Orthogonal confirmation could include targeted resequencing in expanded, clinically harmonized CCA cohorts; protein-level assessments (e.g., TUBB3/FLNC) and locus-specific methylation assays; and isogenic perturbation of sentinel variants (e.g., MAP2K1 p.R49C) in patient-derived organoids and xenografts using CRISPR base editing to quantify pathway output (p-ERK), microtubule dynamics, cytoskeletal mechanics, and cholesterol efflux (ABCA1) with pharmacologic challenge (MEK/ERK inhibitors, taxanes, LXR agonists/statins). The gene discussed is TUBB3; the disease is cholangiocarcinoma.