Highly Specific Droplet-Digital PCR Detection of Universally Methylated Circulating Tumor DNA in Endometrial Carcinoma

Guillaume Beinse; Bruno Borghese; Marie Métairie; Pierre-Alexandre Just; Geoffroy Poulet; Simon Garinet; Beatrice Parfait; Audrey Didelot; Camille Bourreau; Natacha Agueeff; Alexandre Lavollé; Benoit Terris; Charles Chapron; François Goldwasser; Karen Leroy; Helene Blons; Pierre Laurent-Puig; Valérie Taly; Jérôme Alexandre

doi:10.1093/clinchem/hvac020

Highly Specific Droplet-Digital PCR Detection of Universally Methylated Circulating Tumor DNA in Endometrial Carcinoma

Clin Chem. 2022 Jun 1;68(6):782-793. doi: 10.1093/clinchem/hvac020.

Authors

Guillaume Beinse^{1

2}, Bruno Borghese^{1

3}, Marie Métairie^{1

3}, Pierre-Alexandre Just^{4

5}, Geoffroy Poulet¹, Simon Garinet^{1

6}, Beatrice Parfait^{5

7}, Audrey Didelot¹, Camille Bourreau¹, Natacha Agueeff¹, Alexandre Lavollé¹, Benoit Terris^{4

5}, Charles Chapron^{3

5}, François Goldwasser^{2

5

8}, Karen Leroy^{5

6}, Helene Blons^{5

6}, Pierre Laurent-Puig^{1

5

6}, Valérie Taly¹, Jérôme Alexandre^{1

2}

Affiliations

¹ Centre de Recherche des Cordeliers, « Equipe labélisée Ligue Contre le Cancer », CNRS SNC 5096, Sorbonne Université, Université de Paris, INSERM, Paris, France.
² Department of Medical Oncology, Hopital Cochin, Institut du Cancer Paris CARPEM, AP-HP, APHP Centre, Paris, France.
³ Department of Gynecological Surgery, Institut du Cancer Paris CARPEM, AP-HP, APHP Centre, Hopital Cochin, Paris, France.
⁴ Department of Pathology, Institut du Cancer Paris CARPEM, AP-HP, APHP Centre, Hopital Cochin, Paris, France.
⁵ Université de Paris, Paris, France.
⁶ Department of Biology, Institut du Cancer Paris CARPEM, AP-HP, APHP Centre, Hopital Européen Georges Pompidou, Paris, France.
⁷ Institut du Cancer Paris CARPEM, AP-HP, Fédération des Centres de Ressources Biologiques- Plateforme de Ressources Biologiques APHP, Centre de Ressources Biologiques-Site Cochin, Hôpital Cochin, Paris, France.
⁸ Université de Paris, URP 4466, Metheho, Paris, France.

PMID: 35323926
DOI: 10.1093/clinchem/hvac020

Abstract

Background: No circulating biomarker is available for endometrial carcinoma (EC). We aimed to identify DNA positions universally hypermethylated in EC, and to develop a digital droplet PCR (ddPCR) assay for detection of hypermethylated circulating tumor DNA (meth-ctDNA) in plasma from patients with EC.

Methods: DNA positions hypermethylated in EC, and without unspecific hypermethylation in tissue/cell types releasing circulating cell-free DNA in plasma, were identified in silico from TCGA/Gene Expression Omnibus (GEO) data. A methylation-specific ddPCR (meth-ddPCR) assay following bisulfite conversion of DNA extracted from plasma was optimized for detection of meth-ctDNA according to dMIQE guidelines. Performances were validated on a retrospective cohort (n = 78 tumors, n = 30 tumor-adjacent tissues), a prospective pilot cohort (n = 33 stage I-IV patients), and 55 patients/donors without cancer.

Results: Hypermethylation of zinc finger and SCAN domain containing 12 (ZSCAN12) and/or oxytocin (OXT) classified EC samples from multiple noncancer samples with high diagnostic specificity/sensitivity [>97%; area under the curve (AUC) = 0.99; TCGA/GEO tissues/blood samples]. These results were confirmed in the independent retrospective cohort (AUC = 0.99). Meth-ddPCR showed a high analytical specificity (limit of blank = 2) and sensitivity (absolute lower threshold of detection = 50 pgmethDNA/mLplasma). In the pilot cohort, meth-ctDNA was detected in pretreatment plasma samples from 9/11 and 5/20 patients with advanced and non-advanced EC, respectively. 2 of 9 patients had ctDNA detected after macroscopic complete surgery and experienced progression within 6 months. No healthy donors had any copy of hypermethylated DNA detected in plasma.

Conclusions: Meth-ddPCR of ZSCAN12/OXT allows a highly specific and sensitive detection of ctDNA in plasma from patients with EC and appears promising for personalized approaches for these patients.

Keywords: DNA methylation; digital droplet PCR; endometrial carcinoma; molecular characterization; personalized medicine.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Biomarkers, Tumor / genetics
Circulating Tumor DNA* / genetics
Endometrial Neoplasms* / diagnosis
Endometrial Neoplasms* / genetics
Female
Humans
Polymerase Chain Reaction / methods
Prospective Studies
Retrospective Studies

Substances

Biomarkers, Tumor
Circulating Tumor DNA