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e15549 Background: Liquid biopsy is a candidate for detection of minimal residual disease (MRD) in early colorectal cancer. Detection of circulating tumor (ct)DNA in early stage cancer is challenging given the low abundance of ctDNA molecules. We developed a tissue agnostic MRD Test to optimize sensitivity and specificity of ctDNA detection by integrating tumor-specific genomic mutation and DNA methylation signatures coupled to noise-reduction of non-tumor background cfDNA signals. Methods: This MRD Test was developed following CLIA, Nex-StoCT Working Group, and AMP/CAP guidance and validation principles. Using a single input plasma sample, data from somatic and epigenomic targeted capture panels are integrated to generate a final test result: ctDNA detected or ctDNA not detected. ctDNA detected is defined by the presence of mutation or epigenomic classifier inputs exceeding a defined threshold. Quality control (QC) measures were implemented in both process and final sequencing results. Analytical specificity, sensitivity, and accuracy were determined using 130 samples. Results: The MRD Test specificity was 95% (70/74) using colonoscopy screened negative samples.A retrospective review of the clinical histories of these false positive samples showed high risk clinical features such as smoking, family history of CRC, and sub-optimal bowel prep prior to the colonoscopy. Analytical sensitivity (LoD) was established by diluting late stage CRC samples at two clinically relevant cfDNA inputs at three dilutions (30ng or 10ng, ranging 0.1% to 0.75% tumor fraction). The 95% LoD was determined to be 0.3% for 10ng and < 0.1% for 30ng, the lowest level tested for each input. Accuracy was determined by diluting advanced CRC samples to 0.5-0.75% MAF tested at 30ng or 10ng cfDNA with a detection result of 100% (38/38). Conclusions: We present a CLIA validated assay with performance characteristics sufficient to detect residual disease in Stage II/III CRC post-curative intent therapy. This assay is currently being used in multiple interventional clinical trials.

Background: Late stage therapy selection is advancing beyond the detection of classical driver alterations (SNV, Indel, CNV, Fusion) to the use of aggregate biomarkers comprising combinations of SNVs, Indels, and fusions, that reflect the underlying mutational process, indicate the cellular dysfunction, and suggest the neoantigen signature. GuardantOMNI (OMNI), is a highly sensitive 500-gene cfDNA sequencing test designed to detect primary somatic mutations as well as aggregate biomarkers including TMB and DDR pathway defects. The newest version of OMNI introduces MSI biomarker detection, which is an indication for the first FDA-approved pan-cancer therapy. Here we present the first analytical validation study of OMNI MSI-H detection. Methods: Probabilistic models for MSI detection were designed to interrogate instability across >1300 loci, differentiating PCR and sequencing induced artifacts from true biological signals using molecular barcoding as implemented in the Guardant digital sequencing platform. Cut-offs were selected based on the analysis of the training healthy donor cohort and locked prior to validation. Analytical performance was assessed using pre-characterized cell lines, cancer-free donor-samples, and clinical patient samples. Qualitative and quantitative orthogonal confirmation was provided by next-generation sequencing (NGS) and published information. Results: 350 samples were processed for this study, using both 5ng and 30ng cfDNA input levels, and all samples passed sequencing QC metrics established prior to testing. Cell line-based dilution studies performed in triplicate spanning 5ng and 30ng input range as well as in silico simulation demonstrated 95% limit of detection (LoD) at 0.1% Analysis of 121 healthy donor and 16 MSS advanced cancer patient samples identified no false positives (FP). In 240 patient samples with orthogonal confirmation of MSI status from tissue or panel NGS, GuardantOMNI demonstrated 100% analytic sensitivity (3/3) and 100% analytic specificity (237/237) with a PPV of 1 and and NPV 1. Retrospective analysis of 2000+ clinical samples detected MSI high in 16% of colorectal cancers, 4% of prostate cancers, and 0.6% of non-small cell lung cancers. Conclusions: Here we have demonstrated the performance of MSI-H detection with GuardantOMNI (OMNI), which has the potential to accelerate clinical trial enrollment, research and discovery with a single, non-invasive blood sample. Citation Format: Jing Zhao, Alexander Artyomenko, Carlo Artieri, John Latham, Stephen R. Fairclough, Catalin Barbacioru, Elena Helman, John Strickler, Darya Chudova, Richard Lanman, AmirAli Talasaz. Analytical validation of MSI High detection with GuardantOMNI [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1675.