Lab validation of an ultrasensitive ctDNA pan-cancer MRD assay using whole-genome sequencing.
Authors: Kandasamy Ravi , White Eric , Strycharz Joseph P. , Hansen Sherry , Hamby Claude , Waterhouse Sarah , Salkeld Ashley , Mannke Kailyn , Valliere Cecille , Phillips Samuel , Ramaraj Nidhi , Stanley Christine , Ananth Uma , Deochand Sunil , Veksler Ronel , Lauterman Tomer , Afterman Danielle , Tavassoly Iman , Oklander Boris , Zviran Asaf 
Published: June 2022

e13582

Background: Minimal residual disease (MRD) monitoring using liquid biopsy for solid tumors requires a highly sensitive and specific assay that can overcome the limitation of low abundance cfDNA in a standard blood draw. We developed a whole-genome sequencing (WGS)-based assay to detect the presence of circulating tumor DNA (ctDNA) in plasma. The C2i assay is a tumor-informed assay that uses personalized tumor signature, advanced noise models, and artificial intelligence (AI) modalities to interrogate plasma for the presence of ctDNA longitudinally. Methods: The C2i test was developed in accordance with CAP/CLIA and New York state validation principles. We used contrived samples to establish analytical validation of the assay performance, which was then validated with a large clinical cohort of early-stage patients across various cancer types. Briefly, aggregated tumor signatures derived from cancer cell lines were fragmented and spiked into a contrived healthy plasma pool; the mixed samples were used to assess the presence of tumor DNA signature down to tumor fractions of 10e-4. Positive samples are identified by tumor-derived variants detected above the noise levels. Noise modeling was established using a panel of normal (PON) approach. We assessed the analytical sensitivity, specificity, and accuracy using 348 contrived samples derived from five different cancer cell lines. Reproducibility and precision were assessed with multiple replicates, and statistical concordance was reported. This validation was complemented by a cohort of 200 patients and ̃1000 plasma samples across a variety of cancer types including, NSCLC, MIBC, CRC, GBM, Breast Cancer, and a mixture of other cancer types. Results: Cancer cell lines, representing the five most prevalent disease indications, used for determining analytical sensitivity are as follows: CRC HT-29, Breast SK-BR3, Bladder HT-1376, Lung HCI-H526, and Prostate LNCaP. The cell line DNA was enzymatically fragmented and size-selected to mimic ctDNA. This ctDNA was spiked into cfDNA extracted from healthy volunteers at various dilution levels, varying from 10e0 to 10e-4. The 95% probability of detecting ctDNA was established at 10e-4. The reproducibility of tumor signature between replicates was assessed to be greater than 90%. The assay was performed using both normal and maximum input amounts. These performance estimates were then validated on a cohort of plasma collected from early-stage (stage I-III) patients across various cancer types. Conclusions: C2i MRD test is an ultrasensitive pan-cancer MRD monitoring assay used in several clinical trials across the world. We present an extensive analytical and clinical validation of the assay supporting its high performance.