Molecular analysis of centrifugation supernatant fluid from pancreaticobiliary duct samples can improve cancer detection

Objective: We aimed to supplement microscopic examination of biliary cytobrush specimens to improve sensitivity by mutational profiling of: (1) selected cells microdissected from cytology slides and (2) corresponding cell-free DNA in residual supernatant fluid. Study Design: From 43 patients with brushings of bile or pancreatic duct strictures, DNA was extracted from microdissected cells and 1-2 ml of cytocentrifugation supernatant fluid. Mutational analysis targeted 17 genomic sites associated with pancreaticobiliary cancer, including sequencing for KRAS point mutation and loss of heterozygosity (LOH) analysis of microsatellites located at 1p, 3p, 5q, 9p, 10q, 17p, 17q, 21q, and 22q. Results: Mutations were found in 25/28 patients with malignancy, and no mutations were found in 5/5 patients with benign surgical results. The cell-free supernatant fluid generally contained higher levels and quality of DNA, resulting in increased detection of mutations in most patients. KRAS mutations only occurred in patients with pancreatic cancer. Mutational profiling of supernatant fluid specimens resulted in high sensitivity and specificity for malignancy, improving the detection of malignancy over cytology alone. Conclusion: Brush cytology specimens yielded supernatant fluid enriched with DNA, probably from actively proliferating cells. Mutational profiling can enhance the cytologic evaluation and characterization of specimens suspected to contain pancreatic or bile duct cancer.