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 STDHOXB4 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 (ExtF) 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 STDHOXB4 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 STDHOXB4 (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.
Zhang et al. (Tue,) studied this question.