title: New DNA Methylation Markers for Pancreatic Cancer: Discovery, Tissue Validation, and Pilot Testing in Pancreatic Juice.
abstract: OBJECTIVE
  Discriminant markers for pancreatic cancer detection are needed. We sought to identify and validate methylated DNA markers for pancreatic cancer using next-generation sequencing unbiased by known targets.
  
  
  METHODS
  At a referral center, we conducted four sequential case-control studies: discovery, technical validation, biologic validation, and clinical piloting. Candidate markers were identified using variance-inflated logistic regression on reduced-representation bisulfite DNA sequencing results from matched pancreatic cancers, benign pancreas, and normal colon tissues. Markers were validated technically on replicate discovery study DNA and biologically on independent, matched, blinded tissues by methylation-specific PCR. Clinical testing of six methylation candidates and mutant KRAS was performed on secretin-stimulated pancreatic juice samples from 61 patients with pancreatic cancer, 22 with chronic pancreatitis, and 19 with normal pancreas on endoscopic ultrasound. Areas under receiver-operating characteristics curves (AUC) for markers were calculated.
  
  
  RESULTS
  Sequencing identified >500 differentially hyper-methylated regions. On independent tissues, AUC on 19 selected markers ranged between 0.73 and 0.97. Pancreatic juice AUC values for CD1D, KCNK12, CLEC11A, NDRG4, IKZF1, PKRCB, and KRAS were 0.92*, 0.88, 0.85, 0.85, 0.84, 0.83, and 0.75, respectively, for pancreatic cancer compared with normal pancreas and 0.92*, 0.73, 0.76, 0.85*, 0.73, 0.77, and 0.62 for pancreatic cancer compared with chronic pancreatitis (*, P = 0.001 vs. KRAS).
  
  
  CONCLUSIONS
  We identified and validated novel DNA methylation markers strongly associated with pancreatic cancer. On pilot testing in pancreatic juice, best markers (especially CD1D) highly discriminated pancreatic cases from controls.
authors.affiliation: Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota. Kisiel.john@mayo.edu.
authors.forename: John B
authors.initials: JB
authors.lastname: Kisiel
authors.affiliation: Division of Gastroenterology and Hepatology, Mayo Clinic, Jacksonville, Florida.
authors.forename: Massimo
authors.initials: M
authors.lastname: Raimondo
authors.affiliation: Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota.
authors.forename: William R
authors.initials: WR
authors.lastname: Taylor
authors.affiliation: Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Tracy C
authors.initials: TC
authors.lastname: Yab
authors.affiliation: Department of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, Minnesota.
authors.forename: Douglas W
authors.initials: DW
authors.lastname: Mahoney
authors.affiliation: Department of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, Minnesota.
authors.forename: Zhifu
authors.initials: Z
authors.lastname: Sun
authors.affiliation: Department of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, Minnesota.
authors.forename: Sumit
authors.initials: S
authors.lastname: Middha
authors.affiliation: Department of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, Minnesota.
authors.forename: Saurabh
authors.initials: S
authors.lastname: Baheti
authors.affiliation: Center for Individualized Medicine, Mayo Clinic, Rochester, Minnesota.
authors.forename: Hongzhi
authors.initials: H
authors.lastname: Zou
authors.affiliation: Anatomic Pathology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Smyrk
authors.affiliation: Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Boardman
authors.affiliation: Department of Epidemiology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Gloria M
authors.initials: GM
authors.lastname: Petersen
authors.affiliation: Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota.
authors.forename: David A
authors.initials: DA
authors.lastname: Ahlquist
mesh_terms: D000368:Aged; D019540:Area Under Curve; D014408:Biomarkers, Tumor; D016022:Case-Control Studies; D018899:CpG Islands; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D057890:Epigenomics / Q000379:methods; D005260:Female; D018628:Gene Dosage; D006801:Humans; D008297:Male; D008875:Middle Aged; D009367:Neoplasm Staging; D010189:Pancreatic Juice; D010190:Pancreatic Neoplasms / Q000175:diagnosis / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA
chemical_list: D014408:Biomarkers, Tumor