Table_2_Development of a Metastasis-Related Immune Prognostic Model of Metastatic Colorectal Cancer and Its Usefulness to Immunotherapy.docx

Background: Post-surgical recurrence of the metastatic colorectal cancer (mCRC) remains a challenge, even with adjuvant therapy. Moreover, patients show variable outcomes. Here, we set to identify gene models based on the perspectives of intrinsic cell activities and extrinsic immune microenvironment to predict the recurrence of mCRC and guide the adjuvant therapy.
Methods: An RNA-based gene expression analysis of CRC samples (total = 998, including mCRCs = 344, non-mCRCs = 654) was performed. A metastasis-evaluation model (MEM) for mCRCs was developed using the Cox survival model based on the prognostic differentially expressed genes between mCRCs and non-mCRCs. This model separated the mCRC samples into high- and low-recurrence risk clusters that were tested using machine learning to predict recurrence. Further, an immune prognostic model (IPM) was built using the COX survival model with the prognostic differentially expressed immune-related genes between the two MEM risk clusters. The ability of MEM and IPM to predict prognosis was analyzed and validated. Moreover, the IPM was utilized to evaluate its relationship with the immune microenvironment and response to immuno-/chemotherapy. Finally, the dysregulation cause of IPM three genes was analyzed in bioinformatics.
Results: A high post-operative recurrence risk was observed owing to the downregulation of the immune response, which was influenced by MEM genes (BAMBI, F13A1, LCN2) and their related IPM genes (SLIT2, CDKN2A, CLU). The MEM and IPM were developed and validated through mCRC samples to differentiate between low- and high-recurrence risk in a real-world cohort. The functional enrichment analysis suggested pathways related to immune response and immune system diseases as the major functional pathways related to the IPM genes. The IPM high-risk group (IPM-high) showed higher fractions of regulatory T cells (Tregs) and smaller fractions of resting memory CD4+ T cells than the IPM-low group. Moreover, the stroma and immune cells in the IPM-high samples were scant. Further, the IPM-high group showed downregulation of MHC class II molecules. Additionally, the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm and GDSC analysis suggested the IPM-low as a promising responder to anti-CTLA-4 therapy and the common FDA-targeted drugs, while the IPM-high was non-responsive to these treatments. However, treatment using anti-CDKN2A agents, along with the activation of major histocompatibility complex (MHC) class-II response might sensitize this refractory mCRC subgroup. The dysfunction of MEIS1 might be the reason for the dysregulation of IPM genes.
Conclusions: The IPM could identify subgroups of mCRC with a distinct risk of recurrence and stratify the patients sensitive to immuno-/chemotherapy. Further, for the first time, our study highlights the importance of MHC class-II molecules in the treatment of mCRCs using immunotherapy.

背景：转移性结直肠癌（metastatic colorectal cancer, mCRC）的术后复发仍是临床难题，即便接受辅助治疗亦难以有效规避。此外，不同患者的临床结局存在显著异质性。本研究旨在基于肿瘤固有细胞活性与外在免疫微环境的视角，构建基因预测模型，以实现mCRC术后复发风险预测，并辅助指导临床治疗方案选择。
方法：本研究对结直肠癌（colorectal cancer, CRC）样本开展基于RNA的基因表达谱分析，共纳入998例样本，其中mCRC样本344例，非转移性结直肠癌样本654例。基于mCRC与非mCRC间的预后差异表达基因，本研究采用Cox生存模型构建了转移性结直肠癌转移评估模型（metastasis-evaluation model, MEM）。该模型将mCRC样本划分为高复发风险亚组与低复发风险亚组，并通过机器学习方法验证其复发预测效能。进一步，基于两个MEM风险亚组间的预后差异表达免疫相关基因，本研究采用Cox生存模型构建了免疫预后模型（immune prognostic model, IPM）。随后对MEM与IPM的预后预测效能开展分析与验证；此外，利用IPM评估其与免疫微环境及免疫治疗、化疗的响应性关联；最后通过生物信息学方法分析IPM所包含的三个基因的表达失调机制。
结果：研究发现，免疫应答下调与术后复发风险升高相关，而该过程受MEM相关基因（BAMBI、F13A1、LCN2）及其对应的IPM相关基因（SLIT2、CDKN2A、CLU）调控。本研究通过mCRC样本构建并验证了MEM与IPM，二者可在真实世界队列中有效区分患者的高低复发风险分层。功能富集分析显示，IPM相关基因主要富集于免疫应答及免疫系统疾病相关通路。IPM高风险亚组（IPM-high）较IPM低风险亚组（IPM-low）具有更高的调节性T细胞（regulatory T cells, Tregs）浸润比例，而静息记忆CD4+ T细胞浸润比例更低；此外，IPM-high样本中的肿瘤间质与免疫细胞浸润程度较低。进一步研究发现，IPM-high亚组的主要组织相容性复合体II类（major histocompatibility complex class II, MHC II）分子表达下调。此外，肿瘤免疫功能异常与排斥（Tumor Immune Dysfunction and Exclusion, TIDE）算法及癌症药物敏感性基因组学（Genomics of Drug Sensitivity in Cancer, GDSC）分析结果显示，IPM-low亚组对抗CTLA-4治疗及FDA批准的常见靶向药物具有良好响应性，而IPM-high亚组对上述治疗均无响应。不过，采用抗CDKN2A药物治疗联合主要组织相容性复合体II类通路激活，或可使该难治性mCRC亚组对治疗产生响应。MEIS1基因功能异常或为IPM相关基因表达失调的潜在机制。
结论：IPM可有效区分具有不同复发风险的mCRC亚组，并可对免疫/化疗敏感患者进行分层；此外，本研究首次证实了主要组织相容性复合体II类分子在mCRC免疫治疗中的关键作用。