e16018 Background: UC liver metastases are associated with a low response rate to immune checkpoint blockade (ICB) and short survival. We sought to identify tumor specific factors promoting resistance to ICB. Methods: DEPArray is a dielectrophoresis based cell sorting platform for isolating immunohistochemically-defined cell populations from formalin-fixed paraffin-embedded (FFPE) tissue. Available FFPE tissue blocks of mUC liver metastases from patients (pts) treated with ICB were selected for analysis. Following histologic evaluation, tissue blocks were digested to yield cellular suspensions for immunophenotypic DEPArray sorting by keratin (K) and vimentin (V) to identify carcinoma cells and stromal cells, respectively. Cell pools were further analyzed for recurrent mutations and copy number alterations by a targeted OncoSeek panel and by MiSeq. Results: 12 unique patient samples were profiled. Median age was 73 (range 65-89), 11/12 male, 7 (58%) were platinum-refractory, and 4 (33%) received only ICB. 7 (58%) biopsies were obtained prior to initiation of ICB (1PR, 1SD, 5PD). Routine H&E sections generally revealed tumors completely devoid of immune cells, but admixed with atypical mesenchymal cells ranging from 5% to 60% of cellular composition. DEPArray sorting by K and V identified three discrete cell populations: K+/V- carcinoma cells, K-/V+ stromal cells, and K+/V+ cells that exhibited mesenchymal morphology. Of 8 samples with adequate cell counts (range 75-154 cells), next-generation sequencing (NGS) identified that K+/V- carcinoma cells and K+/V+ double positive cells shared unique somatic alterations in 7 (88%) pts, including point mutations in FGFR3, ERBB2, FBXW7, PIK3CA, FLT3 and STK11, and amplification of CDK4. In one liver responder to ICB, V+ stromal cells carried a putative germline TP53 R248W mutation. Conclusions: Archival FFPE tissue from mUC liver metastases can be digitally sorted by DEPArray to yield pools of ~100 cells suitable for NGS. UC liver metastases exhibit a population of K+/V+ cells with clonal somatic alterations suggestive of an EMT cancer cell phenotype that may be playing a role in ICB resistance.