Large healthy donor dataset demonstrates high analytical specificity and repeatability for the NeXT Personal molecular residual disease assay.
Authors: Navarro Fabio , Bartha Gabor , Lyle John , Harris Jason , Northcott Josette , Norton Dan , Pyke Rachel Marty , Abbott Charles , Chen Rui , Boyle Sean Michael , Haudenschild Christian , Santiago Rose , Nelson Christopher , Huo Yelia , Chinnappa Manju , Zhang Qi , Chen Richard 
Published: June 2023

e15031 Background: Residual and recurrent cancer detection using circulating cell-free DNA (cfDNA) in liquid biopsy samples has proven promising in the past decade, with emerging technologies driving increasingly lower limits of detection (LOD). At ultra-high detection sensitivity levels, it is critical to warrant a high specificity to ensure that the detected signals are true measurements of the residual disease. Methods: The molecular residual disease (MRD) detection application of the NeXT Personal platform relies on up to 1,800 personalized somatic variants identified by whole genome sequencing (WGS) of the patient-specific tumor compared with the matched normal sample. Under the assumption that individuals with undetectable cancer should have negative MRD detection, we evaluated the MRD analytical specificity of the assay by measuring background noise using over 200 patient-specific panel designs to a large cohort of healthy donors that should display no tumor signals at any MRD target loci. These panels were designed and manufactured based on a pan-cancer cohort of tumor-normal paired samples whole genome data. Then, those designed panels were evaluated on cfDNA extracted from the plasma of over 200 unique healthy donors. 30-50 ng (median 50 ng) of cfDNA was used for the assay for each sample. Furthermore, six of the 200 patient panels were used to measure assay repeatability by hybridizing to at least 28 different healthy donors per panel. Results: The pan-cancer cohort used for panel design was composed of predominantly stage II ~ IV patients from 12 distinct tumor types. Across all patient samples, no MRD was detected, resulting in 100% specificity (95% confidence interval: 98.23-100%). In addition to the 100% specificity observed, the median anticipated limit of detection (LOD) as calculated by the number of targets and the number of molecules at each target was 2.6 PPM, with the lowest LOD being 1.4 PPM. The average observed unexpected signal was nearly one order of magnitude lower than the average LOD of each panel. Lower LODs were associated with larger panels (up to 1,800 mutation targets) and higher tumor purity. No correlation was observed between noise and tumor type of the designed panels or healthy plasma donor age. Further, we observed 100% repeatability across the six panels that were each applied to at least 28 unique plasma donors, with MRD not being detected in any of the replicates. Conclusions: The large healthy donor screening study demonstrated that our assay maintains excellent specificity and repeatability while still achieving ultra-sensitive detection. In all cases, the observed error-rates in the panels were very low and did not affect detection specificity. Overall these landmark specificity and repeatability results lend further confidence to the robustness of the analytical performance of NeXT Personal.