Evaluating Cell-Free DNA Quantification and Integrity in Breast Cancer Using Fluorometric, Electrophoretic and PCR-Based Platforms.

Cell-free DNA (cfDNA) has emerged as a promising liquid biopsy biomarker; however, differences in analytical methods may influence cfDNA quantification and integrity assessment. This study compared plasma cfDNA concentration and integrity between treatment-naïve breast cancer (BC) patients (n = 100) and healthy controls (HC; n = 70) using complementary analytical platforms. cfDNA concentration was measured by Qubit fluorometry, TapeStation (TS) electrophoretic profiling, and ALU-based quantitative PCR (qPCR). An exploratory genomic DNA (gDNA) correction of Qubit-derived cfDNA concentration was performed using TS-derived fragment-size information. cfDNA integrity was assessed using the ALU-derived DNA Integrity Index (DII (ALU); ALU247/ALU115) and the TS-derived DNA Integrity Index (DII (TS)), and methods were compared for their correlation and agreement. Qubit- and TS-based cfDNA measurements were significantly higher in BC patients than in HC. Among the ALU-based qPCR measures, although ALU115 did not differ significantly between groups, ALU247 and DII (ALU) were significantly higher in BC patients, indicating an enrichment of longer cfDNA fragments. Exploratory gDNA correction did not substantially alter the observed differences. Significant positive correlations were observed among the cfDNA quantification methods. Supporting these findings, agreement analysis demonstrated improved agreement following gDNA correction. In contrast, cfDNA integrity assessment was method-dependent: although DII (ALU) was significantly higher in BC patients, DII (TS) did not differ significantly between groups, and no significant correlation or agreement was observed between the two indices, indicating that the two methods evaluate different lengths of cfDNA fragments. These findings demonstrate that complementary analytical platforms consistently differentiated BC patients from HC based on cfDNA concentration, whereas cfDNA integrity assessment depended on the analytical method employed. Together, these findings provide methodological insights into cfDNA quantification and fragmentation assessment and support the complementary use of fluorometric, electrophoretic, and ALU-based qPCR approaches for comprehensive cfDNA characterization.