Evaluation of a novel method for rapid collection of microgram amounts of cell free DNA.

Abstract

e15048 Background: Cell free DNA (cfDNA) and cell free cancer DNA (ctDNA) have become common place for monitoring the progression of mutations, the presence of minimal residual disease (MRD), and early recurrence (ERD) of cancer. cfDNA is being explored for many novel uses in cancer and other fields. It is currently on the market, although not FDA approved, for multiple cancer early detection (MCED). Finding cancer at curable stages requires detecting very rare fragments of cfDNA. Since a single cell worth of DNA is ~6 pg, to detect this in a million-fold excess of normal DNA, which advanced analytic methods are capable of, ~ 6 ug of DNA is required. Current methods typically isolate only 10s of ng per sample. There is a need for isolating more cfDNA to improve sensitivity of MRD, ERD and MCED. Urine has successfully been used as an alternative to blood for cfDNA. Urine is the obvious choice for urogenital tract cancer, but collection of trans-renal DNA has allowed detection and monitoring of internal cancers including glioma. Isolation from urine has been problematic as there is a rapid, potentially biased, DNA loss within minutes of voiding. Current kits isolate predominantly nucleosomal size (166 bp) DNA, which comes from urothelium, rather than trans-renal DNA that is under 100 bp. Some kits harvest no DNA of this size, others contain it as a minor ( < 30%) component. Typically, 10 mls are processed, some kits allow up to 40 mls to be handled. Methods: We evaluated "uProcess" a method that is purported to immediately stabilize urine cfDNA, and within minutes process complete voids of up to 250 mls, separate the small trans-renal cfDNA and discard both the large urine volume and nucleosomal/urothelial DNA. The cfDNA is trapped in a filter cartridge. A home user may perform this simple initial step. Isolation of the DNA in the lab takes 10 minutes and does not require any instruments. Results: We found that isolation of 1.4 ug per 100 mls of urine was typical. It was stable at RT in the cartridge for 4 days. The purified cfDNA from normal individuals shows a peak at 80-100 bp and virtually no material at 166 bp. PCR from ng amounts of DNA detected unique sequences. Illumina NGS on NovaSeq X following library preparation using the Ovation Ultralow V2 kit resulted in %Q30 bases of 89.3%. Conclusions: Frequent monitoring of ctDNA, at higher sensitivity, is possible with this integrated system. The larger amounts of cfDNA obtained by uProcess may allow for higher clinical sensitivity of current tests, use of tests requiring more DNA, and sample banking. It may enable daily monitoring of initial cancer therapy and between visit monitoring for ctDNA. At home use may facilitate compliance, especially of older patients, those with limited mobility, and those in rural locations throughout the world. Rapid instrument free lab methods may allow low-resource settings to participate. Increased participation in MCED screening programs might occur if home collected urine is used.