：弥漫性大B细胞淋巴瘤（DLBCL）是成人中最常见的淋巴瘤类型。肿瘤微环境与肿瘤预后及免疫检查点阻断疗法（ICBT）密切相关。本研究旨在探究肿瘤微环境（TME）的免疫学和预后特征，以及调控机制。方法：从GEO数据库获取DLBCL患者的基因表达谱和临床数据。采用ESTIMATE、CIBERSORT和ssGSEA分析，利用一致性聚类确定的免疫亚型，探究微环境特征和调控机制。通过GSEA展示富集通路差异。利用WCGNA识别的与CD8+ T细胞相关的枢纽基因，通过GO和KEGG展示其生物学功能。基于枢纽基因的免疫相关基因评分（IRGSs）用于评估免疫亚型和ICBT的预测，并使用回顾性分析进行验证。最后，筛选预后基因以构建风险模型。结果：一致性聚类将DLBCL患者分为两个亚型，预后和免疫微环境存在显著异质性。低免疫浸润与不良预后相关。高免疫浸润的亚型C1富集于多个免疫通路。我们观察到DLBCL中两种常见的突变基因（B2M和EZH2）与MHC-I和微环境密切相关。我们的进一步分析表明，MYD88L265P可能是亚型C1中TLR信号通路激活的主要原因。与CD8+ T细胞相关的枢纽基因（SH2D1A、CD8A、GBP2、ITK、CD3D、RORA、IL1R2、CD28、CD247、CD3G、PRKCQ、CXCR6和CD3E）被用于建立IRGS，证明其为免疫亚型的准确预测工具，且高IRGS组的患者更有可能从ICBT中获益。回顾性分析显示，对PD-1抑制剂产生反应的组别中，绝对淋巴细胞计数（ALC）较高。最后，基于CD3G和CD3D两个基因构建了风险模型，低风险组预后较好。结论：具有高度异质性的DLBCL免疫分类是预后和ICBT的有力预测因子。IRGS被证明是区分免疫亚型的可靠工具，可作为基因表达谱的替代品。