Aggressive PDACs show hypomethylation of repetitive elements and the execution of an intrinsic IFN program linked to a ductal cell-of-origin. Aggressive PDACs show hypomethylation of repetitive elements and the execution of an intrinsic IFN program linked to a ductal cell-of-origin

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer embedded in an extensive desmoplastic stroma. Since this challenges the molecular analyses of bulk tumor samples, we FACS-purified epithelial cells from PDAC and healthy human pancreas and performed genome-wide transcriptome and DNA methylome analyses. Clustering based on DNA methylation revealed two distinct groups of PDAC with different methylation levels at genomic regions encoding repeat elements. Methylationlow tumors showed higher expression of endogenous retroviral (ERV) transcripts and a strong engagement of the dsRNA sensing machinery. This results in the cell intrinsic activation of an interferon response signature (IFNsign), leading to the reprogramming of stromal cells towards a pro-tumorigenic microenvironment and poor patient outcome. Methylationlow/IFNsignhigh and Methylationhigh/IFNsignlow PDAC cells harbored distinct lineage traits specific for normal ductal or acinar pancreatic epithelial cells at the methylation and transcriptional level. Moreover, ductal-cell-derived KrasG12D/Trp53-/- mutant mouse PDACs showed higher expression of IFNsign compared to tumors initiated by the same drivers in acinar cells. Collectively, our data point to two distinct origins and etiology of human PDACs, with the aggressive Methylationlow/IFNsignhigh tumor subtype potentially targetable by agents blocking cell intrinsic IFN-signaling. Overall design: WGBS analysis of the epithelial cells isolated from 7 human PDAC tumors and 6 normal distal adjacent pancreas tissue. Epithelial cells were isolated from the human tissue using flow cytometry and defined as EpCAM+/CD45-. Data were processed as previously described (Wang et al. Nat Protocol, 2013): Sequencing reads were aligned using BWA with human reference genome version hg19/GRCh37. Duplicate reads were discarded. Methylation calling was performed as previously (Delacher and Imbusch et al. Nat Immunology, 2017). The raw counts of methylated and unmethylated reads for each CpG site from different libraries were merged for each sample. *** Submitter declares that the raw data is deposited in the European Genome-phenome Archive (EGA) due to patient privacy concerns. EGA accession number is EGAS00001004660. ***

胰腺导管腺癌（Pancreatic ductal adenocarcinoma, PDAC）是一种高侵袭性癌症，被包裹于广泛的促结缔组织增生间质中。由于这一特征给实体瘤混合样本的分子分析带来了极大挑战，我们通过荧光激活细胞分选（FACS）从胰腺导管腺癌患者及健康人胰腺组织中分离纯化上皮细胞，并开展了全基因组转录组与DNA甲基化组分析。
基于DNA甲基化谱的聚类分析显示，可将胰腺导管腺癌分为两类具有显著差异的亚型，其编码重复序列的基因组区域甲基化水平各不相同。甲基化水平较低的肿瘤亚型中，内源性逆转录病毒（ERV）转录本的表达量显著升高，且双链RNA（dsRNA）感知通路被强烈激活。这会触发细胞固有干扰素应答特征（IFNsign）的激活，进而促使间质细胞重编程为促肿瘤微环境，最终导致患者预后不良。
甲基化低/干扰素应答特征高与甲基化高/干扰素应答特征低的胰腺导管腺癌细胞，在甲基化与转录层面分别呈现出与正常胰腺导管上皮细胞或腺泡上皮细胞高度相似的谱系特异性特征。此外，由导管细胞来源的KrasG12D/Trp53-/-突变构建的小鼠胰腺导管腺癌模型，其干扰素应答特征的表达量高于以腺泡细胞为起源、携带相同驱动突变的肿瘤模型。
综上，本研究数据表明人类胰腺导管腺癌存在两种截然不同的起源与病因学机制，其中侵袭性较强的甲基化低/干扰素应答特征高肿瘤亚型，或可通过阻断细胞固有干扰素信号通路的药物实现靶向干预。
整体实验设计：对从7例人类胰腺导管腺癌组织及6例正常远端毗邻胰腺组织中分离得到的上皮细胞开展全基因组亚硫酸氢盐测序（WGBS）分析。上皮细胞通过流式细胞术从人体组织中分离，以EpCAM+/CD45-作为鉴定标记。数据处理流程参照已发表方法（Wang等，《自然·实验方案》（Nature Protocols），2013年）：使用BWA工具将测序读段比对至人类参考基因组hg19/GRCh37版本，移除重复读段；甲基化位点鉴定流程参照已发表方法（Delacher与Imbusch等，《自然·免疫学》（Nature Immunology），2017年）；将不同文库中每个CpG位点的甲基化与未甲基化读段原始计数按样本进行合并。
提交者声明，出于患者隐私保护考量，原始数据已上传至欧洲基因组-表型组档案库（EGA），其收录编号为EGAS00001004660。