DNA methylation analysis of peripheral blood mononuclear cells in diagnosing breast cancer from benign breast lesions

With the increasing incidence of breast lesions, the differential diagnosis between benign lesions and breast cancer (BCa) has become a big challenge. Host peripheral blood mononuclear cells (PBMCs) could undergo changes in DNA methylation upon disease progression. However, the clinical value of DNA methylation of PBMCs in differentiating benign lesions and BCa is still unclear.

DNA of PBMCs was isolated and the methylation status of PBMCs in patients with BCa and benign breast nodules was detected by using Illumina Infinium methylation EPIC array. The specific methylation targets were validated by pyrosequencing, Targeted Bisulfite Sequencing Assay, and Multiplex Methylation PCR Assay(MMPA).

cg26977936, cg23351954, and cg27209741 were validated as differentially methylated and showed the potential diagnostic values (sensitivity and specificity were 90.0%/53.3%, 43.3%/90.0%, 90.0%/43.3%, respectively). Moreover, a diagnostic model was established using these 3 CGs through logistic regression analysis, and the AUC reached 0.837. Next, this diagnostic model was validated in another, independent cohort with Targeted Bisulfite Sequencing Assay, and the clinical value in distinguishing benign and malignant breast disease was also confirmed (AUC = 0.827, P < 0.05). Finally, to better meet the need for the clinical test, we further validated the differential diagnostic efficacy of the 2 hypermethylated DMPs by establishing a Multiplex Methylation PCR Assay by coupling the 5’-flap endonuclease activity of Taq DNA polymerase and molecular beacon reporters.

DNA methylation changes in PBMCs showed great potential in discriminating BCa patients from breast benign lesion patients and may serve as a novel predictor in clinical applications.