Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Biotechnical Methods Section

Simultaneous detection of NPM1 and FLT3-ITD mutations by capillary electrophoresis in acute myeloid leukemia

A Corrigendum to this article was published on 25 April 2007


Mutations in the Nucleophosmin (NPM1) gene have been recently described to occur in about one-third of acute myeloid leukemias (AML) and represent the most frequent genetic alteration currently known in this subset. These mutations generate an elongated NPM1 protein that localizes aberrantly in the cytoplasm. In analogy with Flt3 alterations, NPM1 mutations are mostly detectable in AML with normal karyotype and their recognition may be relevant to identify distinct response to treatment. Hence, in addition to conventional karyotyping and RT-PCR of fusion genes, combined analysis of both Flt3 and NPM1 mutations will be increasingly relevant in the genetic diagnosis work-up of AML. We developed a multiplex RT-PCR assay followed by capillary electrophoresis to simultaneously analyze NPM1 and Flt3 gene alterations (NFmPCR assay). The assay was validated in leukemic cell RNAs extracted from 38 AML patients, which had been previously characterized for Flt3 status by conventional RT-PCR. Direct sequencing of NPM1 RT-PCR products was carried out in 15 cases to verify results obtained by capillary electrophoresis. Both NPM1 sequencing and conventional RT-PCR Flt3 results showed 100% concordance with the results of the NFmPCR assay. We suggest that this assay may be introduced in routine analysis of genetic alterations in AML.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1

Accession codes




  1. Cheson BD, Bennett JM, Kopecky KJ, Buchner T, Willman CL, Estey EH et al. International working group for diagnosis, standardization of response criteria, treatment outcomes, and reporting standards for the rapeutic trials in acute myeloid leukemia. Revised recommendations of the international working group for diagnosis, standardization of response criteria, treatment outcomes, and reporting standards for therapeutic trials in acute myeloid leukemia. J Clin Oncol 2003; 15: 4642–4649.

    Article  Google Scholar 

  2. Falini B, Mecucci C, Tiacci E, Alcalay M, Rosati R, Pasqualucci L, et al, GIMEMA Acute Leukemia Working Party. Cytoplasmic nucleophosmin (NPM) identifies a subtype of acute myelogenous leukemia with a normal karyotype and NPM1 gene mutations. N Engl J Med 2005; 352: 254–266.

    Article  CAS  Google Scholar 

  3. Thiede C, Steudel C, Mohr B, Schaich M, Schakel U, Platzbecker U et al. Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis. Blood 2002; 99: 4326–4335.

    Article  CAS  Google Scholar 

  4. Gilliland DG, Griffin JD . The roles of FLT3 in hematopoiesis and leukemia. Blood 2002; 100: 1532–1542.

    Article  CAS  Google Scholar 

  5. Abu-Duhier FM, Goodeve AC, Wilson GA, Gari MA, Peake IR, Rees DC et al. FLT3 internal tandem duplication mutations in adult acute myeloid leukaemia define a high-risk group. Br J Haematol 2000; 111: 190–195.

    Article  CAS  Google Scholar 

  6. Rombouts WJ, Lowenberg B, van Putten WL, Ploemacher RE . Improved prognostic significance of cytokine-induced proliferation in vitro in patients with de novo acute myeloid leukemia of intermediate risk: impact of internal tandem duplications in the Flt3 gene. Leukemia 2001; 15: 1046–1053.

    Article  CAS  Google Scholar 

  7. Martinelli G, Piccaluga PP, LoCoco F . FLT3 inhibition as tailored therapy for acute myeloid leukemia. Haematologica 2003; 88: 4–8.

    PubMed  Google Scholar 

  8. Noguera NI, Breccia M, Divona M, Diverio D, Costa V, De Santis S et al. Alterations of the FLT3 gene in acute promyelocytic leukemia: association with diagnostic characteristics and analysis of clinical outcome in patients treated with the Italian AIDA protocol. Leukemia 2002; 16: 2185–2189.

    Article  CAS  Google Scholar 

  9. Nishimura Y, Ohkubo T, Furuichi Y, Umekawa H . Tryptophans 286 and 288 in the C-terminal region of protein B231 are important for its nucleolar localization. Biosci. Biotechnol Biochem 2002; 66: 2239–2242.

    Article  CAS  Google Scholar 

  10. Chomczynsky P, Sacchi N . Single step method of RNA isolation by acid guanidium thiocyanate – phenol chloroform extraction. Anal Biochem 1987; 162: 156–159.

    Google Scholar 

  11. van Dongen JJ, Macintyre EA, Gabert JA, Delabesse E, Rossi V, Saglio G et al. Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia. Leukemia 1999; 13: 1901–1928.

    Article  CAS  Google Scholar 

  12. Liu QR, Chan PK . Characterization of seven processed pseudogenes of nucleophosmin/B23 in the human genome. DNA Cell Biol 1993; 12: 149–156.

    Article  CAS  Google Scholar 

Download references


This work was supported by MIUR Cofin 2003, and AIRC. At the time of the study, NIN was on leave of absence from the Department of Chemical Biochemistry (Hematology) Universidad Nacional de Rosario (Argentina) and EA was on leave of absence from the Division of Hematology of the University of Palermo (Italy). NIN is a member of Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET), Argentina.

Author information

Authors and Affiliations


Corresponding author

Correspondence to F Lo-Coco.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Noguera, N., Ammatuna, E., Zangrilli, D. et al. Simultaneous detection of NPM1 and FLT3-ITD mutations by capillary electrophoresis in acute myeloid leukemia. Leukemia 19, 1479–1482 (2005).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


  • NPM1
  • FLT3
  • acute myeloid leukemia
  • AML genetic characterization

This article is cited by


Quick links