DataSheet_1_Mutational signature and clonal relatedness of recurrent urothelial carcinomas with aristolochic acid.docx

Urothelial carcinomas (UCs) are malignant tumors that arise from the lower and upper urinary tract and are characterized by multiple recurrences. Aristolochic acid (AA) is a potent nephrotoxin and human carcinogen associated with UC. East Asian populations with a high UC prevalence have an unusual genome-wide AA-induced mutational pattern. To address the genomic differences and clonal relatedness between primary and recurrent tumors in the UCs with AA pattern, we investigated the genomic differences and tumor microenvironment (TME) of AA and non-AA UCs. 17 UC patients were recruited, with nine documented AA exposure. Eleven of them showed recurrence. After-surgery tissues of primary and paired recurrent tumors were collected. Capture-based targeted deep sequencing was performed using a commercial panel consisting of 520 cancer-related genes. Tumor-infiltrating lymphocytes (TILs) were identified with an immunofluorescence-based microenvironment analysis panel (MAP). Hierarchical clustering based on the COSMIC signatures confirmed two significant subtypes: AA Sig and non-AA Sig. AA Sig was associated with AA-containing herbal drug intake, recurrence, and higher tumor mutation burden (TMB). The clonal architecture of UCs revealed three types of clonal evolution patterns. Non-AA Sig cohort showed shared clonal origin of primary and recurrent tumors. AA Sig showed heterogeneity and had multiple independent origins. Recurrent tumors as second primary tumors in AA Sig showed immunoreactive TME, indicating a better response with immune checkpoint inhibitor therapy. The AA mutational signature and unique immune profiles are helpful molecular markers to distinguish AA exposure from other carcinogens. These results also provide new insights into the origin of recurrent UCs that could affect treatment strategies.

尿路上皮癌（Urothelial carcinomas, UCs）是起源于上下尿路的恶性肿瘤，以多次复发为典型特征。马兜铃酸（Aristolochic acid, AA）是一种强效肾毒素及人类致癌物，与UC的发生密切相关。在UC患病率较高的东亚人群中，存在独特的全基因组AA诱导突变特征。为阐明携带AA突变特征的UC患者中，原发与复发肿瘤的基因组差异及克隆相关性，本研究对比分析了AA暴露型与非AA暴露型UC的基因组特征及肿瘤微环境（Tumor microenvironment, TME）。本研究共招募17例UC患者，其中9例有明确的AA暴露史，11例出现肿瘤复发。术后采集患者的原发肿瘤及配对复发肿瘤组织，采用包含520个癌症相关基因的商业捕获试剂盒开展基于捕获的靶向深度测序；同时通过基于免疫荧光的微环境分析试剂盒（immunofluorescence-based microenvironment analysis panel, MAP）鉴定肿瘤浸润淋巴细胞（Tumor-infiltrating lymphocytes, TILs）。基于COSMIC特征（COSMIC signatures）的分层聚类分析确认了两个显著亚型：AA特征亚型（AA Sig）与非AA特征亚型（non-AA Sig）。AA特征亚型与含AA的中草药摄入、肿瘤复发及更高的肿瘤突变负荷（Tumor mutation burden, TMB）显著相关。UC的克隆结构可分为三种克隆进化模式：非AA特征亚型队列中，原发与复发肿瘤具有共同克隆起源；而AA特征亚型队列则表现出肿瘤异质性，原发与复发肿瘤具有多个独立克隆起源。AA特征亚型队列中的复发肿瘤属于第二原发肿瘤，其肿瘤微环境呈免疫反应性，提示此类患者可从免疫检查点抑制剂治疗中获得更优应答。AA突变特征及独特的免疫谱可作为区分AA暴露与其他致癌物的有效分子标志物，本研究结果还为阐明复发性UC的起源提供了新见解，或将为后续治疗策略的制定提供参考。