RNA-seq profiling of upper tract urothelial carcinoma: bladder cancer consensus classification relevance, molecular heterogeneity and differential immune signatures

Analyses of large transcriptomics datasets of muscle-invasive bladder cancer (MIBC) has led to a consensus classification. Molecular subtypes of upper tract urothelial carcinomas (UTUC) are less known. Our objective was to determine the relevance of consensus classification in UTUCs by characterizing a novel cohort of surgically treated ≥pT1 tumors.Subtype IHC markers GATA3-CK5/6-TUBB2B in multiplex, CK20, p16 and Ki67, MMR proteins, PD-L1 IHC were evaluated. Heterogeneity was assessed morphologically and/or with subtype IHC. FGFR3 mutations were identified by pyrosequencing. We performed 3’RNA-seq, including with multisampling in heterogeneous cases. Consensus classes, unsupervised groups, and microenvironment cell abundance were determined using gene expression.Most of the 66 patients were men (77.3%), with pT1 (n=23, 34.8%) or pT2-4 stage UTUC (n=43, 65.2%). FGFR3 mutations and dMMR status were identified in 40% and 4.7% of cases, respectively. Consensus subtypes robustly classified UTUCs and reflected intrinsic subgroups. All pT1 tumors were classified as Luminal papillary (LumP). Combining our consensus classification results to that of previously published UTUC cohorts, LumP tumors represented 57.2% of ≥pT2 UTUC, which was significantly higher than in MIBC. Ten patients (15.2%) harbored areas of distinct subtypes. Consensus classes were associated with FGFR3 mutations, stage, morphology and IHC. The majority of LumP tumors were characterized by low immune infiltration and PD-L1 expression, in particular if FGFR3 mutated. 3' Tag RNA-sequencing of 87 upper tract urothelial carcinoma samples from 66 patients

针对肌肉浸润性膀胱癌（muscle-invasive bladder cancer, MIBC）的大型转录组学（transcriptomics）数据集开展的分析已形成共识分类方案。上尿路尿路上皮癌（upper tract urothelial carcinomas, UTUC）的分子亚型仍有待进一步阐明。本研究旨在通过表征一组全新的手术治疗≥pT1期肿瘤队列，明确该共识分类方案在上尿路尿路上皮癌中的应用价值。我们对多重免疫组化（immunohistochemistry, IHC）标志物GATA3-CK5/6-TUBB2B、CK20、p16、Ki67、错配修复（mismatch repair, MMR）蛋白及PD-L1进行了免疫组化检测。通过形态学观察和/或亚型免疫组化分析评估肿瘤异质性；采用焦磷酸测序（pyrosequencing）技术检测FGFR3突变；对样本开展3'RNA测序（3'RNA-seq），针对存在异质性的病例进行多次采样。基于基因表达数据，我们确定了肿瘤的共识亚型、无监督聚类组别以及微环境细胞丰度。本队列共纳入66例患者，其中男性占比77.3%；肿瘤分期为pT1者23例（34.8%），pT2-4期者43例（65.2%）。分别在40%和4.7%的病例中检测到FGFR3突变与错配修复缺陷（deficient MMR, dMMR）状态。共识亚型可稳定区分上尿路尿路上皮癌，并反映其内在亚群特征。所有pT1期肿瘤均被归类为腔面乳头状（Luminal papillary, LumP）亚型。将本研究的共识分类结果与已发表的上尿路尿路上皮癌队列数据整合后发现，腔面乳头状亚型肿瘤占≥pT2期上尿路尿路上皮癌的57.2%，该比例显著高于肌肉浸润性膀胱癌。10例患者（15.2%）的肿瘤中存在不同亚型的区域。共识亚型与FGFR3突变、肿瘤分期、组织学形态及免疫组化标志物显著相关。多数腔面乳头状亚型肿瘤以低免疫浸润和低PD-L1表达为特征，若同时携带FGFR3突变则该特征尤为突出。本研究对66例患者的87份上尿路尿路上皮癌样本完成了3'端标签RNA测序（3' Tag RNA-sequencing）。