Robust Consensus Molecular Subtyping of Muscle-Invasive Bladder Cancer Via 3' RNA Sequencing of Formalin-Fixed...

Transcriptome-based tumor classification has enhanced the molecular characterization of muscle-invasive bladder cancer (MIBC) subtypes. However, the degraded nature of formalin-fixed paraffin-embedded (FFPE) material and the expensive sequencing costs for routine use have limited the use of subtypes in clinical and trial settings. Here, we present an optimized analysis workflow for MIBC molecular subtype prediction from FFPE samples. FFPE material from 240 MIBC samples was sequenced using QuantSeq 3' mRNA sequencing with unique molecular identifiers (UMIs) and analyzed via a customized RNA-Seq pipeline. The association of consensus subtypes with histology and immunohistochemical expression of core basal/luminal protein markers was assessed. In addition, subtype robustness was explored by simulating scenarios at lower sequencing depths and without UMIs. Five MIBC consensus subtypes were identified in the cohort. The basal/squamous group showed higher expression of KRT14, KRT5, and CD44, and was mainly divergent squamous. Vice versa, luminal, and stroma-rich subtypes had conventional urothelial or urothelial subtype histology, with higher expression of KRT20, FOXA1, and GATA3. The neuroendocrine-like samples had small cell neuroendocrine histology and were negative for luminal/basal markers. Subtype calling from 24 matched fresh-frozen samples analyzed with full-length RNA-Seq showed 87.5% agreement. Furthermore, the subtypes were robust to decreasing sequencing depths and to the absence of UMIs. Taken together, we provide a robust and cost-effective workflow for MIBC consensus molecular subtyping from FFPE-derived RNA. This workflow can be easily implemented as a molecular pathological assay for patient care, clinical trials, and translational research.