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Mesothelin is a potential marker of pancreatic adenocarcinoma that was recently identified by serial analysis of gene expression. We evaluated the sensitivity and specificity of mesothelin as a marker of pancreatic adenocarcinoma on destained Papanicolaou (Pap) smears and unstained cellblocks from 28 patients using a monoclonal antibody to mesothelin. Intensity and proportion of staining was semiquantitatively graded on a scale of 1-3, and as 0%, 1 to <10%, 10-50%, or >50%. Positive staining for mesothelin was seen in 64% of the direct smears and in 36% of cell block sections. Focal positivity for mesothelin was noted in benign pancreatic tissue in one of 10 cases. Staining was most often focal (<50% of cells) in both direct smears and cell block sections. The overall sensitivity and specificity of mesothelin as a marker for pancreatic adenocarcinoma were 68% and 90%, respectively. Sensitivity was higher in Pap smears than in cell block sections (64% versus 36%). The presence of occasional mesothelin expression in benign tissue, its very focal expression in malignant tissue may limit the utility of mesothelin as a marker of pancreatic adenocarcinomas in fine-needle aspiration (FNA) specimens.