Molecular Genetic Tests for FLT3, NPM1, and CEBPA in Acute Myeloid Leukemia

  • Qing Zhang
  • Shaochun Bai
  • Gail H. Vance
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 999)

Abstract

Patients with acute myeloid leukemia (AML) and a normal karyotype constitute the single largest cytogenetic group of AML. It is important to identify prognostic markers that predict patients’ outcome more precisely. The presence of mutations in FLT3 (FMS-like tyrosine kinase 3), NPM1 (Nucleophosmin), and CEBPA (CCAAT/enhancer-binding protein alpha) genes hold prognostic significance in patients with AML and normal cytogenetics. Therefore, mutation identification may help to optimize therapeutic approaches in this group of patients. Polymerase chain reaction (PCR)-based fragment length analysis for mutations in FLT3 and NPM1 has been shown to be a fast and sensitive method, while nucleotide sequencing represents a gold standard for CEBPA heterogeneous mutational screening. We describe both fragment length assay and sequencing methods for mutational analysis of these three genes.

Key words

Acute myeloid leukemia (AML) FLT3 NPM1 CEBPA PCR Capillary electrophoresis DNA sequencing Mutational detection 

Notes

Acknowledgment

The authors wish to thank Dr. Stephen Dlouhy for his review of the manuscript.

References

  1. 1.
    Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C, Harrison G et al (1998) The importance of diagnostic cytogenetics on outcome in AML: analysis of 1612 patients entered into the MRC AML 10 trail: the medical research council adult and children’s leukaemia working parties. Blood 92:2322–2333PubMedGoogle Scholar
  2. 2.
    Grimwade D, Hills RK, Moorman AV, Walker H, Chatters S, Goldstone AH et al (2010) Refinement of cytogenetic classification in acute myeloid leukemia: determination of prognostic significance of rare recurring chromosomal abnormalities among 5876 younger adult patients treated in the United Kingdom Medical Research Council trials. Blood 116:354–365PubMedCrossRefGoogle Scholar
  3. 3.
    Gulley ML, Shea TC, Fedoriw Y (2010) Genetic tests to evaluate prognosis and predict therapeutic response in acute myeloid leukemia. J Mol Diagn 12:3–16PubMedCrossRefGoogle Scholar
  4. 4.
    Rau R, Brown P (2009) Nucleophosmin (NPM1) mutations in adult and childhood acute myeloid leukaemia: towards definition of a new leukaemia entity. Hematol Oncol 27:171–181PubMedCrossRefGoogle Scholar
  5. 5.
    Marcucci G, Haferlach T, Dohner H (2011) Molecular genetics of adult acute myeloid leukemia: prognostic and therapeutic implications. J Clin Oncol 29(5):475–486PubMedCrossRefGoogle Scholar
  6. 6.
    Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H et al (eds) (2008) WHO classification of tumours of haematopoietic and lymphoid tissues. Lyon, IARC, pp 120–122Google Scholar
  7. 7.
    Dohner H, Estey EH, Amadori S, Applebaum FR, Buchner T, Burnett AK et al (2010) Diagnosis and management of acute myeloid leukemia in adults. Recommendations from an international expert panel, on behalf of the European Leukemia Net. Blood 115:453–474PubMedCrossRefGoogle Scholar
  8. 8.
    Rosnet O, Mattei M-G, Marchetto S, Birnbaum D (1991) Isolation and chromosomal localization of a novel FMS-like tyrosine kinase gene. Genomics 9:380–385PubMedCrossRefGoogle Scholar
  9. 9.
    Turner AM, Lin NL, Issarachai S, Lyman SD, Broudy VC (1996) FLT3 receptor expression on the surface of normal and malignant hematopoietic cells. Blood 88:3383–3390PubMedGoogle Scholar
  10. 10.
    Whitman SP, Ruppert AS, Radmacher MD, Mrózek K, Paschka P, Langer C et al (2008) FLT3 D835/I836 mutations are associated with poor disease-free survival and a distinct gene-expression signature among younger adults with de novo cytogenetically normal acute myeloid leukemia lacking FLT3 internal tandem duplications. Blood 111:1552–1559PubMedCrossRefGoogle Scholar
  11. 11.
    Bacher U, Haferlach C, Schnittger S, Kern W, Kroeger N, Zander AR et al (2009) Interactive diagnostics in the indication to allogeneic SCT in AML. Bone Marrow Transplant 43:745–756PubMedCrossRefGoogle Scholar
  12. 12.
    Chang JH, Olson MO (1990) Structure of the gene for rat nucleolar protein 23. J Biol Chem 265:18227–18233PubMedGoogle Scholar
  13. 13.
    Falini B, Nicoletti I, Martelli MF, Mecucci C (2007) Acute myeloid leukemia carrying cytoplasmic/mutated nucleophosmin (NPMc  +  AML): biologic and clinical features. Blood 109:874–885PubMedCrossRefGoogle Scholar
  14. 14.
    Szankasi P, Jama M, Bahler DW (2008) A new DNA-based test for detection of nucleophosmin exon 12 mutations by capillary electrophoresis. J Mol Diagn 10:236–241PubMedCrossRefGoogle Scholar
  15. 15.
    Falini B, Martelli MF, Bolli N, Bonasso R, Ghia E, Pallotta MT et al (2006) Immuohistochemistry predicts nucleophosmin (NPM) mutations in acute myeloid leukemia. Blood 108:1999–2005PubMedCrossRefGoogle Scholar
  16. 16.
    Hendricks-Taylor L, Bachinski LL, Siciliano MJ, Fertitta A, Trask B, de Jone P et al (1992) The CCAAT/enhancer binding protein (C/EBP alpha) gene (CEBPA) maps to human chromosome 19q13 and related nuclear factor NF-IL6 (C/EBP beta) gene (CEBPB) maps to human chromosome 20q13.1. Genomics 14:12–17PubMedCrossRefGoogle Scholar
  17. 17.
    Koschmieder S, Halmos B, Levantini E, Tenen DG (2009) Dysregulation of the C/EBPα differentiation pathway in human cancer. J Clin Oncol 27:619–628PubMedCrossRefGoogle Scholar
  18. 18.
    Renneville A, Roumier C, Biggio V, Nibourel O, Boissel N, Fensux P et al (2008) Cooperating gene mutations in acute myeloid Leukemia: a review of the literature. Leukemia 22:915–931PubMedCrossRefGoogle Scholar
  19. 19.
    Pabst T, Eyholzer M, Fos J, Mueller BU (2009) Heterogeneity within AML with CEBPA mutatons; only CEBPA double mutations, but not single CEBPA mutations are associated with favorable prognosis. Br J Cancer 100:1343–1346PubMedCrossRefGoogle Scholar
  20. 20.
    Hackanson B, Bennett KL, Brena RM, Jiang J, Claus R, Chen SS et al (2008) Epigenetic modification of CCAAT/enhancer binding protein a expression in acute myeloid leukemia. Cancer Res 68:3142–3151PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Qing Zhang
    • 1
  • Shaochun Bai
    • 1
  • Gail H. Vance
    • 1
  1. 1.Department of Medical and Molecular GeneticsIndiana University School of MedicineIndianapolisUSA

Personalised recommendations