Enrichment of mutated DNA enables ultra-sensitive ctDNA detection in NSCLC using shallow targeted sequencing.

Liquid biopsy assays using next-generation sequencing have emerged as a promising tool in cancer diagnostics to aid detection of minimal residual disease (MRD) and treatment monitoring. A key concern is the trade-off between sensitivity and sequencing depth, with ultra-sensitive techniques suffering from high costs. To this end, we sought to evaluate the analytical performance of Enspyre, a novel ctDNA enrichment method with the potential to achieve ultra-high sensitivity at a significantly reduced sequencing depth. Tumour and, where available, matched normal tissue from 8 lung cancer patients underwent whole-genome sequencing to identify somatic variants. Custom capture panels designed against patient-specific variants were used to run the Enspyre assay on 72 samples (9 per subject) consisting of patient-derived plasma diluted into a background of healthy donor plasma. An additional 8 healthy donor samples were run as negative controls. Plasma samples were analysed using a proprietary algorithm to estimate ctDNA levels in each sample. Enspyre reached 100% (6/6) detection rate down to 10 parts per million (ppm) and was able to call MRD in samples as low as 5 ppm, while maintaining a false positive rate of zero. This was achieved despite very low DNA input (median: 7.67 ng), without the use of molecular barcodes for error suppression and a median of only 9.6 million read pairs per sample. Given its low sequencing requirements, Enspyre could make liquid biopsy-based MRD and treatment monitoring more accessible by bringing down costs and allowing for use of smaller benchtop sequencing machines. While larger studies are needed to robustly establish the assay's analytical performance, the results described here showcase Enspyre's promise in cancer diagnostics.