An Allele-Specific RT-PCR Assay to Detect Type A Mutation of the Nucleophosmin-1 Gene in Acute Myeloid Leukemia

Abstract

Nucleophosmin-1 (NPM1) mutations represent the most frequent gene alteration in acute myeloid leukemia (AML). The most common NPM1 mutation type, accounting for 75 to 80% of cases , is referred to as mutation A (NPM1-mutA). These NPM1 alterations have been shown to possess prognostic significance because they appear to identify patients who will benefit from chemotherapy. Given the high prevalence and stability of these mutations over the course of disease , NPM1 mutations may serve as ideal targets for minimal residual disease (MRD) assessment in AML. Current detection methods are costly , require sophisticated equipment , and are often not sufficiently sensitive. We report here an allele-specific (ASO)-RT-PCR assay that enables rapid and sensitive detection of NPM1-mutA. A semi-nested ASO-PCR method was also designed to increase the sensitivity of our assay for the monitoring of MRD. We analyzed bone marrow cells collected from 52 patients with AML at presentation. NPM1-mutA was detected in leukemic cells from 21 patients. Nucleophosmin-1 (NPM1) is a nucleocytoplasmic shuttling protein that plays a key role in a variety of cellular processes including promotion of ribosome biogenesis, maintenance of genomic stability, regulation of transcription, and modulation of tumor-suppressor transcription factors.1 The NPM1 gene is one of the most frequent targets of genetic alterations in hematopoietic tumors as it has been shown to be involved in several gene fusions resulting from chromosome translocations and in other subtle mutations detected in lymphomas and acute leukemia. 2In early 2005, Falini et al 3 reported that NPM1 gene mutations, mainly consisting of small nucleotide insertion/ deletions, occur frequently in acute myeloid leukemia (AML) and are strongly associated with normal karyotype. Over 40 different types of mutations in the NPM1 locus have been described so far, which result in the formation of different mutant proteins. These alterations consist of the insertion/deletion of short nucleotide stretches (4 or 10 bp) at positions 956 through 971 in NPM1 exon 12 that lead to an open reading frame shift resulting in different protein variants containing novel C terminus portions.3-5 The most common NPM1 mutation type, accounting for 75% to 80% of cases, is referred to as mutation A (NPM1-mutA) 3 and consists of a duplication of a TCTG tetranucleotide at position 956 to 959 of the reference sequence (GenBank accession number NM_002520).

A monoclonal antibody against mutated nucleophosmin 1 for the molecular diagnosis of acute myeloid leukemias

“…Correspondence: Pier Giuseppe Pelicci, European Institute of Oncology at the IFOM-IEO Campus, Via Adamello, 16, 20139 Milan, Italy; e-mail: piergiuseppe.pelicci@ifom-ieo-campus.it.…”

“…7 Currently available methods to detect NPM1 mutations include DNA sequencing, real-time quantitative polymerase chain reaction (PCR), denaturing high-performance liquid chromatography, capillary electrophoresis, locked nucleic acid-mediated PCR clamping, and allele-specific reverse-transcribed PCR assays. [10][11][12][13][14][15][16][17][18] Although highly specific, these methods are expensive and laborious. Alternatively, NPM1 mutations can be identified indirectly, using antibodies that recognize epitopes that are common for both the wild-type (wt) and mutant NPM1 proteins and allow recognition of the cytoplasmic dislocation of NPM1.…”

Detection of Nucleophosmin 1 Mutations by Quantitative Real-Time Polymerase Chain Reaction Versus Capillary Electrophoresis: A Comparative Study

“…23 A number of methods have been developed to rapidly detect NPM1 mutations in genomic DNA and/or mRNA specimens. 7,17,[24][25][26][27] The most widely adopted screening method, routinely used in our laboratory, is polymerase chain reaction (PCR) amplification of the mutational hotspot region followed by capillary electrophoresis analysis. This approach yields semiquantitative results.…”

“…Recently, several groups have designed quantitative real-time polymerase chain reaction (qPCR)-based assays for detecting single or multiple common NPM1 mutations in AML. [25][26][27] These methods offer the advantage of reliable quantification of mutated NPM1 and, therefore, would be useful for providing a quantitative measurement of disease burden and for assessing MRD after therapy. [25][26][27] The clinical applications of these assays, however, and whether they are ready to replace conventional capillary electrophoresis assays are still being debated.…”

Establishment of a quenching probe method for detection of NPM1 mutations in acute myeloid leukemia cells

“…In addition, the sensitivity of this method is low, with the lower limit of detection beginning at ~20% (10). Therefore, polymerase chain reaction (PCR) methods such as electrophoresis (11), melting curve analysis (12), high-resolution melting (HRM) analysis which detects the different melting temperatures of the PCR products (13), locked nucleic acid clamp-mediated PCR (10,14), and capillary electrophoresis (15,16) have been investigated as potentially more sensitive methods for detection of this 4 bp mutation.…”