Identification of Mutations Coexisting with FLT3 and NPM1 Mutations in AML Patient Samples Using the MyAML™ Targeting Sequencing Strategy
Authors: Carson Andrew R , Patay Bradley A , Graham Suzanne M , Cubbon Andrew Ross , Stenzel Timothy T , Miller Jeffrey Edward 
Published: October 2019

Abstract
Introduction: Acute Myeloid Leukemia (AML) is the most common acute leukemia in adults, with roughly 19,000 new diagnoses expected yearly in the United States. Mutations in fms-related tyrosine kinase 3 (FLT3) and nucleophosmin (NPM1) are observed in over one third of all AML patients. These mutations include internal tandem duplications within the juxtamembrane domain (FLT3-ITD; 15-30% of patients) and substitutions within the tyrosine kinase domain (FLT3-TKD; 5-10% of patients) of FLT3, along with 4bp insertions (15-30% of patients) within the C-terminal domain of NPM1. These mutations have significant impacts on prognosis; patients with FLT3-ITDs have poor prognosis while patients with NPM1 mutations without an associated FLT3-ITD mutation have better long-term outcomes. Since characterization of these mutations is critical for accurate therapeutic decisions, assays have been developed to accurately identify these mutations in AML patients. However, these assays lack greater context because they do not identify coexisting mutations in other AML associated genes. As such, they fail to characterize additional prognostic markers that may more fully predict and stratify AML patients’ disease progression. To investigate the limitations of AML individual mutations assays, we identified coexisting mutations in 22 AML patients with known FLT3 and NPM1 mutations using the MyAML™ targeted sequencing panel.
Methods: Isolated DNA from 22 AML samples with known FLT3-ITD, FLT3-TKD and NPM1 mutation status was sheared then hybridized to MyAML oligonucleotide baits comprised of exons (coding and non-coding) and breakpoint hotspots from 194 genes known or predicted to be involved in AML pathogenesis. Targeted loci were sequenced on an Illumina MiSeq utilizing v3 chemistry with the 600-cycle kit. By indexing two samples per flowcell, we were able to sequence 12.6 to 32.9 M unique reads per sample, providing an average depth of 985x across the ~3.5Mb target. Using a custom bioinformatics pipeline, we performed mutation detection analyses to identify single nucleotide variants, indels, and structural variant breakpoints. We also calculated variant allelic frequencies to investigate potential aneuploidy, loss of heterozygosity and clonality.
Results: Using individual PCR with capillary electrophoresis (PCR/CE) assays, 14 FLT3-ITD, 7 FLT-TKD and 10 NPM1 mutations were initially detected in the 22 AML patient samples. These mutations were 100% concordant with results from the MyAML sequencing data. However, while the individual PCR/CE assays were limited to detecting specific mutations in two genes, the MyAML panel detected 4,172 protein altering variants in 155 of the 192 additionally targeted genes. These include 35 potential mutations in five key AML genes: 13 in DNMT3A, 5 in IDH1, 8 in IDH2, 4 in KIT and 5 in CEBPA. Eight of the 22 patients contained at least two potentially pathogenic mutations in these five genes, with one patient containing mutations in four of the genes. Interestingly, while some co-existing mutations appear to have the same mutant to wild-type allelic ratio as the main FLT3 or NPM1 mutations, others have distinct ratios that may suggest the presence of subclonal cellular populations. For example, we identified an NPM1 Mutation A (c.859_860insTCTG; p.W288fs*>9; COSM158604) in 40.0% of a patient’s sequencing reads, while a co-existing CEPBA missense mutation (c.961A>G; p.N321D; COSM96570) was only present in 4.3% of the sequencing reads.
Conclusions: While individual assays for mutations in FLT3, NPM1 and other common AML genes are useful for patient stratification and prognosis, it is crucial to understand these mutations in a greater genomic context. As more AML-related mutations are detected, such as resistance mutations to treatments with novel tyrosine kinase inhibitors, it becomes increasing important to fully characterize a patient’s tumor genome in order to successfully classify and treat their disease. In addition, as more AML patient samples are sequenced, relationships between mutations and their clonal populations can be elucidated, potentially leading to more effective combination therapies. MyAML targeted gene sequencing is the most comprehensive AML specific assay for the identification of somatic and germline driver mutations in their clonal context for the prediction of recurrence and response to various treatment regimens.

Disclosures
Patay: Genection, Inc.: Consultancy. Cubbon:LabPMM LLC: Employment. Stenzel:Invivoscribe, Inc.: Employment. Miller:Invivoscribe, Inc.: Employment, Equity Ownership.