Cell-free DNA promoter hypermethylation as a diagnostic marker for pancreatic ductal adenocarcinoma – An external validation study

Stine D. Henriksen*, Benjamin E. Stubbe, Poul H. Madsen, Julia S. Johansen, Benny V. Jensen, Carsten P. Hansen, Martin N. Johansen, Inge S. Pedersen, Henrik Krarup, Ole Thorlacius-Ussing

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Background: We recently identified a diagnostic prediction model based on promoter hypermethylation of eight selected genes in plasma cell-free (cf) DNA, which showed promising results as a diagnostic biomarker for pancreatic ductal adenocarcinoma (PDAC). The aim of the present study was to validate this biomarker profile in an external patient cohort and examine any additional effect of serum CA 19-9. Methods: Patients with PDAC (n = 346, stage I-IV) and chronic pancreatitis (n = 25) were included. Methylation-specific PCR of a 28-gene panel was performed on serum cfDNA samples. The previously developed diagnostic prediction model (age>65 years, BMP3, RASSF1A, BNC1, MESTv2, TFPI2, APC, SFRP1 and SFRP2) was validated alone and in combination with serum CA 19-9 in this external patient cohort. Results: Patients with PDAC had a higher number of hypermethylated genes (mean 8.11, 95% CI 7.70–8.52) than patients with chronic pancreatitis (mean 5.60, 95% CI 4.42–6.78, p = 0.011). Validation of the diagnostic prediction model yielded an AUC of 0.77 (95% CI 0.69–0.84). The combination of serum CA 19-9 and our test had an AUC of 0.93 (95% CI 0.89–0.96) in the primary study and 0.85 (95% CI 0.79–0.91) in the validation study. Conclusion: In this validation study, PDAC was associated with a higher number of hypermethylated genes in serum cfDNA than chronic pancreatitis. Our diagnostic test was superior to the predictive value of serum CA 19-9 alone in both the primary and the validation study. The combination of our test with CA 19-9 may serve as a clinically useful diagnostic biomarker for PDAC.

Original languageEnglish
Issue number6
Pages (from-to)1081-1091
Number of pages11
Publication statusPublished - Sept 2021

Bibliographical note

Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.


  • Biomarker
  • Cell-free DNA
  • Epigenetic
  • Methylation
  • Pancreatic cancer


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