Evaluation of uProcess for detection of oncogene sequences in buffer and urine by qPCR following 3-minute capture and 5- minute purification procedure

Cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA), found in blood and urine, have firmly established themselves as key analytes for the diagnosis of various cancers and defining therapy. Primary sequence and epigenetic markers within cfDNA are key in screening and monitoring for cancer. However, both screening and long-term monitoring have two major limitations - test sensitivity and individual participation. Even as NGS, ddPCR, and other methods continue to improve, they require large amounts of cfDNA to reliably detect rare ctDNA fragments. For example, a single BioRad ddPCR reaction can analyze 1 ug of short cfDNA, but a single tube of blood might yield on the order of 10ng, thus cutting potential sensitivity 100x. Additionally, participation is typically a severe impediment to screening and long-term monitoring. Historically, this has been a major obstacle, especially for screening. Travel to clinics, pain/difficulty of phlebotomy, and patient aversion to it limit participation. Urine offers a far more accessible alternative, but ctDNA concentrations are lower and current urine-based cfDNA/ctDNA collection methods have yet to achieve yields adequate for highly sensitive testing. A ug of cfDNA is required for detection at the 0.001% level. Herein, we test and optimize a system (uProcess) designed for at-home urine filtration; it was previously demonstrated to capture over 2 ug of cfDNA from 200 mL of urine on a mail-in filter cartridge. Compared to previous versions, we have reduced the elution volume 5-fold from 2.5 to 0.5 mls, allowing concentrated samples for downstream processing. We demonstrated that the improved device can efficiently concentrate oncogene sequences, in buffer or urine, into a small elution volume in under five minutes vs. typical procedures taking 90-120 minutes. We then detected KRAS and TP53 oncogenes by qPCR, underscoring that the method produces ctDNA suitable for downstream usage. Together, these findings support the potential of uProcess to significantly expand access to cfDNA/ctDNA testing, improve sensitivity, shorten cfDNA purification time, and overcome longstanding barriers to cancer screening or monitoring.