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Introduction: Accurate diagnosis, prognosis and treatment of hematologic disorders from both myeloid and lymphoid origin requires assessing somatic mutation status in an increasingly large number of genes. Here we present the results of the assay development and validation of a targeted next generation sequencing panel which provides full exon sequencing of 141 genes associated with myeloid and lymphoid malignancies, as well as the ability to detect clinically relevant copy number alterations (CNAs) and certain sub-gene level CNAs. Methods: Based on review of clinical guidelines, 141 genes including 72 myeloid and 84 lymphoid associated genes were selected. The assay interrogates all coding exons of the 141 genes to detect single nucleotide variants (SNVs) and insertions/deletions (indels). The assay also identifies CNAs in 16 genes, FLT3 internal tandem duplicates (ITDs), and select non-coding pathogenic variants. The custom hybrid capture-based assay utilizes genomic libraries created from 250 ng gDNA extracted from peripheral blood or bone marrow followed by sequencing on Illumina sequencers. Concordance studies were performed on clinical samples previously assessed using an orthogonal NGS-based laboratory developed test for SNVs/Indels or digital multiplexed ligation-dependent probe amplification for CNAs. In addition, analytical sensitivity was assessed using cell line dilution series and specificity by reference samples from Genome in a Bottle (GIAB). Small indels were defined as Results: Interim estimates of positive percentage agreement (PPA) and specificity have been determined for SNV/Indels, FLT3-ITDs, and CNVs, with further testing ongoing. PPA of SNVs was 100% (218/218 SNVs; n = 134 samples). PPA was also 100% for both small indels (21/21; n = 14 samples) and long indels (6/6; n = 6 samples). A dilution series of 8 positive clinical samples at 4 different dilutions showed SNV and small indel detection of 100% at allele frequency (AF) ≥ 3%. Long indel detection was 87.5% at AF ≥ 5%. FLT3-ITD was evaluated with 2 cell line samples at 5 different dilutions with 100% detection at AF ≥ 5%. PPA of CNAs were 95.7% (157/164) in 46 clinical samples. CNA detection was also evaluated with 8 cell line samples at 4 different dilutions showing 92.3% detection at a copy number ratio threshold ± 0.15. A preliminary estimate of specificity was determined using 4 replicates of the GIAB sample, NA12878, and was >99.99% for SNVs and small indels (n=6.9 × 105). Finally, a model for gender classification was created using a training cohort of 70 clinical samples. The performance was evaluated on a test set of 144 clinical samples with 100% PPA (144/144). Conclusions: Taken together, these data demonstrate that the developed NGS assay is a sensitive, specific, and accurate assay with high clinical utility to inform treatment decision making in hematologic malignancies. Citation Format: Grant Hogg, Tong Liu, Helen Cao, Adib Shafi, Ashraf Shabaneh, John Howitt, Amanda Williamson, Rachel Dango, Xiaojun Guan, Heidi Hoffmann, Michael Mooney, John Pruitt, Scott Parker, Stan Letovsky, Li Cai, Eric A. Severson, Shakti Ramkissoon, Anjen Chenn, Marcia Eisenberg, Eyad Almasri, Taylor Jensen. Development of a mutation profiling NGS assay to facilitate clinical decisions in hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6220.

By analyzing COVID-19 sequential COVID-19 test results of patients across the United States, we herein attempt to quantify some of the observations we've made around long-term infection (and false-positive rates), as well as provide observations on the uncertainty of sampling variability and other dynamics of COVID-19 infection in the United States. Retrospective cohort study of a registry of RT-PCR testing results for all patients tested at any of the reference labs operated by Labcorp® including both positive, negative, and inconclusive results, from March 1, 2020 to January 28, 2021, including patients from all 50 states and outlying US territories. The study included 22 million patients with RT-PCR qualitative test results for SARS-CoV-2, of which 3.9 million had more than one test at Labcorp. We observed a minuscule 115 days, which could account for false positives, long-haulers, and/or reinfection but is indistinguishable in the data. In observing repeat-testing, for patients who have a second test after a first RT-PCR, 30% across the cohort tested negative on the second test. For patients who test positive first and subsequently negative within 96 h (40% of positive test results), 18% of tests will subsequently test positive within another 96-h span. For those who first test negative and then positive within 96 h (2.3% of negative tests), 56% will test negative after a third and subsequent 96-h period. The sudden changes in RT-PCR test results for SARS-CoV-2 from this large cohort study suggest that negative test results during active infection or exposure can change rapidly within just days or hours. We also demonstrate that there does not appear to be a basal false positive rate among patients who test positive >115 days after their first RT-PCR positive test while failing to observe any evidence of widespread reinfection.