FLT3 Mutation Analysis – ITD and D835/836 Codon Analysis in Acute Myeloid Leukemia

Background

FLT3 (fms-like tyrosine kinase 3) is a receptor tyrosine kinase expressed in early hematopoietic progenitor cells, and may play a role in stem cell survival. FLT3 is overexpressed in Acute Myeloid Leukemia (AML). Mutations of the FLT3 gene that lead to constitutive activation of the kinase and autonomous, cytokine independent cell proliferation has prognostic significance of poor outcome. Adult studies show a prevalence of 20-35% for internal tandem duplication (ITD) and an additional 7% for point mutations in the loop domain. The ITD occur in a juxtamembrane coding domain and are always in-frame. Varying allelic ratios of ITD have shown to carry prognostic significance, though a clinically useful threshold has not been established. Activation loop mutations occur at codons 835 and 836 altering the coding for the amino acid aspartic acid.

Clinical Utility

Detection of the D835 mutation and/or internal tandem duplication the FLT3 gene are tested in acute myelocytic leukemia especially in conjunction with NPM1 testing to provide prognostic information.

Methodology

Loci Tested: FLT3 Internal tandem duplication and D835 mutation. Fluorescent labeled primers are used to amplify the kinase (D835) and juxtamembrane (ITD) regions of the FLT3 gene in two distinct PCR reactions. Restriction enzyme digestion and fragment analysis re used to detect the ITD and/or D835 mutation.

Specimen Requirements

  1. Peripheral Blood: 3-5 ml, collected in EDTA (purple top) tube, store at room temperature 24 hours
  2. Bone Marrow: 0.5-1 ml, collected in EDTA (purple top)tube, store at room temperature, 24 hours
  3. Unacceptable Specimens: Frozen blood or bone marrow specimens are unacceptable as are tissue samples that have undergone a freeze/thaw cycle(s). Bone marrow/ Peripheral blood specimens post treatment with very low blast count.

Performed

Twice per month

Reported

Within 1-3 weeks of receipt

CPT

81245

Shipment Must Include

Specimen
Requisition form
Patient pathology report

References

  1. Murphy, K. et al, (2003), Detection of FLT3 Internal Tandem Duplication and D835 Mutations by a Multiplex Polymerase Chain Reaction and Capillary Electrophoresis Assay, J.Mol.Diagn., vol 5(2), pp.96-102.
  2. Meshinichi, S. et al, (2006), Clinical implications of FLT3 mutations in pediatric AML, Blood, vol 108(12), pp. 3654-3661.