Flt3itd and flt3tkd d835 16 6% flt3tkd d835 only 5% prior aml lines of therapy 1 75 30%. Smith cc, american society of clinical oncology educational book. Mutations within the flt3gene are of growing importance for classification, risk assessment and therapeutic targeting in acute. Patients were characterized at the molecular level with regard to flt3 itd, mll ptd, npm1, cebpa, and flt3 tkd d835 mutations, as described previously. Since the food and drug administration approval of imatinib for treatment of chronic myeloid leukemia in 2001, tyrosine kinase inhibitors tkis have become a mainstay in the care of many malignancies. The main objective of the study was to evaluate the associations between mcm7 rs2070215, rs1527423, and rs1534309 single nucleotide polymorphisms snps and acute myeloid leukemia aml risk and prognosis. As this variant is analogous to the human flt3 f691l gatekeeper mutation we. Mutations in this gene are common in acute myeloid leukemia aml and screening for mutations in this gene has been recommended by the world health organization in patients with aml, particularly in cases of cytogenetically normal aml cnaml. Discovery and functional validation of novel pediatric specific. Restrict the view to a region of the gene by dragging across the histogram to highlight the region of interest, or by using the sliders in the filters panel to the left.
In addition, research performed over the last few years has led to the. Activating mutations in fmslike tyrosine kinase 3 flt3, including internal. Peripheral blood involvement is frequent, while infiltration of organs, most ominously the brain andor lung is rare and seen most often in patients with high blood blast counts eg, 50. Receptor tyrosine kinases transmit signals from the cell surface into the cell through a process called signal transduction. Mutations in d835 and i836 remove a naturally occurring ecorv restriction. In our cohort of aml cases, we detected a total of 14 hotspot mutations. The prognostic relevance of flt3 d835i836 mutations flt3tkd in cytogenetically.
Flt3 is a gene change, or mutation, in leukemia cells. Onethird of acute myeloid leukemia patients have mutations of this gene, and the majority of these mutations involve an internal tandem duplication in the juxtamembrane region of flt3, leading to. Inhibitory concentration 50 ic 50 for proliferation of baf3 cells expressing flt3 itd d835 mutants profiled for the clinically active flt3. Inhibitory concentration 50 ic 50 for proliferation of baf3 cells expressing flt3itd d835 mutants profiled for the clinically active. It is structurally homologous to stem cell factor scf and colony stimulating factor 1. These mutations are displayed at the amino acid level across the full length of the gene by default. Crystal structure analysis of flt3 variants was used to assess the potential. We have purified to homogeneity and partially sequenced a soluble form of the flt3 flk2 ligand produced by mouse thymic stromal cells. Gilteritinib for treatment of pediatric patients with flt3.
Here, we demonstrate that cotreatment with jq1 and the flt3 tyrosine kinase inhibitor tki ponatinib or ac220. Cd5 is the receptor for the cytokine flt3 ligand flt3l. Acute myeloid leukemia aml results from accumulation of abnormal myeloblasts, most commonly in the bone marrow, leading to bone marrow failure and death. Clinical evaluation of panel testing by nextgeneration. Downstream molecular pathways of flt3 in the pathogenesis. No mutations have been identified in any cases with diagnoses other than aml. Flt3itd mutations are more frequent within younger adult patients, those with normal karyotype nk, and are associated with proliferative aml eg, higher white blood cell and blast %, and increased risk of relapse leading to decreased os. Frequency and prognostic relevance of flt3 mutations in. Addgene alerts receive email alerts when new plasmids with this gene become available. Flt3 is a class iii receptor tyrosine kinase rtk structurally related to the receptors for platelet derived growth factor pdgf, colony stimulating factor 1 csf1, and kit ligand kl these rtk contain five immunoglobulinlike domains in the extracellular region and an intracelular tyrosine kinase domain splitted in two by a specific. Fmsrelated tyrosine kinase 3 ligand flt3lg is a protein which in humans is encoded by the flt3lg gene flt3 ligand fl is a hematopoietic four helical bundle cytokine.
