Abstract 4628: Validation of an automated, scalable comprehensive genomic profiling assay for hematologic malignancies
Authors: Hogg Grant , Liu Tong , Cao Helen , Shafi Adib , Shabaneh Ashraf , Williams Jon , Howitt John , Williamson Amanda , Dango Rachel , Guan Xiaojun , Hoffmann Heidi , Mooney Michael , Pruitt John , Parker Scott , Dong Henry , Letovsky Stan , Cai Li , Severson Eric A. , Ramkissoon Shakti H. , Chenn Anjen , Eisenberg Marcia , Caveney Brian , Almasri Eyad , Jensen Taylor J. 
Published: March 2024

Abstract
Optimal diagnosis, prognosis, and treatment selection for hematologic malignancies requires assessment of somatic mutations across a subset of clinically relevant genes. Here we present the analytical validation results of an expanded targeted next generation sequencing (NGS) panel of 141 clinically relevant genes for myeloid and lymphoid malignancies. Based on review of clinical guidelines, 141 genes including 79 myeloid and 84 lymphoid associated genes were selected. The expanded hematological panel interrogates all coding exons of the 141 genes to detect single nucleotide variants (SNVs) and insertions/deletions (indels). The panel also identifies copy number aberrations (CNAs) in 16 genes, FLT3 internal tandem duplicates (ITDs), select non-coding pathogenic variants, and patient sex. The custom hybrid capture-based assay utilizes genomic libraries created from 250 ng gDNA extracted from peripheral blood, bone marrow, or cell suspensions, followed by sequencing on Illumina® instruments. Concordance studies were performed on clinical samples previously assessed using an orthogonal NGS-based laboratory developed test for SNVs/indels/FLT3-ITDs or digital multiplexed ligation-dependent probe amplification for CNAs. In total, 308 patient samples were evaluated and included 72 (23.4%) acute myeloid leukemia and 58 (18.8%) myelodysplastic syndromes samples among other indications, of which 240 (77.9%) were bone marrow specimens. Analysis of concordance demonstrated a positive percent agreement (PPA) of 99.7% for SNVs (750/752), 99.5% for indels &lt;25bp (206/207), and 95.7% for indels ≥ 25bp (22/23). Variants were from 45 genes including TP53, NPM1, and IDH1/2. PPA was 100% for FLT3-ITDs (38/38; size range 18-300 bp) and sex (294/294). For CNAs, PPA was 96.4% (132/137) and negative percent agreement (NPA) was 98.3% (931/947). Assay precision was determined using three replicates of 15 clinical samples for both intra and inter-assay precision using multiple operators, instruments, and reagent lots. 100% concordance was observed for SNVs (36/36), indels (11/11), a 24 bp FLT3-ITD (1/1), CNAs (24/24), and sex (15/15). Analytical specificity was assessed using 5 replicates of NA12878 showing specificity &gt;99.99% for SNVs/indels with variant allele frequency ≥ 3%, and &gt;99.99% for CNAs with copy number ≤0.85 or ≥1.15. Dilution series to determine analytical sensitivity are in progress. This study highlights the analytical validation of the expanded NGS panel for the detection of clinically informative genomic alterations in hematologic malignancies. Results from this validation study, when complete, will include at least 576 samples plus orthogonal testing. These data describe the performance of the assay to enable a comprehensive evaluation of genomic alterations using a single sample, further facilitating the use of broad NGS assays in patients with hematologic malignancies.
Citation Format: Grant Hogg, Tong Liu, Helen Cao, Adib Shafi, Ashraf Shabaneh, Jon Williams, John Howitt, Amanda Williamson, Rachel Dango, Xiaojun Guan, Heidi Hoffmann, Michael Mooney, John Pruitt, Scott Parker, Henry Dong, Stan Letovsky, Li Cai, Eric A. Severson, Shakti H. Ramkissoon, Anjen Chenn, Marcia Eisenberg, Brian Caveney, Eyad Almasri, Taylor J. Jensen. Validation of an automated, scalable comprehensive genomic profiling assay for hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4628.