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 mutationspecific primer combined with a wild-type blocking oligonucleotide that amplifies D816V at the level of 1% mutant allele in DNA extracted from formalinfixed , 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

Reactivating PP2A by FTY720 as a Novel therapy for AML with C‐KIT tyrosine kinase domain mutation

“…However, patients with C-KIT/TKD frequently occur resistance to these drugs [Corless et al, 2006;Kosmider et al, 2007;Piccaluga et al, 2007;Verstovsek et al, 2008;Chevallier et al, 2009;Ustun et al, 2009]. Herein we show that FTY720 can induce toxic effects in both primary leukemic cells from AML patients and the AML cell lines harboring C-KIT/TKD or C-KIT/ WT.…”

<b><i>KIT</i></b> D816V Mutation-Positive Cell Fractions in Lesional Skin Biopsies from Adults with Systemic Mastocytosis

“…Büttner et al [7] detected a codon 816 mutation in 6 of 6, Longley et al [8] detected D816V in 11 of 11, Yanagihori et al [9] detected a codon 816 mutation in 4 of 4, and Garcia-Montero et al [3] detected a D816V mutation in 10 of 10 adult cases of sporadic SM. However, several studies have also reported significant proportions of KIT codon 816 mutation-negative skin biopsies from adult sporadic mastocytosis, including Verzijl et al [16] and Corless et al [17] where 5 of 8 and 9 of 13 cases, respectively, were tested mutation-negative. The cases testing mutation-negative in skin had not been confirmed mutation-positive in other tissues.…”

“…However, with the known high fraction of mutation-positive adults with mastocytosis, most of these cases were likely to carry the mutation but tested negative due to insufficient analytical sensitivity [16]. The allele-specific PCR used by Corless et al [17] was reported to detect 1% mutation-positive alleles (= 2% mutation-positive cells) in formalin-fixed paraffin-embedded derived DNA as used in the study, and should, based on the present results, be expected to reliably detect the mutation in most cases. However, it is possible that the analytical sensitivity has been reduced below the expected value due to limiting amounts of amplifiable template DNA, which is a known problem with formalin-fixed paraffin-embedded skin and commonly observed in our laboratory [T. Kristensen, unpubl.…”

“…At present, very little data has been published on the occurrence of the KIT D816V mutation in skin in SM patients. A few reports confirm the presence of the mutation in lesional skin of adults with SM, but studies where the majority of cases shows a mutation-negative result have also been published [3,7,8,9,16,17]. None of the studies on KIT mutation in skin report quantitative data and basic descriptive pathobiological data such as mutation levels and whether the high variation in the mutation-positive cell fraction detected in PB and BM is also observed in lesional skin remains unknown.…”

Myelomastocytic Leukemia With Aberrant CD25 Expression: Case Report and Review of the Literature

“…[5][6][7][8][9][10] Myelomastocytic leukemia is distinct from SM with associated clonal hematologic nonemast cell lineage disease (SM-AHNMD), mast cell leukemia, and AML with tryptase expression, KIT (v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog; c-KIT gene) mutation at codon 816, or both. 5,[11][12][13][14][15][16] This report presents a patient with myelomastocytic leukemia that had aberrant CD25 expression. It describes the clinicopathologic features of this patient and provides a focused review of the literature on this rare variant of AML.…”