Diagnostic utility of targeted next-generation sequencing in indeterminate pancreaticobiliary cytology and small biopsy specimens: a prospective cohort study

Pancreaticobiliary cytology is a highly specific but variably and often insufficiently sensitive diagnostic method. This study evaluated the utility of next-generation sequencing (NGS) in difficult-to-classify pancreaticobiliary specimens. A total of 136 patients were prospectively included, with cytology or small biopsy specimens from the pancreas (n = 74), extrahepatic biliary tree (n = 56), pancreatic duct (n = 4), gallbladder (n = 1), and peripancreatic lymph node (n = 1). All cases were diagnosed by cytopathologists with expertise in pancreaticobiliary (cyto)pathology using 2022 WHO Reporting System for Pancreaticobiliary Cytopathology. Large scale pan-cancer panel was used for NGS testing. Forty-five cases (33,1%) were non-analyzable by NGS, and no genetic changes were found in 21 cases (23,1% of analyzable cases). Separate acquisition and storage of cytology/biopsy materials in RNAlater solution significantly decreased the rate of non-analyzable samples (23.3% versus 44.4%; Fisher's exact test: p = 0.0108, OR = 0.379, 95% CI 0.182-0.792). Diagnostic genetic alterations were identified in 70/91 of analyzable cases (76.9%). Suspicion was raised for the presence of germline mutation in 10 patients. Potentially druggable alterations (n = 42) were found in 36/91 patients (39,5%), including mutations in homologous recombination genes, PI3K/AKT/mTOR pathway, Cyclin D1-CDK4/6-Rb pathway, and potentially targetable KRAS mutations (KRAS G12D mutation occurred in 19/41 [46,3%] of KRAS-mutated tumors). Supplementing pancreaticobiliary (cyto)pathology with information from NGS increased the sensitivity for tumor diagnosis from 35.9% to 86.6% (with specificity 100%), improved negative predictive value from 14.2% to 41.2%, and enhanced overall diagnostic accuracy from 42.1% to 87.8%, respectively. In challenging cases, NGS significantly improves preoperative pancreaticobiliary diagnosis and informs clinical decision-making.