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

Abstract Background Cell-free DNA (cfDNA) has emerged as a promising biomarker for cancer detection and monitoring. However, different quantification and integrity assessment methods may yield variable results. This study aimed to compare cfDNA concentration and integrity between breast cancer (BC) patients and healthy controls using three complementary analytical techniques and to evaluate the concordance between these approaches. Methods Plasma cfDNA from BC patients and healthy controls was quantified using Qubit fluorometry, electrophoretic profiling by TapeStation (TS), and ALU-based quantitative PCR (qPCR) assays. cfDNA integrity was assessed using two indices: TS-derived DNA Integrity Index (DII) and ALU-qPCR–derived DII (ALU 247/115 ratio). Spearman correlation analyses were performed to compare results across methods. Results cfDNA concentration was significantly elevated in BC patients compared with controls across all three platforms, even after genomic DNA (gDNA) correction. Qubit, TS, and gDNA-corrected cfDNA concentrations showed strong correlations in both groups, indicating robust cross-method agreement. In contrast, cfDNA integrity showed method-dependent differences: ALU-qPCR–derived DII was significantly higher in tumor samples, whereas TS-derived DII did not differ between groups. Moreover, TS DII and ALU-qPCR DII were not correlated, suggesting that the two methods capture distinct aspects of cfDNA fragmentation. Conclusions cfDNA quantification is consistent across fluorometric, electrophoretic, and qPCR-based methods. However, cfDNA integrity assessments vary substantially by technique. ALU-qPCR appears more sensitive in distinguishing BC- associated fragmentation patterns. Combining cfDNA quantification with ALU-qPCR-based integrity analysis may enhance the diagnostic utility of cfDNA in BC.