Differential methylation of circulating free DNA assessed through cfMeDiP as a new tool for breast cancer diagnosis and detection of BRCA1/2 mutation

Sep 21, 2026

Recent studies highlight cfDNA methylation profiling for detecting breast cancer and its subtypes. This study evaluates whether plasma cfDNA methylation measured with cell-free Methylated DNA Immunoprecipitation and High-Throughput Sequencing (cfMeDIP-seq) can characterize breast cancer in patients with BRCA1/2 germline mutations, supporting early detection and therapy response assessment. Twenty-three BRCA1/2 germline carriers and matched controls were analyzed using extracted 1 mL plasma, shallow whole-genome sequencing, differential methylation region identification, and classifier training for discrimination.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages12

Reading Time30 min

AccessFree to download

Abstract

Background

Methods

Results

Conclusions

Keywords

Background

Breast cancer risk factors and BRCA1/BRCA2 roles

Grisolia et al. Journal of Translational Medicine (2024) 22:938 [https://doi.org/10.1186/s12967-024-05734-2](https://doi.org/10.1186/s12967-024-05734-2) Journal of Translational Medicine RESEARCH Open Access Differential methylation of circulating free DNA assessed through cfMeDiP as a new tool for breast cancer diagnosis and detection of BRCA1/2 mutation Piera Grisolia 1,2†, Rossella Tufano3†, Clara Iannarone2, Antonio De Falco3, Francesca Carlino4,5, Cinzia Graziano2, Raffaele Addeo6, Marianna Scrima2, Francesco Caraglia4, Anna Ceccarelli7, Pier Vitale Nuzzo8, Alessia Maria Cossu2,4, Stefano Forte9, Raffaella Giuffrida9, Michele Orditura4, Michele Caraglia2,4 and Michele Ceccarelli 1* Abstract Background Recent studies have highlighted the importance of the cell-free DNA (cfDNA) methylation profile in detecting breast cancer (BC) and its different subtypes. We investigated whether plasma cfDNA methylation, using cell-free Methylated DNA Immunoprecipitation and High-Throughput Sequencing (cfMeDIP-seq), may be informative in characterizing breast cancer in patients with BRCA1/2 germline mutations for early cancer detection and response to therapy. Methods We enrolled 23 BC patients with germline mutation of BRCA1 and BRCA2 genes, 19 healthy controls without BRCA1/2 mutation, and two healthy individuals who carried BRCA1/2 mutations. Blood samples were collected for all study subjects at the diagnosis, and plasma was isolated by centrifugation. Cell-free DNA was extracted from 1 mL of plasma, and cfMeDIP-seq was performed for each sample. Shallow whole genome sequencing was performed on the immuno-precipitated samples. Then, the differential ly methylated 300-bp regions (DMRs) between 25 BRCA germline mutation carriers and 19 non-carriers were identified. DMRs were compared with tumorspecific regions from public datasets to perform an unbiased analysis. Finally, two statistical classifiers were trained based on the GLMnet and random forest model to evaluate if the identified DMRs could discriminate BRCA-positive from healthy samples. Results We identified 7,095 hypermethylated and 212 hypomethylated regions in 25 BRCA germline mutation carriers compared to 19 controls. These regions discriminate tumors from healthy samples with high accuracy and sensitivity. We show that the circulating tumor DNA of BRCA1/2 mutant breast cancers is characterized by the hypomethylation of genes involved in DNA repair and cell cycle. We uncovered the TFs associated with these DRMs | †Piera Grisolia and RossellaTufano contributed equally to this work. | | --- | | Michele Caraglia and Michele Ceccarell i co-senior authors. | *Correspondence: Michele Ceccarelli[m.ceccarelli@gmail.com](m.ceccarelli@gmail.com) Full list of author information is available at the end of the article © The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit [http://](http://)[ ](http://)[creativecommons.org/l](creativecommons.org/l)icenses/by-nc-nd/4.0/. Grisolia et al. Journal of Translational Medicine (2024) 22:938 Page 2 o

FAQs

What is cfMeDiP-seq and how was it used in this study?

Plasma cell-free methylated DNA immunoprecipitation was combined with high-throughput sequencing (cfMeDIP-seq). The authors extracted cfDNA from 1 mL plasma and performed sequencing for each sample to detect differential methylation regions.

How were participants grouped for differential methylation analysis?

The study enrolled 23 breast cancer patients with BRCA1/2 germline mutations and 19 healthy controls without BRCA1/2 mutations, plus two additional healthy individuals carrying BRCA1/2 mutations. Differentially methylated 300-bp regions were identified for BRCA mutation carriers versus non-carriers.

What classification performance did the identified DMRs achieve?

Two statistical classifiers trained on the identified DMRs were used to discriminate BRCA-positive from healthy samples. The reported performance includes an AUC of 0.95, sensitivity of 88%, and specificity of 94.74%.

Sep 21, 2026

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