Features of tumor microenvironment in HER2-positive urothelial carcinoma and its implications for immunotherapy resistance

BACKGROUND: HER2 overexpression or ERBB2 amplification represents an important molecular feature of urothelial carcinoma (UC) and has been associated with differential responses to immune checkpoint inhibitors (ICIs). However, the tumor microenvironmental (TME) features associated with HER2-defined UC and their potential relationship with ICI responsiveness remain incompletely characterized. METHODS: Tumor tissues from 52 UC patients treated with ICIs monotherapy at our center underwent multiplex immunofluorescence (IF) staining to evaluate immune cell infiltration and immune checkpoint protein expression. In parallel, transcriptomic and somatic mutation data from 408 UC cases in The Cancer Genome Atlas (TCGA) were analyzed. Differential gene expression and pathway enrichment analyses were performed based on HER2 copy number status (GISTIC score ≥ 2 for amplification). The xCell algorithm was used to estimate immune cell infiltration in bulk RNA-seq data. RESULTS: In the institutional cohort, HER2-positive tumors showed numerically lower objective response rate (ORR) and disease control rate (DCR) than HER2-negative tumors, although these differences were not statistically significant. Multiplex IF analysis revealed reduced infiltration of CD4⁺ T cells, CD8⁺ T cells, and CD20⁺ B cells in HER2-positive tumors. PD-1 and STING expression were also lower in HER2-positive tumors, whereas LAG-3, TIM-3, CTLA-4, and PD-L1 showed non-significant downward trends. In the TCGA-BLCA cohort, ERBB2-amplified tumors showed downregulation of immune-related pathways and reduced inferred infiltration of multiple immune cell populations, including T cells, dendritic cells, B cells, NK cells, and macrophages. CONCLUSIONS: HER2-positive UC in the institutional IHC-defined cohort and ERBB2-amplified UC in the TCGA cohort were associated with reduced adaptive immune infiltration and attenuated immune-related signaling. These findings support the presence of an immune-cold phenotype in HER2-defined UC and provide hypothesis-generating evidence for further investigation of biomarker-guided combination strategies integrating HER2-targeted therapy with immune checkpoint blockade.