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. 2009 Sep;11(5):458-63.
doi: 10.2353/jmoldx.2009.090043. Epub 2009 Jul 30.

Rapid detection of KIT mutations in core-binding factor acute myeloid leukemia using high-resolution melting analysis

Oscar Fuster  1 , Eva Barragán, Pascual Bolufer, José Cervera, Maria José Larráyoz, Antonio Jiménez-Velasco, Joaquín Martínez-López, Ana Valencia, Federico Moscardó, Miguel Angel Sanz
Affiliations

Rapid detection of KIT mutations in core-binding factor acute myeloid leukemia using high-resolution melting analysis

Oscar Fuster et al. J Mol Diagn. 2009 Sep.

Abstract

The most frequent KIT mutations reported in core-binding factor acute myeloid leukemia are point mutations and insertions/deletions in exons 17 and 8. The vast majority of KIT mutation detection procedures are time-consuming, costly, or with a high lower limit of detection. High-resolution melting (HRM) is a gene scanning method that combines simplicity and rapid identification of genetic variants. We describe an HRM method for the simultaneous screening of exons 8 and 17 KIT mutations and report the results obtained in 69 core-binding factor acute myeloid leukemia patients. Mutation detection was compared with sequencing as the gold standard. The HRM method used high-resolution melting master reagents (Roche) and the LightCycler 480 (Roche) platform. HRM was reproducible, showed a lower limit of detection of 1%, and discriminated all patients with mutated KIT from controls without false positive or false negative results. Additionally, most of the mutations were differentiated from the other mutations. KIT mutations were present in 15.9% of patients, showing a higher incidence in inv(16) (25.8%) than in t(8;21) (7.9%). The presence of a KIT mutation was associated with a high white blood cell count, and adult patients with an exon 17 mutation had a higher incidence of relapse. These findings verify that HRM is a reliable, rapid, and sensitive method for KIT mutation screening. Furthermore, our study corroborates the unfavorable prognosis associated with exon 17 KIT mutations.

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Figures

Figure 1
Figure 1
HRM method. Examples of difference plots obtained for wild-type (WT) DNA and different exon 8 (A) and exon 17 (B) KIT mutations.
Figure 2
Figure 2
Lower limit detection of HRM assay for exons 8 and 17 KIT mutations. Differential plots obtained with the exon 8 mutation T417_D419delinsS (A) and exon 17 mutation D820G (B) serially diluted in wild-type DNA.
Figure 3
Figure 3
RFS in adult patients according to mutations in KIT exon 17.
See this image and copyright information in PMC

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