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. 2019 Sep;114(9):1539-1549.
doi: 10.14309/ajg.0000000000000284.

Novel Methylated DNA Markers Discriminate Advanced Neoplasia in Pancreatic Cysts: Marker Discovery, Tissue Validation, and Cyst Fluid Testing

Shounak Majumder  1 , William R Taylor  1 , Tracy C Yab  1 , Calise K Berger  1 , Brian A Dukek  1 , Xiaoming Cao  1 , Patrick H Foote  1 , Chung Wah Wu  1 , Douglas W Mahoney  2 , Harry R Aslanian  3 , Carlos Fernández-Del Castillo  4 , Leona A Doyle  5 , James J Farrell  3 , William E Fisher  6 , Linda S Lee  5 , Yvonne N Lee  7 , Walter Park  7 , Clifton Rodrigues  4 , Bonnie Elyssa Gould Rothberg  3 , Ronald R Salem  3 , Diane M Simeone  8 , Sumithra Urs  8 , George Van Buren  6 , Thomas C Smyrk  9 , Hatim T Allawi  10 , Graham P Lidgard  10 , Massimo Raimondo  1 , Suresh T Chari  1 , Michael L Kendrick  11 , John B Kisiel  1 , Mark D Topazian  1 , David A Ahlquist  12
Affiliations

Novel Methylated DNA Markers Discriminate Advanced Neoplasia in Pancreatic Cysts: Marker Discovery, Tissue Validation, and Cyst Fluid Testing

Shounak Majumder et al. Am J Gastroenterol. 2019 Sep.

Abstract

Objectives: Pancreatic cystic lesions (PCLs) may be precancerous. Those likely to harbor high-grade dysplasia (HGD) or pancreatic cancer (PC) are targets for surgical resection. Current algorithms to predict advanced neoplasia (HGD/PC) in PCLs lack diagnostic accuracy. In pancreatic tissue and cyst fluid (CF) from PCLs, we sought to identify and validate novel methylated DNA markers (MDMs) that discriminate HGD/PC from low-grade dysplasia (LGD) or no dysplasia (ND).

Methods: From an unbiased whole-methylome discovery approach using predefined selection criteria followed by multistep validation on case (HGD or PC) and control (ND or LGD) tissues, we identified discriminant MDMs. Top candidate MDMs were then assayed by quantitative methylation-specific polymerase chain reaction on archival CF from surgically resected PCLs.

Results: Of 25 discriminant MDMs identified in tissue, 13 were selected for validation in 134 CF samples (21 cases [8 HGD, 13 PC], 113 controls [45 ND, 68 LGD]). A tree-based algorithm using 2 CF-MDMs (TBX15, BMP3) achieved sensitivity and specificity above 90%. Discrimination was significantly better by this CF-MDM panel than by mutant KRAS or carcinoembryonic antigen, with areas under the receiver operating characteristic curve of 0.93 (95% confidence interval: 0.86-0.99), 0.71 (0.57-0.85), and 0.72 (0.60-0.84), respectively. Cutoffs for the MDM panel applied to an independent CF validation set (31 cases, 56 controls) yielded similarly high discrimination, areas under the receiver operating characteristic curve = 0.86 (95% confidence interval: 0.77-0.94, P = 0.2).

Discussion: Novel MDMs discovered and validated in tissue accurately identify PCLs harboring HGD/PC. A panel of 2 MDMs assayed in CF yielded results with potential to enhance current risk prediction algorithms. Prospective studies are indicated to optimize and further evaluate CF-MDMs for clinical use.

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Conflict of interest statement

CONFLICTS OF INTEREST

Guarantor of the article: David A. Ahlquist, MD.

Potential competing interests: Mayo Clinic has licensed intellectual property to Exact Sciences on molecular markers and sample processing techniques for multiple cancers and precancers, including pancreatic cancer. As co-inventors on licensed technologies, several co-authors (D.A.A., J.B.K., S.M., W.R.T., D.W.M., and T.C.Y.) could share potential future royalties to Mayo Clinic from Exact Sciences in accordance with institutional policy and oversight. G.P.L. and H.T.A. are Exact Sciences employees. Exact Sciences provided assay materials and partial funding but had no role in the protocol design, study execution, or analysis of data. The other authors of this manuscript have no conflicts of interest to declare.

Figures

Figure 1.
Figure 1.
Study flow diagram. HGD, harbor high-grade dysplasia; LGD, low-grade dysplasia; MDM, methylated DNA marker; RRBS, reduced representation bisulfite sequencing.
Figure 2.
Figure 2.
Discrimination of selected MDMs assayed from cyst fluid. (a) Decision tree classification with marker cutoffs using the top 2 markers TBX15 and BMP3. (b) Heat matrix for cyst fluid MDMs among cases and controls with sensitivity of individual MDMs at 90% specificity. The color ranges from yellow to green with “more green” indicating further distance from the specificity cutoff. HGD, harbor high-grade dysplasia; LGD, low-grade dysplasia; MDM, methylated DNA marker.
Figure 3.
Figure 3.
Distributions of 2 discriminant methylated DNA markers in cases and controls. (a) Distributions in tissue by different grades of neoplasia from biological mucinous neoplasm. (b) Distributions in CF from cases (HGD or cancer) and controls (LGD or no dysplasia). CF, cyst fluid; HGD, harbor high-grade dysplasia; IPMN, intraductal papillary mucinous neoplasm; LGD, low-grade dysplasia.
Figure 4.
Figure 4.
Findings in Fukuoka subsets. (a) Proportion of cases (HGD or cancer) and controls (LGD or no dysplasia) across the 3 Fukuoka risk groups. (b) Distributions of TBX15 and BMP3 across Fukuoka categories. (c) Sensitivity of the MDM panel at a specificity cutoff of 90% in the different Fukuoka risk groups. HGD, harbor high-grade dysplasia; LGD, low-grade dysplasia; MDM, methylated DNA marker.
Figure 5.
Figure 5.
Comparative AUCs for TBX15, BMP3 (individually and in combination based on the predicted probability from rPart decision tree [indicated by asterisk]), mutant KRAS and cyst fluid CEA for distinguishing cases (HGD or cancer) and controls (LGD or no dysplasia). AUC, areas under the receiver operating characteristic curve; HGD, harbor high-grade dysplasia; LGD, low-grade dysplasia; rPart, regression partition tree.
Figure 6.
Figure 6.
Comparative results of CF pilot and validation study. (a and b) CF distributions of TBX 15 and BMP 3 from cases (HGD or cancer) and controls (LGD or no dysplasia) in pilot study and subsequent validation. (c) Sensitivities and specificities of the 2-MDM panel. (d) AUCs for the 2-MDM panel in independent pilot and validation sets. AUC, areas under the receiver operating characteristic curve; CF, cyst fluid; HGD, harbor high-grade dysplasia; LGD, low-grade dysplasia; MDM, methylated DNA marker.
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