New DNA Methylation Markers for Pancreatic Cancer: Discovery, Tissue Validation, and Pilot Testing in Pancreatic Juice

Abstract

Purpose 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. Experimental Design At a referral center, we conducted four sequential case-control studies: discovery, technical validation, biological 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 6 methylation candidates and mutant KRAS was performed on secretin-stimulated pancreatic juice samples from 61 pancreatic cancer patients, 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 – 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 to normal pancreas and 0.92*, 0.73, 0.76, 0.85*, 0.73, 0.77 and 0.62 for pancreatic cancer compared to chronic pancreatitis (*p=0.001 vs KRAS). Conclusion 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.

Pancreatic Cancer Cell Fraction Estimation in a DNA Sample

“…Third, our present criterion for the isolation of fraction marker genes might have been too strict. Indeed, although BNC1, ADMTS1, and CD1D have been reported to be accurate in the detection of pancreatic cancers [37,38], these genes were excluded from the candidate marker genes in this study. Specifically, ADMTS1 showed a low incidence of methylation (β > 0.3) in the cancer tissues (0-2 of 22 tissues).…”

Epigenetics of gastrointestinal diseases: notes from a workshop

“…The use of these samples allows for a non-invasive approach to assess targeted aberrancies of DNA methylation. Multi-target stool DNA assays detect 94% of Stage I-II colorectal cancer (CRC) and 70% of advanced adenomas with high-grade dysplasia; the assay technology used in the COLOGUARD system developed in collaboration with Exact Sciences (Madison, WI) is expected to allow for detection of DNA methylation patterns in other GI cancers as well [36]. Dr. Kisiel’s group has shown that aberrant DNA methylation patterns in stool is more broadly informative and sensitive for detection of neoplasia than individual DNA mutations.…”

Genomics in Primary and Secondary Prevention of Pancreatic Cancer

“…Kisiel et al [38] identified a methylomics profile in pancreatic juice that discriminated well PC from chronic pancreatitis and normal pancreas with AUCs up to 0.92 for CD1D . Unfortunately, the collection of pancreatic juice requires an aggressive maneuver that cannot be implemented in a population screening program.…”