Background: Pancreatic cancer remains a deadly disease, and the vast majority of pancreatic cancer patients are not candidates for treatment with curative intent. Erlotinib, an EGFR-specific tyrosine kinase inhibitor, was approved recently for use in patients with pancreatic cancer. Somatic mutations in the EGFR gene appear to predict survival and response to tyrosine kinase inhibitor therapy in a subset of patients with non-small-cell lung cancer (NSCLC). Methods: The purpose of this study was to characterize EGFR mutations in pancreatic adenocarcinoma. EGFR TK mutations were analyzed in 9 pancreatic carcinoma cell lines and 31 clinical specimens from patients with pancreatic cancer who underwent resection. Using laser capture microdissection, tumor cells from patients were harvested selectively for genomic DNA extraction. Mutations were examined by direct sequencing of exons 18-21. Results: Of 9 pancreatic cancer cell lines, 6 had either 2454G>A or 2361G>A mutations in exon 20. Of 31 patients, 25 patients had 2361G>A in exon 20, and 1 patient had 2508C>T in exon 21. All were silent mutations. Conclusions: The EGFR tyrosine kinase domain is highly conserved in pancreatic cancer. The association among EGFR mutation status, clinical prognosis, and response to anti-EGFR therapy described in NSCLC may not be applicable to pancreatic cancer. This observation does not diminish the possible role of anti-EGFR therapy in pancreatic cancer because there remains a need to explore alternative explanations for pancreatic cancer aberrant EGFR pathway activation such as ligand overexpression, gene amplification, and loss of inhibitory mechanisms. © 2007 Mosby, Inc. All rights reserved.