Analytical validation of OriMIRACLE S, a personalized and tumor-informed approach to minimal residual disease surveillance.

Abstract

e15013 Background: The presence of minimal residual disease (MRD) after curative-intent therapies in solid tumors is recognized as a risk factor for relapse. Glycoprotein-based tumor markers lack sensitivity and specificity for clinical application. Circulating tumor DNA (ctDNA) based MRD detection which can identify disease relapse ahead of radiological imaging has shown promising performance. The objective of this study was to develop and validate OriMIRACLE S (Minimal Residual Circulating Nucleic Acid Longitudinal Detection in Solid Tumor), a highly sensitive and specific tumor-informed assay for MRD detection via plasma cell-free DNA. Methods: Tumor-specific somatic single nucleotide variants (SNVs) were identified by whole exome sequencing of tumor tissue and matched germline DNA. Clonal SNVs were selected by OriSelector algorithm for patient-specific multiplex PCR-based NGS assay for MRD tracking. The limit of detection (LOD), specificity, sensitivity, repeatability, and reproducibility of this assay were determined experimentally with a series of standard references and in silico. Standard references were developed as following for assessing assay performance. The somatic variant references were two commercialized standards each containing five positive sites at variant allele frequency [VAF] of 0.1% and 1%, and a wild type sample. The single nucleotide polymorphism (SNP) references were made by blending three gDNA samples (160 sites) with gDNA samples without these SNPs to VAFs of 0%, 0.005%, 0.01%, 0.02%, 0.05% and 0.1%. And cfDNA references were made by titrating the clinical cfDNA mixture from five patients containing 31 positive sites with mononucleosomal HEK293 DNA to VAFs of 0.01-0.3% and 0.1-3%. Results: Site level sensitivity of ctDNA samples were 68.8% to 100% with 10 to 50ng input ctDNA (3,000 to 15,000 haploid genome equivalents). Site level reproducibility and repeatability were 98.0% and 97.0% using somatic variant references, respectively. The detection of three positive sites was sufficient to achieve a nearly 100% overall sample level sensitivity and specificity (6,000 haploid genome equivalents of gDNA samples) determined either by calculating a binomial probability or in silico with customized panels have 21 to 30 variants. The LOD of this assay was as low as 0.01% with the algorithm of OriCleverClover which could filter process and sequencing-related artifacts. Conclusions: OriMIRACLE S has been analytically validated as a highly sensitive and specific method to detect low quantities of ctDNA in plasma. The low limit of detection at a VAF of only 0.01% with low DNA input. OriMIRACLE S has wide-ranging potential applications in the clinical setting and drug development in a real-world scenario.