Background: TERT is a catalytic subunit of telomerase, the unique enzyme that confers immortality to cells and is expressed in >90% of cancer cells. Mutations in the TERT promoter region (pTERTmut) are the most prevalent noncoding mutations in cancer. Recent data suggest pTERTmut are associated with improved outcomes in patients with UC treated with immune checkpoint inhibitors. We evaluated the molecular and immune landscape of UC with and without pTERTmut. Methods: UC tissue samples were analyzed for DNA alterations (NextSeq, 592 Genes; NovaSeq, WES) and mRNA expression (NovaSeq, WTS). Immune cell fraction was calculated by QuantiSeq (Finotello 2019, Genome Medicine). PD-L1 expression was assessed by immunohistochemistry (IHC) (Caris Life Sciences, Phoenix, AZ). MSI/MMR was tested by fragment analysis, IHC and NGS. TMB-H was based on a cut-off of > 10 mut/MB. We compared alterations between samples with and without detected pathogenic pTERTmut. Significance was determined by Mann–Whitney U, X², and Fischer-Exact and p adjusted for multiple comparisons (q) was < 0.05 using Benjamini-Hochberg. Results: Overall, 1686 UC samples were analyzed, 1166 from primary lesions and 499 from a lymph node or metastatic site. pTERTmut was present in 68% of primary and 61% of metastatic tumors, and correlated with modest increase of TERT expression (1.18 fold, p=0.015). pTERTmut was associated with less frequent alterations in TP53, KMT2D, CCND1, MYC, KEAP1 and less MSI/dMMR. By contrast, pTERTmut was associated with more frequent alterations in ARID1A, TSC1, PIK3CA and TMB-H (all q<0.05). Over 41% of pTERTmut were TMB-H. TERTp mutations were not associated with FGFR alterations. The frequency of co-occurring mutations was similar by specimen site. In evaluating the immune landscape (Table), pTERTmut was associated with higher expression of PD-L1 (IHC, mRNA), PD-L2 (mRNA) and TIM3 (mRNA) in tumors from primary sites (all p and q<0.05), but not in metastatic sites. Investigation of tumor-associated immune cells demonstrated that pTERTmut correlated with higher percentage of M1-macrophages and CD8+ T cells in primary tumors, and was inversely-correlated with NK cells in metastatic sites (all q<0.05). Conclusions: This is the largest analysis looking at the molecular and immune landscape of pTERTmut UC tumors. We observed differential patterns of DNA alterations and tumor immune microenvironment based on pTERTmut status. Further work is needed to understand differences in these molecular cohorts and the association of these data with clinical outcomes.

*q<0.05.