Cells expressing wild-type FLT-3 were exposed to 10 ng/mL FLT-3 ligand (R&D Systems) for 10 minutes before harvest. To test TKI inhibition, the cells were exposed to a range of concentrations of drug for 1 hour before harvest. The cells were grown in cytokine-free medium for 4 hours and then harvested for cell lysis and phospho-western analysis. 11īa/F3 cells stably expressing FLT-3 constructs were washed to remove rm IL-3. Although databases such as the Catalog of Somatic Mutations in Cancer (COSMIC) provide repositories for such information, a systematic approach has not been undertaken to couple these correlative data with validated testing. With this new level of precision, a need has arisen for more complete information regarding the biological and clinical significance to assist clinicians in interpreting this new wealth of potentially treatment-informing data. With these new technologies, it is now possible to examine FLT-3 and other genes with nucleotide level resolution and identify gene derangements that previous PCR-based techniques would not have detected. The recent advent of next-generation sequencing (NGS) has provided clinicians with a new way of examining the FLT-3 status of patients with AML. 10 FLT-3/ITD mutations are identified by the presence of an additional PCR product longer than the wild-type FLT-3 fragment, with the relative abundance of that additional fragment allowing for direct measurement of the variant allele frequency. The traditional diagnostic method for detecting FLT-3/ITD mutations is polymerase chain reaction (PCR) amplification of the FLT-3 JMD. With the promise of FLT-3 TKI therapy becoming a practical reality and given the role of FLT-3 as one of the most risk-defining mutations in AML, the proper identification of targetable FLT-3 mutations is becoming increasingly critical. It is likely that, as NGS becomes more commonly used in the diagnosis of patients with AML, these and other activating mutations will be discovered with increasing frequency. Our results suggest a new class of FLT-3 mutations that may have an impact on patient care and highlight the increasing importance of a systematic understanding of FLT-3 mutations other than ITD.
These mutations target key residues in the JMD involved in the interactions within FLT-3 that regulate its activation. We present the results of a systematic analysis that identified Y572Δ, E573Δ, and S574Δ as similarly activating and targetable deletions located in the FLT-3 juxtamembrane domain (JMD). This mutation is activating, drives downstream signaling comparable to FLT-3/ITD, and can be targeted using available FLT-3 TKIs. We present a novel mutation, FLT-3/Q575Δ, identified in a patient with AML through next-generation sequencing (NGS). The most common mutation is the internal tandem duplication (ITD). The FMS-like tyrosine kinase 3 (FLT-3) is the most frequently mutated gene in acute myeloid leukemia (AML), a high-risk feature, and now the target of tyrosine kinase inhibitors (TKIs), which are approved and in development.
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