The flt3 gene provides instructions for making a protein called fmslike tyrosine kinase 3 flt3, which is part of a family of proteins called receptor tyrosine kinases rtks. To test the gene patterns of flt3itd mutations and d835, primers designed to. Flt3 is activated by binding the fmsrelated tyrosine kinase 3 ligand to the extracellular domain, which induces homodimer formation in the plasma membrane leading to autophosphorylation of the receptor. The flt3d324n variant is a functionally silent polymorphism in. Detects the flt3 d835 i836 exon 20 tyrosine kinase domain variant. Description fms related receptor tyrosine kinase 3 also known as cd5, flk2, flk2, stk1 species homo sapiens entrez id 2322. We profiled all d835 substitutions previously reported to cause flt3 tki resistance in patients 1, 5, 6, as well as d835 mutations occurring in patients as cataloged in the sanger cosmic database or the cancer genome atlas. D835 civic clinical interpretation of variants in cancer. Detailed annotation on the structure, function, physiology, pharmacology and clinical relevance of drug targets.
Pretreatment somatic mutations influence acute myeloid leukemia aml pathogenesis and responses to chemotherapy. We assessed the features of patients n 178 harboring. In synergy with other growth factors, flt3 ligand stimulates the proliferation and differentiation of various blood cell. Two basic categories of mutation are recognized in flt3. Ligand for flt3 flk2 receptor tyrosine kinase regulates growth of haematopoietic stem cells and is encoded by variant rnas. Flt3 mutation testing in acute myeloid leukemia genetics.
Molecular diagnostics lab flt3 mutational analysis pcr. The role of targeted therapy in the management of patients. Flt3 d835 master mix tests for tkd region mutations. Flt3 d835i836 mutations are associated with poor diseasefree. Flt3 internal tandem duplication itd and tyrosine kinase. Binary alignment map files of all variant calls and breakpoints. The secondary objectives were to assess if any relationships existed between the mentioned snps and flt3, dnmt3a, npm1 mutations with clinical outcomes and. Internal tandem duplication of the flt3 gene is a novel modality of.
Flt3 is expressed on early hematopoietic progenitor cells and supports growth and differentiation within the hematopoietic system 1,2. Flt3 d835i836 tkd blood laboratory services directory. Recently, treatment with bromodomain and extraterminal protein antagonist ba such as jq1 has been shown to inhibit growth and induce apoptosis of human acute myelogenous leukemia aml cells, including those expressing flt3itd. Aml patients with flt3 internal tandem duplication itd mutations have. Hybridization capturebased next generation sequencing reliably. Evolution of flt3itd and d835 activating point mutations in relapsing acute myeloid leukemia and response to salvage therapy article in leukemia research 2810. In acute myeloid leukemia aml, activating mutations in the fmslike tyrosine kinase 3 flt3 gene result in survival and proliferation of leukemic blasts and are. Promotes activation of ras signaling and phosphorylation of downstream kinases, including mapk1erk2 and. Promotes phosphorylation of shc1 and akt1, and activation of the downstream effector mtor.
Evolution of flt3itd and d835 activating point mutations. Flt3 d835 mutations confer differential resistance to type. Activating mutation of d835 within the activation loop of flt3 in human. Briefly, flt3 exon 20 was pcr amplified using the primer pair forward 5. Flt3internal tandem duplication occurs in 2030% of acute myeloid leukemia. The variant allelic frequency compares the amount of itds to the sum of the mutated and wildtype wt forms of the flt3segment the allelic ratio is the ratio of itds to wt forms the flt3itd mutation allelic ratio appears to be prognostically important, but has not been standardized for clinical decision making. Between 20 and 30 percent of people with aml have this mutation. There is a hydrogen bond directly between the carboxyl group and. Servte performed using kinases under license trom invitrogen.
Identification of driver and passenger mutations of flt3. Detection of flt3 internal tandem duplication and d835. Flt3 a gene on chromosome q12 that encodes a classiii receptor tyrosine kinase, which regulates haematopoiesis. Flt3 somatic variants are among the most common driver mutations with the strongest effects on the overall survival in acute myeloid leukemia 1 aml, the most deadly form of leukemia, which is. Plasmids containing this gene, or a homologous gene. In addition to the known flt3 mutations flt3itd, n128 and d835i836, n37. University of north carolina molecular genetics laboratory 11818, p. Tyrosineprotein kinase that acts as cellsurface receptor for the cytokine flt3lg and regulates differentiation, proliferation and survival of hematopoietic progenitor cells and of dendritic cells. Reference compound ic50 for flt3 d835y 109 8 7 6 5 4 0 20 40 60 80 100 120 pp2 ag1478 pdgfr iiii d64406 log compound m % activit y. Genes free fulltext nanopore targeted sequencing for. Alterations of flt3 have been reported at a frequency of 25%35% in adult acute myeloid leukemia aml, the most common being an internal tandem duplication itd and a missense change at amino acid position 835 d835 in the kinase domain international journal of hematology 20 976. Bet protein antagonist jq1 is synergistically lethal with. Establishment of a costeffective method to detect fltitd and d835.
