title: Allele-specific polymerase chain reaction for the imatinib-resistant KIT D816V and D816F mutations in mastocytosis and acute myelogenous leukemia.
abstract: Oncogenic mutations of the receptor tyrosine kinase KIT contribute to the pathogenesis of gastrointestinal stromal tumors, systemic mastocytosis (SM), and some cases of acute myelogenous leukemia (AML). The D816V substitution in the activation loop of KIT results in relative resistance to the kinase inhibitor imatinib (Gleevec). Because this mutation occurs in 80 to 95% of adult SM, its detection has diagnostic and predictive significance. Unfortunately, the fraction of mutation-positive cells in clinical SM samples is often below the 20 to 30% threshold needed for detection by direct DNA sequencing. We have developed an allele-specific polymerase chain reaction assay using a mutation-specific primer combined with a wild-type blocking oligonucleotide that amplifies D816V at the level of 1% mutant allele in DNA extracted from formalin-fixed, paraffin-embedded tissue. There were no amplifications among 64 KIT wild-type tumors and cell lines, whereas all D816V-mutant samples (eight AML and 11 mast cell disease) were positive. Other D816 substitutions associated with resistance to imatinib in vitro are rare in SM. Among these D816F was detectable with the assay whereas D816H, D816Y, and D816G did not amplify. Nine biopsies (bone marrow, skin, or colon) with suspected SM were negative by denaturing high performance liquid chromatography and/or DNA sequencing but positive by allele-specific polymerase chain reaction. Thus, the assay may be useful in confirming the diagnosis of SM.
authors.affiliation: OHSU Dept. of Pathology (L471), 3181 SW Sam Jackson Park Rd., Portland, OR 97239, USA. corlessc@ohsu.edu
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Corless
authors.forename: Patina
authors.initials: P
authors.lastname: Harrell
authors.forename: Mario
authors.initials: M
authors.lastname: Lacouture
authors.forename: Troy
authors.initials: T
authors.lastname: Bainbridge
authors.forename: Claudia
authors.initials: C
authors.lastname: Le
authors.forename: Ken
authors.initials: K
authors.lastname: Gatter
authors.forename: Clifton
authors.initials: C
authors.lastname: White
authors.forename: Scott
authors.initials: S
authors.lastname: Granter
authors.forename: Michael C
authors.initials: MC
authors.lastname: Heinrich
mesh_terms: D000328:Adult; D000368:Aged; D000483:Alleles; D000818:Animals; D001549:Benzamides; D002460:Cell Line; D005260:Female; D011905:Genes, ras; D006801:Humans; D000068877:Imatinib Mesylate; D015470:Leukemia, Myeloid, Acute / Q000175:diagnosis* / Q000235:genetics; D008297:Male; D008415:Mastocytosis / Q000175:diagnosis* / Q000235:genetics; D051379:Mice; D008875:Middle Aged; D009154:Mutation; D010879:Piperazines / Q000627:therapeutic use*; D016133:Polymerase Chain Reaction / Q000379:methods*; D047428:Protein Kinase Inhibitors / Q000627:therapeutic use; D011743:Pyrimidines / Q000627:therapeutic use*; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics*; D012680:Sensitivity and Specificity
chemical_list: D001549:Benzamides; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D000068877:Imatinib Mesylate; D020794:Receptor Protein-Tyrosine Kinases