Development of the cf-MMSP Assay for Enhanced Analytical Sensitivity of Methylated DNA in Breast Cancer Liquid Biopsy.

Abstract

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 the extraction efficiency of three commercial kits using normal plasma spiked with 12.5 to 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, and ZNF671) was amplified from bisulfite-converted DNA using a nested PCR that pairs a methylation-independent external forward primer with a reverse methylation (RM)-specific primer in the first multiplex reaction.

Results: The QIAamp MinElute Virus Spin Kit yielded superior cfDNA recovery of the STD_HOXB4 control (P < 0.0001 vs. both MAGicBead cfDNA Isolation Kit and QIAamp MinElute ccfDNA Mini Kit). The new primer combination significantly lowered cycle threshold 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 fourfold improvement in analytical detection sensitivity. In an evaluation of 21 stage IV breast cancer plasma samples and 20 benign controls, cf-MMSP distinguished cases from controls with 81% sensitivity [95% confidence interval (CI), 58.1%-94.6%], 85% specificity (95% CI, 62.1%-96.8%), and area under the receiver operating characteristic curve = 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 with cMethDNA in stage IV breast cancer.

Impact: cf-MMSP improves low-input methylated cfDNA detection and warrants further validation in earlier-stage breast cancer.