Simple SummaryUrothelial carcinoma of the large, nested variant is a specific histological morphology subtype of urothelial carcinoma. Although it is a rare variant, it requires specific attention due to its bland histology and the fact that it may potentially be missed in routine biopsies. In this study, we identifyFibroblast Growth Factor Receptor-3(FGFR-3) as the most common mutation present in this subtype among other potential targetable mutations. All our cases of this variant also harbored other potentially actionable mutations in other genes, which could also be amenable to novel targeted therapy. Patients with this variant may benefit from additional molecular screening to identify potential therapeutic targets that could improve the clinical outcome of such patients.AbstractThe large, nested variant of urothelial carcinoma (LNVUC) is characterized by bland histomorphology mimicking that of benign von Brunn nests. In the current study, we aimed to investigate theFibroblast Growth Factor Receptor-3 (FGFR-3) activation and missense mutation in 38 cases, including 6 cases diagnosed with LNVUC and 32 with metastatic invasive urothelial carcinoma (UC). Initially, six formalin-fixed paraffin-embedded (FFPE) tissue samples of the LNVUC were subjected to whole-exome sequencing (WES), and then we performed targeted sequencing on 32 cases of metastatic invasive UC of various morphological subtypes, which were interrogated for theFGFR3. Our results revealed 3/6 (50%) LNVUC cases evaluated by WES in our study showed an activating mutation inFGFR-3, 33% showed an activating mutation inPIK3CA, and 17% showed activating mutation inGNASorMRE11. Additionally, 33% of cases showed a truncating mutation inCDKN1B. All LNVUC in our study that harbored theFGFR-3mutation showed additional activating or truncating mutations in other genes. Overall, 6/32 (18.75%) cases of random metastatic invasive UC showed missense mutations of theFGFR-3gene. The LNVUC variant showed the higher incidence ofFGFR-3mutations compared to other types of mutations. Additionally, all LNVUC cases show additional activating or truncating mutations in other genes, thus being amenable to novel targeted therapy.