Background: Presently there is an urgent need for better biomarkers for early pancreatic cancer (PaCa) diagnosis. Hypermethylation of several genes is known to correlate with presence of neoplasia in pancreatic tissue, and tumor DNA is known to be present in bodily fluids. With this background, we wanted to develop and validate a panel of methylated gene markers that would provide clinically relevant information when applied to plasma from those undergoing diagnostic procedures for pancreatic cancer.

Methods: Cell-free DNA was extracted from plasma obtained from 11 PaCa patients, 15 pancreatitis patients and 15 unaffected age-matched controls using either the automated EZ-1 (Qiagen, Valencia, CA) or the manual Charge Switch (Invitrogen, Carlsbad, CA) methods. The DNA was bisulfite treated using EZ DNA Methylation-Gold (Zymo Research, Orange, CA) then analyzed using a panel of quantitative real-time PCR assays to discriminate the methylated C or T that exists after bisulfite conversion, and normalizing the signal to the amplification level of a housekeeping gene (Col2A) to assess methylation of status of 13 genes known to be differentially methylated in pancreatic tissues. Several different combinations of 6 methylated gene markers (3-OST-2, ARF/p14, FOXE1, CDKN2/p16, ppENK, and RASSF1A) showed good discrimination between the 3 groups of patients to warrant further study with an additional 53 plasma specimens (from 21 PaCa patients, 17 pancreatitis patients, and 15 unaffected controls). Receiver operator curves were generated to examine the predictive probability between marker methylation and disease, and reliability of the regression model was evaluated by using the Hosmer-Leme show goodness-of-fit test.

Results: Statistical analysis of the 6 methylated gene markers for all 94 plasma specimens yielded an AUC of 0.638 (95% CI: 0.50-0.78) to differentiate PaCa from pancreatitis, and the goodness-of-fit for the model was good (p=0.697).

Conclusions: Cell-free DNA extracted from plasma is an appropriate sample for the study of gene methylation in PaCa. We utilized real-time assays to detect methylation in 6 target genes and found a good correlation between the methylation status of genes and the presence of PaCa. Further studies are needed to define the clinical utility of this approach. Funding was provided by a grant from the American Cancer Society - Illinois Division (#07-06) and by funds from the Department of Pathology and Laboratory Medicine at NorthShore.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4907.