Promoter hypermethylation of SFRP1 is an allele fraction-dependent prognostic biomarker in metastatic pancreatic ductal adenocarcinoma

Benjamin Stubbe; Malene P Stoico; Simone K Terp; Poul H Madsen; Søren Lundbye-Christensen; Carsten P Hansen; Laurids Ø Poulsen; Louise S Rasmussen; Mette N Yilmaz; Lars H Jensen; Torben F Hansen; Per Pfeiffer; Anders C Larsen; Henrik B Krarup; Inge S Pedersen; Jane P Hasselby; Astrid Z Johansen; Inna M Chen; Julia S Johansen; Ole Thorlacius-Ussing; Stine D Henriksen

doi:10.3389/fonc.2025.1568386

Promoter hypermethylation of SFRP1 is an allele fraction-dependent prognostic biomarker in metastatic pancreatic ductal adenocarcinoma

Front Oncol. 2025 May 26:15:1568386. doi: 10.3389/fonc.2025.1568386. eCollection 2025.

Authors

Benjamin Stubbe^{1

2

3}, Malene P Stoico⁴, Simone K Terp⁴, Poul H Madsen^{3

4}, Søren Lundbye-Christensen^{2

5}, Carsten P Hansen⁶, Laurids Ø Poulsen^{2

7}, Louise S Rasmussen⁷, Mette N Yilmaz⁷, Lars H Jensen⁸, Torben F Hansen⁸, Per Pfeiffer⁹, Anders C Larsen^{1

2

3}, Henrik B Krarup^{3

4}, Inge S Pedersen^{2

3

4}, Jane P Hasselby¹⁰, Astrid Z Johansen¹¹, Inna M Chen¹¹, Julia S Johansen^{11

12

13}, Ole Thorlacius-Ussing^{1

2

3}, Stine D Henriksen^{1

2

3}

Affiliations

¹ Department of Gastrointestinal Surgery, Aalborg University Hospital, Aalborg, Denmark.
² Department of Clinical Medicine, Aalborg University, Aalborg, Denmark.
³ Clinical Cancer Research Center, Aalborg University Hospital, Aalborg, Denmark.
⁴ Department of Molecular Diagnostics, Aalborg University Hospital, Aalborg, Denmark.
⁵ Unit of Research Data and Biostatistics, Aalborg University Hospital, Aalborg, Denmark.
⁶ Department of Surgery, Copenhagen University Hospital - Rigshospitalet, Copenhagen, Denmark.
⁷ Department of Oncology and Clinical Cancer Research Center, Aalborg University Hospital, Aalborg, Denmark.
⁸ Department of Oncology, University Hospital of Southern Denmark, Vejle, Denmark.
⁹ Department of Medical Oncology, Odense University Medical Center, University of Odense, Odense, Denmark.
¹⁰ Department of Pathology, Copenhagen University Hospital - Rigshospitalet, Copenhagen, Denmark.
¹¹ Department of Oncology, Copenhagen University Hospital - Herlev and Gentofte, Herlev, Denmark.
¹² Department of Medicine, Copenhagen University Hospital-Herlev and Gentofte Hospital, Herlev, Denmark.
¹³ Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Abstract

Introduction: Metastatic pancreatic ductal adenocarcinoma (PDAC) is highly lethal. Promoter hypermethylation of SFRP1 (phSFRP1) in cell-free DNA is an established prognostic biomarker in PDAC. We used digital droplet PCR (ddPCR) to examine whether the prognostic impact of phSFRP1 was allele fraction (AF) dependent.

Methods: Prospectively collected plasma samples were analyzed blinded. Dual-strand methylation ddPCR assays were designed for SFRP1, with single-strand assay for the reference gene EPHA3. Patients were stratified into unmethylated SFRP1 (umSFRP1), low phSFRP1 AF (phSFRP1^low), and high phSFRP1 AF (phSFRP1^high). Survival was assessed with Kaplan-Meier curves. The 3-, 6-, and 12-month absolute risk difference (ARD) was calculated, and performance assessed with ROC analyses.

Results: Overall, 354 patients were included. Patients with umSFRP1 (n=137) had a mOS of 9.1 months compared to 7.2 months in phSFRP1^low (n=78) and 3.4 months in phSFRP1^high (n=143, P<0.01). phSFRP1^high was associated with increased mortality at 3 (ARD 26%, 95%CI: 15, 37), 6 (ARD 37%, 95%CI: 26, 48), and 12 months (ARD 23%, 95%CI: 14, 33). phSFRP1^low was associated with increased mortality at 12 months (ARD 13%, 95%CI: 2, 25) but not at 3 (ARD -3%, 95%CI: -13, 8) or 6 months (ARD 3%, 95%CI: -10, 17). phSFRP1 significantly improved performance in predicting mortality compared to only clinical variables (AUC: 0.70-0.71 vs. 0.54-0.57).

Discussion: Patients with phSFRP1^high had significantly shorter survival than phSFRP1^low or umSFRP1, indicating AF-dependent prognostic effects. phSFRP1^low had a worse prognosis than umSFRP1 at only 12 months, indicating dynamic changes. This could help personalize the treatment of PDAC.

Keywords: Sfrp1; biomarker; epigenetics; liquid biopsy; methylation; pancreatic ductal adenocarcinoma.

Copyright © 2025 Stubbe, Stoico, Terp, Madsen, Lundbye-Christensen, Hansen, Poulsen, Rasmussen, Yilmaz, Jensen, Hansen, Pfeiffer, Larsen, Krarup, Pedersen, Hasselby, Johansen, Chen, Johansen, Thorlacius-Ussing and Henriksen.