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JMD 2006, Vol. 8, No. 5
Copyright © 2006 American Society for Investigative Pathology & Association for Molecular Pathology

Allele-Specific Polymerase Chain Reaction for the Imatinib-Resistant KIT D816V and D816F Mutations in Mastocytosis and Acute Myelogenous Leukemia

Christopher L. Corless^*, Patina Harrell, Mario Lacouture, Troy Bainbridge, Claudia Le, Ken Gatter^*, Clifton White, Jr, Scott Granter^¶ and Michael C. Heinrich^||

From the Department of Pathology, * Oregon Health and Science University Cancer Institute, and the Divisions of Dermatology and Hematology and Oncology, ^|| Oregon Health and Science University, Portland, Oregon; the Section of Dermatology, University of Chicago, Chicago, Illinois; and the Department of Pathology, ^¶ Brigham and Women’s Hospital, Boston, Massachusetts

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.

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