BACKGROUND Immunotherapy, including checkpoint inhibition, has remarkably improved prognosis in advanced melanoma. Despite this success, acquired resistance is still a major challenge. The T cell costimulatory receptor TNFRSF9 (also known as 4-1BB and CD137) is a promising new target for immunotherapy and two agonistic antibodies are currently tested in clinical trials. However, little is known about epigenetic regulation of the encoding gene. In this study we investigate a possible correlation of TNFRSF9 DNA methylation with gene expression, clinicopathological parameters, molecular and immune correlates, and response to anti-PD-1 immunotherapy to assess the validity of TNFRSF9 methylation to serve as a biomarker. METHODS We performed a correlation analyses of methylation at twelve CpG sites within TNFRSF9 with regard to transcriptional activity, immune cell infiltration, mutation status, and survival in a cohort of N = 470 melanoma patients obtained from The Cancer Genome Atlas. Furthermore, we used quantitative methylation-specific PCR to confirm correlations in a cohort of N = 115 melanoma patients' samples (UHB validation cohort). Finally, we tested the ability of TNFRSF9 methylation and expression to predict progression-free survival (PFS) and response to anti-PD-1 immunotherapy in a cohort comprised of N = 121 patients (mRNA transcription), (mRNA ICB cohort) and a case-control study including N = 48 patients (DNA methylation, UHB ICB cohort). FINDINGS We found a significant inverse correlation between TNFRSF9 DNA methylation and mRNA expression levels at six of twelve analyzed CpG sites (P ≤ 0.005), predominately located in the promoter flank region. Consistent with its role as costimulatory receptor in immune cells, TNFRSF9 mRNA expression and hypomethylation positively correlated with immune cell infiltrates and an interferon-γ signature. Furthermore, elevated TNFRSF9 mRNA expression and TNFRSF9 hypomethylation correlated with superior overall survival. In patients receiving anti-PD-1 immunotherapy (mRNA ICB cohort), we found that TNFRSF9 hypermethylation and reduced mRNA expression correlated with poor PFS and response. INTERPRETATION Our study suggests that TNFRSF9 mRNA expression is regulated via DNA methylation. The observed correlations between TNFRSF9 DNA methylation or mRNA expression with known features of response to immune checkpoint blockage suggest TNFRSF9 methylation could serve as a biomarker in the context of immunotherapies. Concordantly, we identified a correlation between TNFRSF9 DNA methylation and mRNA expression with disease progression in patients under immunotherapy. Our study provides rationale for further investigating TNFRSF9 DNA methylation as a predictive biomarker for response to immunotherapy. FUNDING AF was partly funded by the Mildred Scheel Foundation. SF received funding from the University Hospital Bonn BONFOR program (O-105.0069). DN was funded in part by DFG Cluster of Excellence ImmunoSensation (EXC 1023). The funders had no role in study design, data collection and analysis, interpretation, decision to publish, or preparation of the manuscript; or any aspect pertinent to the study.

中文翻译：

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全面分析肿瘤坏死因子受体TNFRSF9（4-1BB）DNA甲基化的分子和临床病理特征，免疫浸润以及对黑色素瘤免疫治疗的反应预测。

背景技术包括检查点抑制在内的免疫疗法已显着改善了晚期黑素瘤的预后。尽管取得了成功，但获得的抵抗仍然是一个重大挑战。T细胞共刺激受体TNFRSF9（也称为4-1BB和CD137）是免疫治疗的有希望的新靶标，目前正在临床试验中测试两种激动剂。然而，关于编码基因的表观遗传调控知之甚少。在这项研究中，我们调查了TNFRSF9 DNA甲基化与基因表达，临床病理参数，分子和免疫相关性以及对抗PD-1免疫疗法的反应之间可能的相关性，以评估TNFRSF9甲基化作为生物标志物的有效性。方法我们对TNFRSF9中12个CpG位点的甲基化进行了转录活性的相关分析，从癌症基因组图集获得的N = 470名黑色素瘤患者队列中，免疫细胞浸润，突变状态和存活率。此外，我们使用了定量甲基化特异性PCR来确认N = 115个黑色素瘤患者样本（UHB验证队列）队列中的相关性。最后，我们测试了TNFRSF9甲基化和表达预测无进展生存期（PFS）和抗PD-1免疫疗法反应的能力，该队列由N = 121例患者（mRNA转录），（mRNA ICB队列）和病例对照研究，包括N = 48例患者（DNA甲基化，UHB ICB队列）。结果我们发现在十二个分析的CpG位点中的六个位点（P≤0.005），主要位于启动子侧翼区域，TNFRSF9 DNA甲基化与mRNA表达水平之间存在显着的负相关。与它在免疫细胞中作为共刺激受体的作用相一致，TNFRSF9 mRNA表达和甲基化不足与免疫细胞浸润和干扰素-γ标记呈正相关。此外，升高的TNFRSF9 mRNA表达和TNFRSF9低甲基化与较高的总生存期相关。在接受抗PD-1免疫疗法（mRNA ICB队列）的患者中，我们发现TNFRSF9甲基化过高和mRNA表达降低与不良的PFS和反应相关。解释我们的研究表明TNFRSF9 mRNA的表达受DNA甲基化的调节。观察到的TNFRSF9 DNA甲基化或mRNA表达与对免疫检查点阻滞反应的已知特征之间的相关性表明，TNFRSF9甲基化可在免疫疗法中用作生物标志物。一致地，我们确定了免疫治疗患者中TNFRSF9 DNA甲基化和mRNA表达与疾病进展之间的相关性。我们的研究为进一步研究TNFRSF9 DNA甲基化作为对免疫疗法反应的预测性生物标志物提供了理论依据。资金AF由Mildred Scheel基金会部分资助。SF从波恩大学医院BONFOR计划获得了资助（O-105.0069）。DN部分由DFG卓越免疫感知集群（EXC 1023）资助。资助者在研究设计，数据收集和分析，解释，出版决定或手稿的编写中不起作用；或与研究相关的任何方面。我们的研究为进一步研究TNFRSF9 DNA甲基化作为对免疫疗法反应的预测性生物标志物提供了理论依据。资金AF由Mildred Scheel基金会部分资助。SF从波恩大学医院BONFOR计划获得了资助（O-105.0069）。DN部分由DFG卓越免疫感知集群（EXC 1023）资助。资助者在研究设计，数据收集和分析，解释，出版决定或手稿的编写中不起作用；或与研究相关的任何方面。我们的研究为进一步研究TNFRSF9 DNA甲基化作为对免疫疗法反应的预测性生物标志物提供了理论依据。资金AF由Mildred Scheel基金会部分资助。SF从波恩大学医院BONFOR计划获得了资助（O-105.0069）。DN部分由DFG卓越免疫感知集群（EXC 1023）资助。资助者在研究设计，数据收集和分析，解释，出版决定或手稿的编写中不起作用；或与研究相关的任何方面。资助者在研究设计，数据收集和分析，解释，发表决定或手稿中没有作用；或与研究相关的任何方面。资助者在研究设计，数据收集和分析，解释，出版决定或手稿的编写中不起作用；或与研究相关的任何方面。