A novel melting curve‑based method for detecting c‑kit mutations in acute myeloid leukemia

Lu,Quanyi; Huang,Xiao; Chen,Huaying; Zhao,Xiaomin

doi:10.3892/ol.2014.2128

A novel melting curve‑based method for detecting c‑kit mutations in acute myeloid leukemia

Authors:
- Quanyi Lu
- Xiao Huang
- Huaying Chen
- Xiaomin Zhao
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Affiliations: Department of Hematology, Zhongshan Hospital of Xiamen University, Xiamen, Fujian 361004, P.R. China
Published online on: May 9, 2014 https://doi.org/10.3892/ol.2014.2128
Pages: 99-104

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Abstract

The c‑kit gene encodes a class III tyrosine kinase receptor. Specific somatic mutations in c‑kit have been associated with acute myeloid leukemia (AML) and are markers of a poor prognosis in AML. Various methods have been used to detect the c‑kit gene mutation; however, the suitability of these methods in the clinical management of AML remains unclear. The current study developed a novel method, using modified hybridization probes and melting curve analysis, for detecting c‑kit mutations in exon 17. Dual‑labeled self‑quenched oligonucleotide probes containing two segments, labeled with carboxyrhodamine or hexachlorofluorescein, were designed to detect sequences around the D816 or N820/N822 mutation hot spots in exon 17 of c‑kit. The exon 17 region of c‑kit was amplified by polymerase chain reaction using control plasmids carrying wild‑type or mutant sequences, or genomic DNA derived from AML patients. Melting curve analysis of the amplification products was performed using a self‑quenched probe. The results showed that the detection sensitivity, assayed using mutation‑positive control plasmids, was 10% for the N820G mutation and 5% for the six other mutations; N822K(A), N822K(G), D816V, D816Y, D816H and D816F. In addition, c‑kit mutations were identified in six of the 12 samples from the core‑binding factor (CBF)‑AML patients. This demonstrates that the novel method developed in the present study, is simple, rapid, specific and highly sensitive, and may facilitate the diagnosis and treatment of CBF‑AML.

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1	Döhner K and Döhner H: Molecular characterization of acute myeloid leukemia. Haematologica. 93:976–982. 2008.
2	Beghini A, Peterlongo P, Ripamonti CB, et al: C-kit mutations in core binding factor leukemias. Blood. 95:726–727. 2000.
3	Dombret H, Preudhomme C and Boissel N: Core binding factor acute myeloid leukemia (CBF-AML): is high-dose Ara-C (HDAC) consolidation as effective as you think? Curr Opin Hematol. 16:92–97. 2009.
4	Heinrich MC, Blanke CD, Druker BJ and Corless CL: Inhibition of KIT tyrosine kinase activity: a novel molcular approach to the treatment of KIT-positive malignancies. J Clin Oncol. 20:1692–1703. 2002.
5	Roskoski R Jr: Structure and regulation of Kit protein-tyrosine kinase - the stem cell factor receptor. Biochem Biophys Res Commun. 338:1307–1315. 2005.
6	Mrózek K and Bloomfield CD: Chromosome aberrations, gene mutations and expression changes, and prognosis in adult acute myeloid leukemia. Hematology Am Soc Hematol Educ Program. 169–177. 2006.
7	Nanri T, Matsuno N, Kawakita T, et al: Mutations in the receptor tyrosine kinase pathway are associated with clinical outcome in patients with acute myeloblastic leukemia harboring t(8;21)(q22;q22). Leukemia. 19:1361–1366. 2005.
8	Schnittger S, Kohl TM, Haferlach T, Kern W, Hiddemann W, Spiekermann K and Schoch C: KIT-D816 mutations in AML1-ETO-positive AML are associated with impaired event-free and overall survival. Blood. 107:1791–1799. 2006.
9	Corless CL, Harrell P, Lacouture M, et al: Allele-specific polymerase chain reaction for the imatinib-resistant KIT D816V and D816F mutations in mastocytosis and acute myelogenous leukemia. J Mol Diagn. 8:604–612. 2006.
10	Fuster O, Barragán E, Bolufer P, et al: Rapid detection of KIT mutations in core-binding factor acute myeloid leukemia using high-resolution melting analysis. J Mol Diagn. 11:458–463. 2009.
11	Sotlar K, Escribano L, Landt O, et al: One-step detection of c-kit point mutations using peptide nucleic acid-mediated polymerase chain reaction clamping and hybridization probes. Am J Pathol. 162:737–746. 2003.
12	Paschka P, Marcucci G, Ruppert AS, et al; Cancer and Leukemia Group B. Adverse prognostic significance of KIT mutations in adult acute myeloid leukemia with inv(16) and t(8;21): a Cancer and Leukemia Group B Study. J Clin Oncol. 24:3904–3911. 2006.
13	Huang Q, Liu Z, Liao Y, et al: Multiplex fluorescence melting curve analysis for mutation detection with dual-labeled, self-quenched probes. PloS One. 6:e192062011.
14	Vardiman JW, Thiele J, Arber DA, et al: The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood. 114:937–951. 2009.
15	Higuchi R, Krummel B and Saiki RK: A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 16:7351–7367. 1988.
16	Heckman KL and Pease LR: Gene splicing and mutagenesis by PCR-driven overlap extension. Nat Protoc. 2:924–932. 2007.
17	Kohl TM, Schnittger S, Ellwart JW, Hiddemann W and Spiekermann K: KIT exon 8 mutations associated with core-binding factor (CBF)-acute myeloid leukemia (AML) cause hyperactivation of the receptor in response to stem cell factor. Blood. 105:3319–3321. 2005.
18	Frost MJ, Ferrao PT, Hughes TP and Ashman LK: Juxtamembrane mutant V560GKit is more sensitive to Imatinib (STI571) compared with wild-type c-kit whereas the kinase domain mutant D816VKit is resistant. Mol Cancer Ther. 1:1115–1124. 2002.
19	Wang YY, Zhou GB, Yin T, et al: AML1-ETO and C-KIT mutation/overexpression in t(8;21) leukemia: implication in stepwise leukemogenesis and response to Gleevec. Proc Natl Acad Sci USA. 102:1104–1109. 2005.
20	Lück SC, Russ AC, Du J, et al: KIT mutations confer a distinct gene expression signature in core binding factor leukaemia. Br J Haematol. 148:925–937. 2010.
21	Paschka P, Marcucci G, Ruppert AS, et al; Cancer and Leukemia Group B. Adverse prognostic significance of KIT mutations in adult acute myeloid leukemia with inv(16) and t(8;21): a Cancer and Leukemia Group B Study. J Clin Oncol. 24:3904–3911. 2006.