Pancreatic ductal adenocarcinoma is a disease of extremely poor prognosis for which there are no reliable markers of asymptomatic disease. To identify pancreatic cancer biomarkers, we focused on a genomic interval proximal to the most common fragile site in the human genome, chromosome 3p12, which undergoes smoking-related breakage, loss of heterozygosity, and homozygous deletion as an early event in many epithelial tumors, including pancreatic cancers. Using a functional genomic approach, we identified a seven-gene panel (TNC, TFPI, TGFBI, SEL-1L, L1CAM, WWTR1, and CDC42BPA) that was differentially expressed across three different expression platforms, including pancreatic tumor/normal samples. In addition, Ingenuity Pathways Analysis (IPA) and literature searches indicated that this seven-gene panel functions in one network associated with cellular movement/morphology/development, indicative of a "migration signature" of the 3p pathway. We tested whether two secreted proteins from this panel, tenascin C (TNC) and tissue factor pathway inhibitor (TFPI), could serve as plasma biomarkers. Plasma ELISA assays for TFPI/TNC resulted in a combined area under the curve (AUC) of 0.88 and, with addition of CA19-9, a combined AUC for the three-gene panel (TNC/TFPI/CA19-9), of 0.99 with 100% specificity at 90% sensitivity and 97.22% sensitivity at 90% specificity. Validation studies using TFPI only in a blinded sample set increased the performance of CA19-9 from an AUC of 0.84 to 0.94 with the two-gene panel. Results identify a novel 3p pathway-associated migration signature and plasma biomarker panel that has utility for discrimination of pancreatic cancer from normal controls and promise for clinical application. ©2010 AACR.