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Cystic lesions of the pancreas are increasingly being recognized due to the widespread use of high resolution abdominal imaging. Since certain cyst types are precursors to invasive cancer, this situation presents an opportunity to intervene prior to malignant progression. Effective implementation of that strategy has been hampered by difficulties in clearly distinguishing cystic lesions with no malignant potential from those with malignant potential. Here we explored whether glycosylation variants on specific proteins in cyst fluid samples could serve as biomarkers to aid in this diagnosis.We used a novel antibody-lectin sandwich microarray method to measure the protein expression and glycosylation of mucin (MUC)1, MUC5AC, MUC16, carcinoembryonic antigen, and other proteins implicated in pancreatic neoplasia in cyst fluid samples. Fifty-three cyst fluid samples were obtained from patients with mucinous cystic neoplasms (n = 17), intraductal papillary mucinous neoplasms (n = 15), serous cystadenomas (n = 12), or pseudocysts (n = 9), with confirmation of histologic diagnosis at surgical resection.The detection of a glycan variant on MUC5AC using the lectin wheat-germ agglutinin discriminated mucin-producing cystic tumors (mucinous cystic neoplasms + intraductal papillary mucinous neoplasms) from benign cystic lesions (serous cystadenomas + pseudocysts) with a 78% sensitivity at 80% specificity, and when used in combination with cyst fluid CA 19-9 gave a sensitivity of 87% at 86% specificity. These biomarkers performed better than cyst fluid carcinoembryonic antigen (37%/80% sensitivity/specificity).These results demonstrate the value of glycan variants for biomarker discovery and suggest that these biomarkers could greatly enhance the accuracy of differentiating pancreatic cystic tumors. Validation studies will be required to determine the clinical value of these markers.