Identification of four immune subtypes in locally advanced rectal cancer treated with neoadjuvant chemotherapy for predicting the efficacy of subsequent immune checkpoint blockade. Identification of four immune subtypes in locally advanced rectal cancer treated with neoadjuvant chemotherapy for predicting the efficacy of subsequent immune checkpoint blockade

Introduction: Neoadjuvant chemoradiotherapy (nCRT) was the foundation treatment for locally advanced rectal cancer (LARC). The nCRT can improve the efficacy of immunotherapy on account of its in-situ vaccine effect. Objective: The aim is to identify stable and reliable transcriptome signatures to predict the efficacy of immune checkpoint blockade (ICB) in patients with LARC. Methods: Immunophenotyping was established by using xCell immune cell infiltration abundance and consistent clustering in GSE39582 and verified in several data sets. The effects of immunophenotyping, follicular regulatory T cells, tumor associated fibroblasts (CAF) and tertiary lymphoid structures (TLS) signatures on the efficacy of ICB were analyzed by using IMvigor210, GSE91061 and an independent Daping Hospital (DPH) cohort. Results: There are four stable and repeatable immune subtypes in rectal cancer, among which C1 is low immune infiltration type, C2 is high interstitial infiltration type, C3 is high immune infiltration type and C4 is ion channel type. C2 is mainly characterized by high infiltration CAF, C3 is characterized by high infiltration of cytotoxic T lymphocytes, high expression of PD-L1 and TLS. In rectal cancer patients receiving nCRT, immunophenotyping was not significantly associated with pathological remission rate, but immunophenotyping was an independent prognostic factor of RFS. In IMvigor210 patients treatment with atezolizumab, the pathological remission rates of C1, C2, C3 and C4 were 23.86%, 10.94%, 33.33% and 23.08% respectively (χ2 = 8.981, P = 0.029), which were 11.76%, 50.00%, 42.86% and 0.0% respectively in the GSE91061 patients treatment with nivolumab (Fisher's exact probability, P = 0.018). Both follicular regulatory T cells and CAF showed a further impact on the ICB therapeutic efficacy of C2 and C3 subtype. In addition, both GSE91404 and DPH cohorts showed that nCRT treatment induced a significant increase in the expression of TNFRSF9 and abundance of macrophages in C3 subtype. Conclusion: Our data suggest that there are four immune types of rectal cancer, which are related to the prognosis of patients. Among them, C3 and some C2 subtypes represents the patients who may benefit from ICB after nCRT treatment. Overall design: Daping Hospital (DPH) cohort was comprised of resected tissues from nine LARC patients with complete pathological response (pCR), nine without pathological response (npCR) and biopsy tissues from nine LARC patients prior nCRT. The raw fastq files containing sequence information are not permitted to be submitted according to Chinese law.

引言：新辅助放化疗（neoadjuvant chemoradiotherapy, nCRT）是局部进展期直肠癌（locally advanced rectal cancer, LARC）的标准基础治疗方案，其通过原位疫苗效应可提升免疫治疗的疗效。研究目的：本研究旨在筛选稳定可靠的转录组特征（transcriptome signatures），以预测局部进展期直肠癌患者接受免疫检查点阻断（immune checkpoint blockade, ICB）治疗的疗效。研究方法：通过xCell免疫细胞浸润丰度分析与一致性聚类方法，在GSE39582数据集中构建免疫分型（immunophenotyping），并在多个数据集上完成验证；采用IMvigor210、GSE91061及独立的大坪医院（Daping Hospital, DPH）队列，分析免疫分型、滤泡调节性T细胞（follicular regulatory T cells）、肿瘤相关成纤维细胞（cancer-associated fibroblasts, CAF）及三级淋巴结构（tertiary lymphoid structures, TLS）特征对免疫检查点阻断治疗疗效的影响。研究结果：直肠癌存在四种稳定可重复的免疫亚型：C1为低免疫浸润型，C2为间质高浸润型，C3为高免疫浸润型，C4为离子通道型。其中C2主要以肿瘤相关成纤维细胞高浸润为特征，C3则以细胞毒性T淋巴细胞高浸润、PD-L1高表达及三级淋巴结构富集为特征。在接受新辅助放化疗的直肠癌患者中，免疫分型与病理缓解率无显著关联，但却是无复发生存期（recurrence-free survival, RFS）的独立预后因素。在接受阿替利珠单抗（atezolizumab）治疗的IMvigor210队列患者中，C1、C2、C3、C4亚型的病理缓解率分别为23.86%、10.94%、33.33%及23.08%（χ²=8.981，P=0.029）；而在接受纳武利尤单抗（nivolumab）治疗的GSE91061队列患者中，上述四亚型的病理缓解率分别为11.76%、50.00%、42.86%及0.0%（Fisher确切概率法，P=0.018）。滤泡调节性T细胞与肿瘤相关成纤维细胞均对C2、C3亚型的免疫检查点阻断治疗疗效产生进一步影响。此外，GSE91404与大坪医院队列均显示，新辅助放化疗可诱导C3亚型中TNFRSF9表达水平及巨噬细胞浸润丰度显著升高。研究结论：本研究数据表明，直肠癌存在四种免疫亚型，且与患者预后相关；其中C3亚型与部分C2亚型的患者，在接受新辅助放化疗后可从免疫检查点阻断治疗中获益。整体研究设计：大坪医院（DPH）队列纳入了9例达到病理完全缓解（pathological complete response, pCR）、9例未达到病理完全缓解（non-pathological complete response, npCR）的局部进展期直肠癌患者的术后切除组织，以及9例新辅助放化疗前的局部进展期直肠癌患者的活检组织。根据中国相关法律法规，包含序列信息的原始fastq文件不得提交。