Beronja, SlobodanMills, Claire Burke2026-04-202026-04-202026Mills_washington_0250E_29218.pdfhttps://hdl.handle.net/1773/55425Thesis (Ph.D.)--University of Washington, 2026Local recurrence rates in head and neck squamous cell carcinoma (HNSCC) exceed 50%, potentially due to field cancerization, a phenomenon where tissue that appears normal microscopically actually harbors molecular changes that can give rise to new tumors. The prevailing model assumes these "at-risk" tissues share driver mutations with adjacent tumors, but whether field cancerization depends solely on mutations remains unknown. To comprehensively characterize this phenomenon, we profiled matched tumor, normal-adjacent field (NAF), contralateral normal, and blood samples from HPV-negative HNSCC patients using whole genome sequencing, targeted driver gene sequencing, single-nucleus multiome profiling, and functional cell-fate tracing assays. While histologically normal NAF tissues did harbor expanded mutant clones, these mutations rarely overlapped with paired tumors and were not shared across patients, suggesting features beyond mutations are altered in NAF. Single-cell multiomics revealed that NAF epithelium loses its normal organizational hierarchy: basal stem cell markers persist in cells that should be differentiating, while terminal differentiation programs fail to activate. Proliferating cells appeared in tissue layers normally restricted to post-mitotic cells, a pattern typically seen only in overt dysplasia. Additionally, we identified a distinct "emerging state" cell population enriched in NAF that expresses tumor-associated markers and transcriptionally bridges normal epithelium and tumor, which we spatially identify using immunofluorescence staining of key markers. Together, our findings demonstrate that field cancerization operates through epithelial hierarchy disorganization, disrupting differentiation and generating cells with tumor-like features, all within tissue that appears histologically normal.application/pdfen-USnoneBiologyDentistryTo Be AssignedThesis