Development of the cf-MMSP assay for enhanced analytical sensitivity of methylated DNA in breast cancer liquid biopsy

BACKGROUND: The use of liquid biopsy to analyze cell-free DNA (cfDNA) provides a noninvasive approach for breast cancer detection, but its low abundance necessitates highly sensitive assays. METHODS: We developed cell-free Multiplex Methylation-specific PCR (cf-MMSP) through optimized cfDNA extraction and primer design. We compared extraction efficiency of three commercial kits using normal plasma spiked with 12.5 - 50 copies of fully methylated genomic DNA and 50 copies of STD_HOXB4 plasmid. A nine-gene methylation panel (AKR1B1, COL6A2, HIST1H3C, HOXB4, RASGRF2, RASSF1A, TM6SF1, TMEFF2, ZNF671) was amplified from bisulfite-converted DNA using a nested PCR that pairs a methylation-independent external forward primer (Ext_F) with a reverse methylation-specific primer (RM) in the first multiplex reaction. RESULTS: The QIAamp MinElute Virus Spin Kit yielded superior cfDNA recovery of the STD_HOXB4 control (P < 0.0001 versus both MAGicBead cfDNA Isolation Kit and QIAamp MinElute ccfDNA Mini Kit). The new primer combination significantly lowered Ct values for genes (median 17.9 vs 19.8, P = 0.0001) and STD_HOXB4 (median 16.8 vs 18.8, P < 0.0001), translating to a four-fold improvement in analytical detection sensitivity. In an evaluation of 21 stage IV breast cancer plasma and 20 benign controls, cf-MMSP distinguished cases from controls with 81% sensitivity (95% CI, 58.1-94.6%), 85% specificity (95% CI, 62.1-96.8%) and AUC = 0.89 (P < 0.0001). CONCLUSIONS: Optimization of DNA extraction and primer design in cf-MMSP improved low-input analytical sensitivity while maintaining performance comparable to cMethDNA in stage IV breast cancer. IMPACT: cf-MMSP improves low-input methylated cfDNA detection and warrants further validation in earlier-stage breast cancer.