Analytical validation of NeXT Personal®, an ultra-sensitive personalized circulating tumor DNA assay

// Josette Northcott 1 , Gabor Bartha 1 , Jason Harris 1 , Conan Li 1 , Fabio C.P. Navarro 1 , Rachel Marty Pyke 1 , Manqing Hong 1 , Qi Zhang 1 , Shuyuan Ma 1 , Tina X. Chen 1 , Janet Lai 1 , Nitin Udar 1 , Juan-Sebastian Saldivar 1 , Erin Ayash 1 , Joshua Anderson 1 , Jiang Li 1 , Tiange Cui 1 , Tu Le 1 , Ruthie Chow 1 , Randy Jerel Velasco 1 , Chris Mallo 1 , Rose Santiago 1 , Robert C. Bruce 1 , Laurie J. Goodman 1 , Yi Chen 1 , Dan Norton 1 , Richard O. Chen 1 , * and John M. Lyle 1 , * 1 Personalis, Inc., Fremont, CA 94555, USA * Co-last authors Correspondence to: John M. Lyle, email: john.lyle@personalis.com Keywords: circulating tumor DNA; whole genome sequencing; molecular residual disease; tumor-informed assay; analytical validation Received: December 11, 2023 Accepted: February 12, 2024 Published: March 14, 2024 Copyright: © 2024 Northcott et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT We describe the analytical validation of NeXT Personal ® , an ultra-sensitive, tumor-informed circulating tumor DNA (ctDNA) assay for detecting residual disease, monitoring therapy response, and detecting recurrence in patients diagnosed with solid tumor cancers. NeXT Personal uses whole genome sequencing of tumor and matched normal samples combined with advanced analytics to accurately identify up to ~1,800 somatic variants specific to the patient's tumor. A personalized panel is created, targeting these variants and then used to sequence cell-free DNA extracted from patient plasma samples for ultra-sensitive detection of ctDNA. The NeXT Personal analytical validation is based on panels designed from tumor and matched normal samples from two cell lines, and from 123 patients across nine cancer types. Analytical measurements demonstrated a detection threshold of 1.67 parts per million (PPM) with a limit of detection at 95% (LOD 95 ) of 3.45 PPM. NeXT Personal showed linearity over a range of 0.8 to 300,000 PPM (Pearson correlation coefficient = 0.9998). Precision varied from a coefficient of variation of 12.8% to 3.6% over a range of 25 to 25,000 PPM. The assay targets 99.9% specificity, with this validation study measuring 100% specificity and in silico methods giving us a confidence interval of 99.92 to 100%. In summary, this study demonstrates NeXT Personal as an ultra-sensitive, highly quantitative and robust ctDNA assay that can be used to detect residual disease, monitor treatment response, and detect recurrence in patients.