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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                    <jats:italic>n<\/jats:italic>\n                    \u2009=\u200974), extrahepatic biliary tree (\n                    <jats:italic>n<\/jats:italic>\n                    \u2009=\u200956), pancreatic duct (\n                    <jats:italic>n<\/jats:italic>\n                    \u2009=\u20094), gallbladder (\n                    <jats:italic>n<\/jats:italic>\n                    \u2009=\u20091), and peripancreatic lymph node (\n                    <jats:italic>n<\/jats:italic>\n                    \u2009=\u20091). 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\u2019s exact test:\n                    <jats:italic>p\u2009<\/jats:italic>\n                    =\u20090.0108, OR\u2009=\u20090.379, 95% CI 0.182\u20130.792). 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                    <jats:italic>n<\/jats:italic>\n                    \u2009=\u200942) 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\n                    <jats:italic>KRAS<\/jats:italic>\n                    mutations (\n                    <jats:italic>KRAS<\/jats:italic>\n                    G12D mutation occurred in 19\/41 [46,3%] of\n                    <jats:italic>KRAS<\/jats:italic>\n                    -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.\n                  <\/jats:p>","DOI":"10.1007\/s00428-026-04531-0","type":"journal-article","created":{"date-parts":[[2026,4,30]],"date-time":"2026-04-30T02:36:08Z","timestamp":1777516568000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Diagnostic utility of targeted next-generation sequencing in indeterminate pancreaticobiliary cytology and small biopsy specimens: a prospective cohort study"],"prefix":"10.1007","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4424-7174","authenticated-orcid":false,"given":"Krist\u00fdna","family":"Behensk\u00e1","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0930-7071","authenticated-orcid":false,"given":"Ond\u0159ej","family":"Daum","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2241-1383","authenticated-orcid":false,"given":"Magdal\u00e9na","family":"Daumov\u00e1","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lud\u011bk","family":"Bouchner","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5927-8942","authenticated-orcid":false,"given":"Zden\u011bk","family":"Chud\u00e1\u010dek","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ji\u0159ina","family":"Pintov\u00e1","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-5241-9723","authenticated-orcid":false,"given":"Tom\u00e1\u0161","family":"Van\u011b\u010dek","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Petr","family":"Mart\u00ednek","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4361-7628","authenticated-orcid":false,"given":"Roman","family":"Mezencev","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mihaela","family":"Farcas","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8052-4741","authenticated-orcid":false,"given":"Mari\u00e1n","family":"\u0160vajdler","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,4,30]]},"reference":[{"key":"4531_CR1","doi-asserted-by":"publisher","first-page":"229","DOI":"10.3322\/caac.21834","volume":"74","author":"F Bray","year":"2024","unstructured":"Bray F, Laversanne M, Sung H et al (2024) Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. 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