开发cf-MMSP检测方法以通过液体活检增强乳腺癌中甲基化cfDNA的检测

Background: While mammography remains a standard screening tool for breast cancer, its high false positive rate and invasiveness of subsequent biopsies highlight the need for improved technologies. Liquid biopsy, leveraging circulating cell-free DNA (cfDNA), offers a noninvasive alternative, yet the low levels of cfDNA in blood demand highly sensitive detection techniques. In this study, we sought to improve our two-step lab assay, cMethDNA (1), by testing three commercial DNA isolation kits, and through innovative use of primer combinations to specifically enrich for methylated cfDNA in blood. Methods: Circulating cfDNA was isolated using three kits: QIAamp MinElute Virus Spin Kit (Virus kit), QIAamp MinElute ccfDNA Mini Kit (ccfDNA kit), and MAGicBead cfDNA Isolation Kit. A 9-gene panel consisting of AKR1B1, COL6A2, HIST1H3C, HOXB4, RASGRF2, RASSF1A, TM6SF1, TMEFF2, and ZNF671 (2) was amplified using bisulfite-converted DNA and optimized primer combinations in a cell free, multiplexed, methylation-specific PCR (cf-MMSP) assay. Technical validation of the assay was performed with normal plasma spiked with 12.5-50 copies of fully methylated human genomic DNA and 10 copies of STDHOXB4 plasmid. We quantified relative methylation levels for each gene, and cumulative methylation (CM) of all 9 markers in each sample. The cf-MMSP assay was validated in a pilot study of plasma samples from 21 stage IV breast cancer patients and 20 healthy controls or women with benign disease. Results: The Virus Spin kit outperformed the MAGicBead and ccfDNA kits in cfDNA recovery, showing a significant improvement in the detection of STDHOXB4 reference DNA (p < 0.0001 vs. MAGicBead). Using a combination of a methylation-agnostic (ExtF) and a methylation sequence-specific primer (IntMR) set in the first multiplex PCR step significantly reduced Ct values of target genes (median 17.9 vs. 19.8, p = 0.0001) and STDHOXB4 (median 16.8 vs. 18.8, p < 0.0001), enhancing detection sensitivity by at least 4-fold compared to cMethDNA. In clinical samples, the cf-MMSP assay distinguished stage IV breast cancer patients from controls with 86% sensitivity (95% CI = 65.4-95.0) and 90% specificity (95% CI = 69.9-98.2), achieving an area under the curve (AUC) of 0.8929 (p < 0.0001). Conclusions: The cf-MMSP assay, which included the use of the Virus kit for cfDNA isolation, choice of primers that selectively amplified methylated products, and a single STDHOXB4 standard, significantly reduced assay cost and time and enhanced its sensitivity for detecting methylated cfDNA in plasma from breast cancer patients. Reference: 1) Fackler et al, PMID: 24737128. 2) Fackler et al, PMID: 36046124.