AimsRadiological imaging and morphological assessment of cytology material have limitations for preoperative classification of pancreatic or periampullary lesions, often resulting in surgical resection without definitive diagnosis. Our prospective study aims to define the diagnostic value of targeted next-generation sequencing (NGS) of DNA from cytology material.MethodsPatients with a suspect pancreatic or periampullary lesion underwent standard diagnostic evaluation including preoperative morphological cytology assessment. Treatment options for suspect lesions were surgical exploration with possible resection, follow-up or palliation. The cytology samples were analysed with NGS, in which 50 genes were sequenced for the presence of pathogenic variants. The NGS results were integrated with the clinical information during multidisciplinary team meetings, and changes in the treatment plan were scored. Diagnostic accuracy of NGS analysis (malignancy vs benign disease) was calculated.ResultsNGS results of the cytology samples were confirmed in the resection specimens of the first 10 included patients. The integration of the NGS results led to a change in treatment plan in 7 out of 70 patients (from exploration to follow-up, n=4; from follow-up to exploration and resection, n=2; from palliation to resection, n=1). In four patients, the NGS results were contradictory, but did not affect the treatment plan. In the remaining 59 patients, NGS analysis supported the initial treatment plan. The diagnostic accuracy of NGS analysis was 94% (sensitivity=93%; specificity=100%).ConclusionsNGS can change the treatment plan in a significant portion of patients with suspect pancreatic or periampullary lesions. Application of NGS can optimise treatment selection and diminish unnecessary surgeries.