Validation of itd mutations in flt3 as a therapeutic. Identification of a germline f692l drug resistance variant in cis with. Thus a total of 24 patients had identifiable flt3 mutations 21 itd, 2 d835, 1 both itd and d835, yielding an overall flt3 mutation rate of 24 of 110, 21. Flt3 itd and d835 mutations tend to occur in a mutually exclusive manner. Flt3 is activated after binding with its ligand fl, which results in a cascade of tyrosine autophosphorylation and tyrosine phosphorylation of downstream targets 3. Fl cytokine receptor precursor, tyrosineprotein kinase receptor flt3. Promotes activation of ras signaling and phosphorylation of downstream kinases, including.
The flt3 d835 mutations are also ligandindependent, gainoffunction mutations. Mutations of flt3 comprise one of the most frequently identified types of genetic alterations in acute myeloid leukemia. Flt3 plays a role in early hematopoiesis and flt3 deficient mice have reduced numbers of progenitors of multiple lymphoid lineages mackarehtschian k, et al. However, lengths of the itd mutations 30189 bp, n 4 fell within the range reported to be detectable by pindel 17185 bp, which was one of the variant callers used in this study. Flt3internal tandem duplication and the d835i836 tyrosine kinase. Useful as a prognostic indicator in acute myeloid leukemias.
Flt3 deletion and deletioninsertion mutations were previously reported in cases of pediatric acute lymphoblastic leukemia, but seldom described in adult acute leukemia. Internal tandem duplication mutations in flt3, known to be associated with a poor prognosis in acute myeloid leukaemia, are now shown to be a valid therapeutic target for the disease. Mutations of flt3 were first described in 1997 and account for the most frequent molecular mutations in acute myeloid leukemia aml. Detection of flt3 inframe internal tandem duplications flt3itd mutation is performed as a mailout test sent to a reference laboratory, whereas the assays that detect flt3 point mutations that alter the aspartic acid at position 835 flt3 d835 and the npm1 mutations are performed in the university of iowa molecular pathology laboratory. The results of our clinical experience are summarized in table 1 1. Cluster of differentiation antigen 5 cd5 also known as fms like tyrosine kinase 3 flt3, receptortype tyrosineprotein kinase flt3, or fetal liver kinase2 flk2 is a protein that in humans is encoded by the flt3 gene.
The importance of flt3 mutational analysis in acute. Repression of flt3 by pax5 is crucial for bcell lineage commitment. Flt3 is a cytokine receptor which belongs to the receptor tyrosine kinase class iii. Activating mutations in flt3 are found in approximately 30% of aml patients, representing the most frequent genetic alteration in the disease. Detection of activating mutations in the receptor tyrosine kinase flt3 that occur in 15% of acute myeloid leukemia. Flt3 is an important cytokine receptor involved in normal hematopoiesis. Gene variant descriptions flt3 d835x indicates any flt3 missense mutation that results in the aspartic acid d at amino acid 835 being replaced by a different amino acid. The flt3 gene codes for a protein called flt3 that helps white blood cells grow. Notably, responders included patients who had been resistant to other flt3 inhibitors, such as sorafenib and quizartinib, and patients with both flt3itd and d835 mutations.
Identification of a novel type of itd mutations located in nonjuxtamembrane domains of. The amino acid sequences of the three flt3 itd mutations are shown, with the duplicated region shown below the wildtype sequence. While the biological significance of this type of flt3 mutations is unknown in human disease, a small 10amino acid tyr589 to tyr599 deletion in the juxtamembrane domain of flt3 has been previously shown to lead to. D835 is an integral part of a hydrogen bonding network that seems to stabilize the al backbone in the autoinhibited conformation. A flt3 mutation is a genetic mutation that may be screened during genetic testing, when diagnosed with aml. Mutations of flt3 d835 in exon 20 previously exon 17 were determined as described. Establishing the identification of these two variants from other subtypes of aml.
Erg expression is an independent prognostic factor and. The flt3 protein is found in the outer membrane of certain cell. The gene view histogram is a graphical view of mutations across flt3. Known flt3 itd mutations detected by sanger sequencing in patients 3, 36, 37 and 41 were originally not reported by the ngs panel.
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