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JMD 2006, Vol. 8, No. 2
Copyright © 2006 American Society for Investigative Pathology & Association for Molecular Pathology

Denaturing High-Performance Liquid Chromatography

A Valid Approach for Identifying NPM1 Mutations in Acute Myeloid Leukemia

Giovanni Roti^*, Roberto Rosati^*, Rossella Bonasso, Paolo Gorello^*, Daniela Diverio, Massimo Fabrizio Martelli, Brunangelo Falini, Cristina Mecucci^* for the Gruppo Italiano Malattie Ematologiche dell’ Adulto Working Party

From the Laboratories of Cytogenetics and Molecular Genetics * and Immunohistochemistry, and the Hematology Unit, University of Perugia, Perugia; and the Department of Cellular Biotechnologies and Hematology, La Sapienza University, Rome, Italy

NPM1 gene mutations are the most frequent genetic lesion in the 60% of adult acute myeloid leukemias (AMLs) with normal karyotype and no evidence of typical fusion genes (BCR/ABL1, PML/RARA, AML1/ETO, CBFB/MYH11, DEK/CAN). Using direct sequencing we previously identified six different heterozygous mutants within exon 12 encoding the nucleophosmin C-terminus. Because of these mutations the shuttling protein nucleophosmin is aberrantly delocalized in the cytoplasm of leukemic cells (NPMc+). Here, we designed and tested a denaturing high-performance liquid chromatography (DHPLC) assay to detect NPM1 mutated variants. To assess specificity, sensitivity, reliability, and reproducibility, we analyzed DNA from 120 primary adult AMLs and compared DHPLC results with immunohistochemistry and sequencing. All electropherogram profiles in the 26 NPMc+ leukemias were different from the wild type, indicating 100% sensitivity. Sequencing categorized mutations A, B, and D, and all mutation A cases gave identical elution profiles. The other mutations showed typical chromatograms, with mutations B and D differing for one nucleotide. Elution profiles and sequencing also identified four new variants. Our results suggest that DHPLC detects NPM1mutations as well as direct sequencing and immunohistochemistry, providing a helpful approach in the diagnosis of NPMc+ AML.

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