Breast cancer (BC) is the most prevalent malignancy globally, with early diagnosis crucial for improved survival. Current screening methods like mammography and ultrasound have limitations, including low sensitivity for dense BC and high false-positive rates. This necessitates the development of more accurate and sensitive diagnostic tools. DNA methylation, an epigenetic modification regulating gene expression, plays a significant role in cancer development. Changes in DNA methylation occur in various cancers, even at precancerous or early stages, making it a promising biomarker. Previous research focused on tumor tissue and circulating tumor materials, but these have limitations regarding accessibility, specificity, and abundance. Studies have shown the potential of DNA methylation in peripheral blood cells as complementary cancer biomarkers. This study aimed to determine if DNA methylation alterations in PBMCs could serve as indicators for early BC detection, focusing on tumor immune alterations. The researchers hypothesized that BC-specific DNA methylation patterns in PBMCs could reflect host immune responses and facilitate early BC diagnosis.

Existing literature highlights the importance of early breast cancer detection for improved patient outcomes. Studies have demonstrated the limitations of current screening methods such as mammography and ultrasound, particularly regarding sensitivity in detecting cancers in dense breast tissue and high rates of false positives. The role of DNA methylation in cancer development is well-established, with alterations occurring early in tumorigenesis. While studies have explored DNA methylation in tumor tissue and circulating tumor-derived materials (like circulating tumor cells, circulating tumor DNA, and cell-free DNA) for cancer biomarker development, these approaches face challenges related to invasiveness, low specificity, and low abundance of target molecules. Emerging research has shown the potential of using DNA methylation in peripheral blood cells, specifically PBMCs, as a less invasive and potentially more effective approach for cancer detection. Previous studies have identified cancer-specific methylation changes in PBMCs from various cancers such as hepatocellular carcinoma, prostate cancer, colorectal cancer, and head and neck squamous cell carcinoma. However, the utility of PBMC DNA methylation for early breast cancer detection remained unclear.

This multicenter retrospective study enrolled 820 patients from ten hospitals across six provinces in China between May 2020 and July 2022. After excluding samples due to insufficient data or poor quality, 781 PBMC samples (366 BC, 290 normal controls, 125 other tumors) were used. The study comprised three phases: discovery, validation, and application. In the discovery phase (Cohort I, n=80), genome-wide DNA methylation profiling using Infinium Human Methylation 850 K BeadChip was performed on PBMCs from 50 BC patients and 30 normal controls. Differentially methylated CpG positions (DMPs) were identified based on criteria of |Δβ| ≥ 0.06 and p ≤ 0.01. Candidate markers were further selected using LASSO analysis and other filters, prioritizing those located in the TSS region. The validation phase (Cohort II, n=200) used pyrosequencing and targeted bisulfite sequencing (TBS) to confirm the identified markers. Finally, the application phase (Cohort III, n=501) involved the development and evaluation of a multiplex quantitative methylation-specific PCR assay (BC-mqmsPCR) based on the selected markers. This assay simultaneously detects four methylation markers (cg11754974, cg16652347, cg13828440, and cg18637238) in a single reaction. The performance of BC-mqmsPCR was evaluated in terms of its diagnostic efficiency, particularly for early-stage and minimal tumors, and compared to conventional tumor markers (CEA, CA153, CA125). Statistical analysis included PCA, volcano plots, ROC curves, and comparative analysis between BC-mqmsPCR and traditional tumor markers. A follow-up study of 170 normal controls was also conducted to assess the predictive value of the assay.

Genome-wide DNA methylation analysis revealed 289 DMPs in PBMCs from BC patients compared to controls. Most DMPs (61.2%) were hypomethylated, with an enrichment of genes involved in immune surveillance and immuno-editing. After multi-step validation, four hypermethylated CpG sites (cg11754974, cg16652347, cg13828440, and cg18637238) were selected for the BC-mqmsPCR assay. The BC-mqmsPCR assay demonstrated superior performance in distinguishing BC patients from normal controls (AUC=0.925 in training set, AUC=0.918 in validation set; sensitivity 83.1% and 80.3%, specificity 90.4% and 89.1% respectively) compared to individual marker assays. Importantly, the assay showed excellent diagnostic accuracy for early-stage (AUC=0.940 and 0.927) and minimal tumors (≤1.5 cm, AUC=0.945 and 0.936), outperforming traditional tumor markers (CEA, CA153, and CA125). The BC-mqmsPCR assay successfully identified BC cases missed by conventional imaging and serum marker tests. A follow-up study demonstrated that two out of seventeen patients predicted to be positive by the BC-mqmsPCR test developed BC within one year, suggesting the potential for early detection.

The study's findings demonstrate the potential of a novel, non-invasive, blood-based assay (BC-mqmsPCR) for early breast cancer detection. The superior performance of BC-mqmsPCR, particularly in detecting early-stage and minimal tumors, addresses the critical need for improved early diagnostic tools for BC. This success is attributed to the focus on DNA methylation changes in PBMCs, reflecting the interaction between the tumor and the host immune system. The high sensitivity and specificity of the assay, coupled with its multiplex nature and relative simplicity, suggest its potential for clinical translation. The results support the hypothesis that epigenetic changes in PBMCs can serve as sensitive and specific biomarkers for early BC detection, potentially enhancing early diagnosis and improving patient prognosis. The identification of these four specific methylation markers provides new insights into the epigenetic landscape of breast cancer and its interaction with the immune system.

This study successfully developed and validated a multiplex blood-based assay (BC-mqmsPCR) for early and accurate breast cancer detection. The assay's high sensitivity and specificity, especially for early-stage and minimal tumors, demonstrate its clinical potential. Future research should focus on larger-scale prospective studies to further validate the assay's performance and investigate the underlying biological mechanisms of the identified methylation markers. Longitudinal studies are needed to evaluate the impact of BC-mqmsPCR on patient outcomes and explore its role in risk stratification and monitoring.

This study is retrospective, limiting the generalizability of the findings. The exclusion of patients with inflammatory diseases may restrict the applicability of the assay in certain populations. The relatively short follow-up period in the normal control cohort might underestimate the long-term predictive power of the assay. Further, the biological mechanisms underpinning the observed methylation changes require further investigation. The study also didn't investigate the impact of the methylation changes on recurrence or patient survival due to a lack of follow-up data on BC patients. Prospective studies with longer follow-up are needed to address these limitations and further establish the clinical utility of this assay.