UIHC FLT3 and NPM1 Mutation Detection Assay

The UIHC Molecular Pathology Laboratory announces the implementation of the FLT3 and NPM1 Mutation Detection Assay, which will be available from our regular Molecular Pathology test menu on July 1, 2008. Recurrent mutations in FLT3 and/or NPM1 are now recognized as useful clinical prognostic markers in newly diagnosed cases of acute myeloid leukemia (AML), particularly for those cases of AML which show no other cytogenetic abnormality. These mutations occur in two regions of the FLT3 locus on 13q12: the more common mutation is internal tandem duplication (ITD) of variable size in exons 14-15 encoding the juxtamembrane region of the FLT3 receptor, occurring in ~30% of AML; the other common FLT3 mutation alters an aspartic acid residue (D835) in the tyrosine kinase domain (TKD) seen in ~7% of AML. The FLT3-ITD and FLT3-D835 mutations confer a poorer prognosis. Mutations in exon 12 of the nucleophosmin (NPM1) gene largely involve 4 bp insertions that result in a frameshift affecting subcellular localization of NPM1 protein. The NPM1 mutation, in the absence of FLT3 mutations, confers a more favorable prognosis. Thus, the mutational status for FLT3 and NPM1 may be useful in designing treatment strategies.

The UIHC FLT3 and NPM1 Mutation Detection Assay is a qualitative multiplex fluorescence PCR assay designed to simultaneously detect the FLT3-ITD, FLT3-D835 (TKD) and NPM1 exon 12 mutations common in AML. Following PCR amplification using differentially labeled mutation-specific primers, EcoRV restriction endonuclease digestion is performed for the FLT3-D835 and NPM1 amplicons, which is necessary to identify the FLT3-D835 (digestion of the NPM1 PCR product serve as an internal control). Labeled PCR products are then analyzed by capillary electrophoresis on the ABI 3130. Both wild-type and mutant FLT3-ITD, FLT3-D835 and NPM1 alleles are amplified from high-quality genomic DNA prepared from peripheral blood, purified granulocytes or mononuclear cells, or bone marrow.

The UIHC FLT3 and NPM1 Mutation Detection Assay was developed and validated within the UIHC Molecular Pathology Laboratory, and will be performed weekly as a batch test with a turn-around time of 7 days from receipt of the specimen. This test has not been cleared or approved by the U.S. Food and Drug Administration (FDA); the FDA has determined that such approval is not necessary. The test is used for clinical purposes, and should NOT be regarded as 'investigational' or 'for research.' The University of Iowa Molecular Pathology laboratory is certified under the Clinical Laboratory Improvement Amendment Act of 1988 (CLIA) as qualified to perform high-complexity clinical laboratory testing. Any questions regarding this test may be directed to the UIHC Molecular Pathology Laboratory (384-9568) or to the Molecular Oncology-Hematology section director (Jonathan Heusel, MD, PhD at 335-8217).