Abstract 816: Utility and validation of a comprehensive cost-effective targeted DNA panel including FLT3-ITDs, CALR and CEBPA on a next-generation sequencing (NGS) platform for hematological malignancies
Authors: Ahluwalia Meenakshi , Mondal Ashis , Sahajpal Nikhil , Njau Allan , Jones Kimya , Ahluwalia Pankaj Kumar , Jilani Yasmeen , Kolhe Ravindra 
Published: September 2020

Abstract
A clinical next generation sequencing (NGS) assay comprises various factors taken into consideration including selection of appropriate markers thereby panel size, nucleic acid input, assessment and interpretation, and workflow. The downstream benefits of the development of such panels therefore involve improvement in prognosis, patients' outcome, and benefit from targeted therapy. Myeloid malignancies such as myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS), MDS/MPN, acute myeloid leukemia (AML) are associated with somatic mutations in around 25 to 50 genes. Herein, we describe clinical validation of a DNA based targeted NGS assay utilizing QIAact Myeloid DNA UMI panel in combination with QIAGEN GeneReader NGS system. The assay employs unique molecular index technology (UMI) into a gene specific, primer based target enrichment technology enabling sequencing of specific regions of interest providing an integrated solution by simultaneously assessing many candidate genes for actionable mutations. The QIAact Myeloid DNA UMI panel is a 25 gene panel for markers of known clinical significance, including single nucleotide variants (SNV), and large insertion/deletion mutations. At the lab, 40 individual AML specimens with known mutation profiles were tested. The limit of detection (LOD) was assessed by sequencing Seraseq myeloid mutation DNA mix as positive control. For accuracy, inter-run and intra-run controls were also implemented. All samples were previously characterized by using another 54 gene NGS panel. The samples were prepared using QIAact Myeloid DNA UMI panel kits, sequenced with QIAGEN GeneReader NGS system, and mutations identified using the QIAGEN Clinical Insight (QCI) Analyze software suite, adjusted specifically for variant calling. Variant calling was accurate and reproducible at allele frequencies ≥5%. Limit of detection (LOD) studies also determined that input of 40ng DNA was optimal for high analytical sensitivity. High positive and negative percentage agreement with prior results was observed across all variant categories. The assay was also able to identify 52bp deletion CALR type 1 variant, and CEBPα amplicons mutations which require separate bi-directional Sanger sequencing for identification. Also, the assay was able to identify FLT3 ITDs up to 101bp insertion which are detected by performing PCR separately. Hence, the study presents the data establishing the QIAact Myeloid DNA UMI panel suitable for implementation as a routine clinical NGS test for myeloid malignancies. The optimized chemistry allows high-throughput analytical sensitivity for detection of highly relevant mutations including large Insertion/Deletion (InDel).
Citation Format: Meenakshi Ahluwalia, Ashis Mondal, Nikhil Sahajpal, Allan Njau, Kimya Jones, Pankaj Kumar Ahluwalia, Yasmeen Jilani, Ravindra Kolhe. Utility and validation of a comprehensive cost-effective targeted DNA panel including FLT3-ITDs, CALR and CEBPA on a next-generation sequencing (NGS) platform for hematological malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 816.