A novel extraction method for enrichment of circulating cell-free DNA

Proximal biofluids have shown enriched sensitivity for liquid biopsy but present unique preanalytical challenges. Even in plasma, variations in collection or processing can affect cell-free DNA (cfDNA) isolation and size profiles. These differences may affect downstream sensitivity. Our lab has pioneered the use of lymphatic exudate (“lymph”) from surgical drains as a novel proximal liquid biofluid for MRD detection. We have previously shown that lymph collected 24 hours after surgery identified molecular residual disease (MRD) in head and neck squamous cell carcinoma (HNSCC). This biofluid contains lymphatic fluid, blood, and interstitial fluid, and differs markedly from blood or plasma. cfDNA in lymph displays distinct nucleosomal distribution, including prominent mono- and dinucleosome peaks (“cfDNA fraction”), but most extracellular DNA is >700 bp (“HMW fraction”). Our analysis revealed that tumor-derived DNA is more abundant within the < 700 bp cfDNA fraction. To negate the signal-diluting effects of the HMW fraction, we developed a novel extraction method that simultaneously maximizes cfDNA recovery and selectively depletes HMW DNA. The method utilizes Proteinase K treatment for protein removal, selective bead-based removal of the HMW fraction, and purification of the enriched cfDNA from the supernatant. We benchmarked this method against three commercial cfDNA extraction kits. To characterize all size fractions, we also generated a non-depleted extract using a simplified version of our protocol lacking the selective bead-binding step. We refer to this non-depleted material as “raw cfDNA extract.” Lymph was collected in K2EDTA/Streck blood collection tubes, centrifuged to remove cells and debris, and the supernatant stored at -80 °C (n=36). All 36 samples were processed using our novel extraction method; matched subsets were used for commercial kit comparisons. cfDNA concentration and size distribution were analyzed using Qubit dsDNA and Tapestation cfDNA assays respectively, with cfDNA defined as fragments 50-700 bp. Raw cfDNA extract was highly concentrated (mean 40 ng/µL of extracted lymph, n=9) but displayed variable purity (6-31%). Commercial kits yielded highly variable quantity and purity (cfDNA yield = 0.08-19.88 ng/µL of extracted lymph; purity = 1.3-31%; n = 3, 3 and 2 for kits 1-3). In stark contrast, our novel extraction method delivered high yield and purity consistently across all 36 extractions (mean yield = 1.8 ng/µL, cfDNA purity = 92% [80-98%]). We present an optimized cfDNA extraction method which consistently yields high purity, concentrated cfDNA from biofluids with substantial HMW fractions, dramatically increasing the effective ctDNA yield compared to standard commercial protocols. This technical advancement resolves a critical preanalytical challenge for assays using lymphatic exudate, enabling sensitive clinical monitoring with this readily available proximal biofluid.