Role of novel DNA methylation markers in detection of advanced neoplasia in pancreatic cysts

Feb. 27, 2018

Pancreatic cystic lesions are often detected incidentally on cross-sectional abdominal imaging (such as CT or MRI). A Mayo Clinic study published in Clinical Gastroenterology and Hepatology in 2016 reported a prevalence of 41.6 percent for incidental pancreatic cysts in patients undergoing abdominal magnetic resonance imaging (MRI).

The vast majority of these cysts do not harbor advanced neoplasia and are at a low risk of malignant degeneration. Annual incidence of pancreatic cancer (PC) in patients with pancreatic cystic lesions (PCLs) is less than 1 percent. Given that pancreatic resection is associated with a high degree of morbidity and mortality, current guidelines use a combination of clinical and imaging characteristics to identify "high-risk" and "worrisome" cysts that can be selected for surgical resection.

A significant percentage of cysts surgically resected based on these current prediction algorithms are found to be nondysplastic or harbor low-grade dysplasia and would not have justified surgery if detected preoperatively. Experts recommend reserving surgical resection for patients with cysts harboring high-grade dysplasia (HGD) or early invasive cancer. However, none of the currently available cyst fluid biomarkers can accurately detect the presence of HGD or cancer in pancreatic cysts.

"Currently available risk prediction algorithms for detection of advanced neoplasia in pancreatic cysts lack sufficient sensitivity, specificity and diagnostic accuracy," explains Shounak Majumder, M.D., a Mayo Clinic gastroenterologist in Rochester, Minnesota, whose research focuses on pancreatic diseases. "These issues make assessment of PCLs for malignant potential and surgical case selection extremely challenging."

To address these issues, Dr. Majumder and gastroenterologist David A. Ahlquist, M.D., also from Mayo Clinic's campus in Minnesota, led a research team that conducted two sequential case-control studies designed to discover and validate the performance of a panel of novel methylated DNA markers to accurately identify PCLs with HGD or PC. These studies were partly funded by Exact Sciences, as part of a larger collaboration between Mayo Clinic and Exact Sciences that focuses on biomarker discovery and cancer diagnostics.

Mayo Clinic researchers from all three campuses collaborated on these studies. John B. Kisiel, M.D., Mark D. Topazian, M.D., and Gloria M. Petersen, Ph.D., participated from Mayo Clinic's Minnesota campus. Massimo Raimondo, M.D., participated from Mayo Clinic's Florida campus. Rahul Pannala, M.D., participated from Mayo Clinic's Arizona campus.

Study goals and design

The goal of the first study was to discover and validate biomarker candidates present in pancreatic tissue that discriminate HGD and cancer from normal pancreas and low-grade precursor lesions. Discovery tissue samples (fresh frozen and formalin fixed, paraffin embedded) were chosen from Mayo Clinic institutional cancer registries.

After classification was confirmed by pathologists, samples underwent unbiased whole-methylome sequencing. A Mayo Clinic research team led by Drs. Ahlquist and Majumder then used reduced representation bisulfite sequencing to identify candidate methylated markers capable of distinguishing a case group comprised of high-grade precursor lesions — including intraductal papillary mucinous neoplasms high-grade dysplasia (IPMN-HGD), pancreatic intraepithelial neoplasia-3 (PanIN-3) and invasive pancreatic cancer lesions — from a control group composed of normal pancreas or low-grade precursor lesions including IPMN low-grade dysplasia (IPMN-LGD), PanIN-1 and PanIN-2 lesions.

A panel of top candidate markers from this discovery effort was subsequently validated in an independent tissue set. The top-performing markers were then assayed in pancreatic cyst fluid for a multicenter study. Headquartered at Mayo Clinic's campus in Minnesota, the multicenter study included cyst fluid contributions from researchers at Baylor College of Medicine, Brigham and Women's Hospital, Stanford University, University of Michigan, and Yale University.


During the tissue phase of the study, Mayo researchers successfully identified and validated novel methylated DNA markers in pancreatic tissue that accurately discriminate high-grade precursor lesions and cancer from low-grade precursors and normal pancreas tissue. Results presented at Digestive Diseases Week (DDW) and published in abstract form in Gastroenterology in 2016 show that a panel of top tissue markers yielded an area under the receiver operating characteristic curve (AUC) of 0.90 (95 percent confidence interval 84 to 96 percent).

The second phase of the study also yielded promising results, presented at DDW and published in abstract form in Gastroenterology in 2017. In a multicenter study using 134 archival cyst fluid samples, the top-performing methylated DNA marker showed a sensitivity of 81 percent at a specificity of 85 percent for diagnosis of HGD or cancer using the gold standard of surgical histology. The top four methylation markers individually achieved AUCs of greater than 0.90.

"This is the first study assessing the performance of methylated DNA biomarkers in pancreatic cyst fluid, identified based on tissue discovery and specifically aimed at detecting advanced neoplasia and discriminating between grades of dysplasia in pancreatic cysts," explains Dr. Majumder. "Overall, the detection accuracy for these novel methylated markers was significantly better than that provided by the use of currently available cyst fluid markers. These findings demonstrate that cyst fluid-methylated DNA markers can accurately identify cysts with advanced neoplasia that can be targeted for surgical resection."

Dr. Majumder acknowledges that additional research will need to be conducted to address this study's potential limitations and to further validate the performance of these novel cyst fluid biomarkers in a prospectively collected sample set. "Studying patients who undergo surgery as well as those followed with surveillance imaging in larger prospective trials will help optimize marker combinations and corroborate these findings," explains Dr. Majumder.

For more information

Moris M, et al. Association between advances in high-resolution cross-section imaging technologies and increase in prevalence of pancreatic cysts from 2005 to 2014. Clinical Gastroenterology and Hepatology. 2016;14:585.

Majumder S, et al. 596 Detection of pancreatic high-grade dysplasia and cancer using novel methylated DNA markers: Discovery and tissue validation. Gastroenterology. 2016;150(suppl 1):S120.

Majumder S, et al. Novel DNA methylation markers assayed from cyst fluid accurately detect advanced neoplasia in pancreatic cysts: A multicenter study. Gastroenterology. 2017;152(suppl 1):S148.