Background: Epidermal growth factor receptor (EGFR) expression does not predict response to anti-EGFR therapy with erlotinib in pancreatic cancer patients. In the absence of known pancreatic cancer EGFR activating mutations, we sought to identify alternative factors, such as increased gene copy number (genomic gain) and ligand overexpression, which could be associated with aberrant EGFR pathway activation and improved response to targeted therapy. Methods: EGFR gene copy number was analyzed by fluorescence in situ hybridization in nine pancreatic cancer cell lines and 31 pancreatic cancer surgical specimens. In vitro effects of erlotinib on tumor cell proliferation were tested. Tumor specimen EGFR expression levels were measured by reverse transcriptase-polymerase chain reaction. Expression of stimulating ligand (EGF), phosphorylated receptor (p-EGFR), and activated downstream adaptor proteins (p-Akt and p-ERK), were evaluated by immunohistochemistry and immunoblotting. Results: Pancreatic cancer EGFR genomic gain, in the form of high polysomy, was present in four of nine cell lines and in 10/24 (42%) of patients. Twenty-four patients (77%) expressed EGFR transcript, and of those, half displayed p-EGFR (35% of all patients). A majority of patients demonstrated downstream EGFR pathway activation, with 65% expressing p-Akt and 84% expressing p-ERK. EGFR-expressing tumors also expressed EGF, with exclusive tumor cell localization, suggesting autocrine stimulation of the EGFR pathway. EGF expression level was significantly greater in patients with increased EGFR gene copy number (P = 0.016). Conclusions: Increased EGFR gene copy number and elevated EGF levels are present in a significant proportion of pancreatic cancer patients, and this may reflect increased EGFR pathway dependence with improved sensitivity to EGFR-targeted therapy. © 2007 Elsevier Inc. All rights reserved.