Leukocyte DNA Methylation Signature Differentiates Pancreatic Cancer Patients from Healthy Controls

Katrina S Pedersen,Gloria M Petersen,Martha E Matsumoto,Mariza De Andrade,Hui Tang,Ann L Oberg,Stephen N Thibodeau,William R Bamlet,Liang Wang,Hana Algül

doi:10.1371/journal.pone.0018223

Abstract

Pancreatic adenocarcinoma (PaC) is one of most difficult tumors to treat. Much of this is attributed to the late diagnosis. To identify biomarkers for early detection, we examined DNA methylation differences in leukocyte DNA between PaC cases and controls in a two-phase study. In phase I, we measured methylation levels at 1,505 CpG sites in treatment-naïve leukocyte DNA from 132 never-smoker PaC patients and 60 never-smoker healthy controls. We found significant differences in 110 CpG sites (false discovery rate <0.05). In phase II, we tested and validated 88 of 96 phase I selected CpG sites in 240 PaC cases and 240 matched controls (p≤0.05). Using penalized logistic regression, we built a prediction model consisting of five CpG sites (IL10_P348, LCN2_P86, ZAP70_P220, AIM2_P624, TAL1_P817) that discriminated PaC patients from controls (C-statistic = 0.85 in phase I; 0.76 in phase II). Interestingly, one CpG site (LCN2_P86) alone could discriminate resectable patients from controls (C-statistic = 0.78 in phase I; 0.74 in phase II). We also performed methylation quantitative trait loci (methQTL) analysis and identified three CpG sites (AGXT_P180_F, ALOX12_E85_R, JAK3_P1075_R) where the methylation levels were significantly associated with single nucleotide polymorphisms (SNPs) (false discovery rate <0.05). Our results demonstrate that epigenetic variation in easily obtainable leukocyte DNA, manifested by reproducible methylation differences, may be used to detect PaC patients. The methylation differences at certain CpG sites are partially attributable to genetic variation. This study strongly supports future epigenome-wide association study using leukocyte DNA for biomarker discovery in human diseases.

Highlights

Pancreatic cancer (PaC) is the 10th most common tumor type for men and women in yearly incidence in the United States and the fourth leading cause of cancer mortality [1]
These analyses identified significant differences between PaC patients and controls at 110 CpG sites in 92 independent genes (FDR #0.05). 109 of the 110 significant CpG sites were located on autosomes
In this two phase study, we examined peripheral blood DNA to determine whether differences in methylation at various CpG sites could distinguish between subjects with and without PaC

Summary

Introduction

Pancreatic cancer (PaC) is the 10th most common tumor type for men and women in yearly incidence in the United States and the fourth leading cause of cancer mortality [1]. PaC is associated with a very poor prognosis as it remains one of the most difficult tumors to treat. Much of this may be attributed to the late stage at which cancer is usually detected. Between 1999 and 2006, only 8% of patients were diagnosed, often by incidental finding on radiologic imaging, at a localized stage where immediate surgical resection and subsequent cure could be considered [2]. With few to no characteristic symptoms and insensitive methods for early detection, curative intervention is rare

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