Abstract 7657: Detection of KMT2A-PTDs and KMT2A fusions using next generation sequencing
Authors: Huang Jiajie , Gu Haigang , Orton Janet , Sedova Marina , Marcovitz Amir , Burke Jennifer , Brozio Sarah , Williams Paul , Myrand Scott , Olowo Nate , Broomer Adam , Deal Brendan , Seeger Collyn , Sadis Seth , Rozenzhak Sophie , Hyland Fiona , Liu Guang 
Published: March 2024

Abstract
Introduction: KMT2A (MLL) rearrangements (fusions) is a therapeutic biomarker for Menin inhibitors. KMT2A-PTDs (partial tandem duplications) are considered a prognostic biomarker in myeloid malignancies. KMT2A fusions and PTDs are traditionally detected by RT-qPCR (quantitative real-time PCR). This study investigates KMT2A PTDs levels in healthy donors and myeloid malignancy samples to establish a threshold to report samples with high PTDs, using next generation sequencing (NGS) with OncomineTM Myeloid Assay GX v2. We also report KMT2A fusions in myeloid malignancies.
Methods: We sequenced 8483 research samples with known myeloid malignancies at Sonora Quest LaboratoriesTM and at Thermo Fisher ScientificTM. We acquired 20 healthy donor whole blood samples (total 127 replicates) from StanfordTM Blood Center and Discovery Life SciencesTM and sequenced them at 3 different sites of Thermo Fisher ScientificTM. Samples were sequenced with the Ion TorrentTM GenexusTM 6.6 or Ion GeneStudioTM S5 System. They were profiled for 6 different KMT2A-PTD variants and 199 KMT2A fusion isoforms.
Results: The mean read length of this data set is 90 - 120 bp and the mean mapped fusion reads is 20,000 - 30,000. KMT2A-PTDs were detected in both healthy donors and myeloid samples. Healthy donor PTD read counts were consistently &lt;2000 and averaged 1/3 of myeloid sample PTD read counts. About 33% of myeloid samples had higher PTD read counts than any healthy donor sample. BLAT (BLAST-Like Alignment Tool) analysis confirmed specific exon matching on the KMT2A gene in both cohorts. Among the 8483 myeloid samples, 162 samples contained a total of 5 unique KMT2A PTDs, and 105 samples contained a total of 30 unique KMT2A fusion isoforms with KMT2A-MLLT1 and KMT2A-MLLT3 being the most prevalent KMT2A fusion gene pairs.
Conclusions: We characterize the KMT2A fusions present in myeloid malignant samples. We also describe the abundance of KMT2A PTDs in both healthy donor and myeloid samples, with myeloid cases showing significantly higher PTD read counts. KMT2A PTD read count &gt;2000 is present only in malignant samples but not in healthy donors. This intriguing finding opens opportunities for prospective studies to monitor individuals with elevated PTD levels for myeloid malignancy development and retrospective studies to explore whether healthy donors identified with this alteration years ago after blood donation were subsequently recorded in the national health system with myeloid malignancies.
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Citation Format: Jiajie Huang, Haigang Gu, Janet Orton, Marina Sedova, Amir Marcovitz, Jennifer Burke, Sarah Brozio, Paul Williams, Scott Myrand, Nate Olowo, Adam Broomer, Brendan Deal, Collyn Seeger, Seth Sadis, Sophie Rozenzhak, Fiona Hyland, Guang Liu. Detection of KMT2A-PTDs and KMT2A fusions using next generation sequencing [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 7657.