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Published online before print April 10, 2008
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Technical Advances |
From the Dipartimento di Biopatologia e Diagnostica per Immagini,
* University Tor Vergata, Rome; Dipartimento di Medicina Interna,
University Tor Vergata, Rome; Laboratorio di Ricerca, Istituto di Ricovero e Cura a Carattere Scientifico,
Ospedale Pediatrico Bambino Gesù, Rome; and Dipartimento di Medicina di Laboratorio,
Policlinico Tor Vergata, Rome, Italy
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. Assay specificity was confirmed by capillary electrophoresis and DNA sequencing. ASO-RT-PCR and semi-nested ASO-PCR assays could detect NPM1-mutA with sensitivities of 10–2 and 10–5, respectively. Results obtained here verify that our ASO-RT-PCR assay is a specific and sensitive method for the routine screening of NPM1-mutA, as well as for MRD monitoring of AML patients with this alteration. This method is convenient and easily applicable in countries with limited resources and no access to real-time quantitative PCR-based technologies.
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.2
In early 2005, Falini et al3 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, 4, 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).
To date, NPM1 gene mutations represent the most frequent known genetic abnormality in AML. Moreover, these alterations have been shown to carry prognostic significance because they seem to identify patients with better response to chemotherapy. As a consequence, the analysis of NPM1 mutational status is now recommended for inclusion in the genetic routine characterization of AML.6, 7, 8, 9, 10, 11 Finally, given their high prevalence and stability over the course of the disease, NPM1 mutations may serve as an ideal target for minimal residual disease (MRD) assessment, particularly for patients with AML-normal karyotype.6
Several protocols and techniques have been developed for the detection of NPM1 mutations. These include DNA sequencing, a real-time quantitative PCR assay,6 denaturing high-performance liquid chromatography,7 capillary electrophoresis,8 and locked nucleic acid-mediated PCR clamping.9 Although these methods allow high specificity to assess NPM1 mutational status at diagnosis, most have drawbacks including an elevated cost and requirement of sophisticated equipment and, except for RQ-PCR and locked nucleic acid-PCR clamping, poor sensitivity. To date, a highly specific, low-cost and sensitive method for the detection of NPM1 mutations using non-quantitative PCR has not been developed. In this study we report an allele-specific (ASO)-RT-PCR assay that enables rapid and sensitive detection of the most common NPM1 mutation. This test also seems suitable for assessment of response and longitudinal molecular monitoring of patients with AML harboring NPM1-mutA.
Materials and Methods
Patients and Samples
After informed consent, bone marrow cells were collected at presentation from 52 patients with AML diagnosed at the Institute of Hematology, Department of Biopathology of the University Tor Vergata of Rome. Total RNA was extracted from Ficoll-Hypaque-isolated mononuclear cells using the method of Chomczynski and Sacchi.12
RNA was reverse-transcribed using random hexamer primers as previously described.13
RNA was chosen as starting material (instead of DNA) because of the need to routinely amplify fusion genes (AML1/ETO, BCR/ABL [Abelson gene], CBFbeta/MYH11, and others) in the diagnostic workup of AML, which can only be feasible using cDNA.
Three cDNA aliquots were used for ASO-RT-PCR and semi-nested ASO-PCR, sequencing, and capillary electrophoresis experiments, respectively.
ASO-RT-PCR
With the aim of analyzing the presence of type A mutation in NPM1 exon 12, we used an ASO-RT-PCR strategy. A forward primer (NPM-A; Table 1
) was designed to specifically amplify NPM1 exon 12 only if the mutA was present. This primer in fact contains an intentional mismatch at the third nucleotide from the 3' end to improve specificity. The amplified region includes the insertion of a TCTG tetranucleotide at positions 956 to 959 of the reference sequence. In this region NPM1 and its seven pseudogenes14
are highly homologous. Therefore, to rule out the amplification of pseudogenes, we used a previously described reverse primer (NPM-REV-6) excluding the amplification of pseudogenes.8
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Preheating of the mixture at 95°C for 7 minutes was followed by 35 cycles of 30 seconds at 95°C, 45 seconds at 67°C, and 45 seconds at 72°C. A final extension of 7 minutes was performed at 72°C on a Gene Amp PCR System 2400 (Perkin Elmer, Emeryville, CA). PCR products were visualized by electrophoresis on 2% (w/v) agarose gel. As internal PCR control, we used ABL amplification with the same ASO-RT-PCR conditions but using the primers ABL-A2B-5' and ABL-A3E-3'.
To assess the detection threshold of our assay, we performed serial dilution experiments using RNA obtained from an AML patient’s marrow with >95% leukemic cells and harboring the NPM1-mutA, with RNA extracted from the bone marrow of a patient with NPM1-w/t (wild-type) AML (dilution ratios NPM1-mutA/NPM1-w/t: 1/10, 1/102, 1/103, 1/104, 1/105, and 1/106).
Semi-Nested ASO-PCR
A semi-nested ASO-PCR strategy was designed to increase the sensitivity of our assay for the monitoring of MRD. For this purpose, we designed an internal specific forward primer for NPM1-mutA (NPM-AN). A 0.5-µl aliquot of the first step ASO-RT-PCR was amplified in a total volume of 25 µl of the reaction mixture containing 10 pmol of each primer, NPM-AN and NPM-REV-6, 1x PCR buffer, 2.5 mmol/L MgCl2, 5 mmol/L deoxynucleoside-5'-triphosphates, and 0.7 U of Taq polymerase (Applied Biosystems). Preheating of the mixture at 95°C for 7 minutes was followed by 35 cycles of 30 seconds at 95°C, 45 seconds at 67°C, and 45 seconds at 72°C. A final extension of 7 minutes was performed at 72°C on a Gene Amp PCR System 2400 (Perkin Elmer). PCR products were visualized by electrophoresis on a 2% (w/v) agarose gel.
Capillary Electrophoresis of NPM1 RT-PCR Products
To independently validate the specificity of the ASO-RT-PCR and NPM1-mutA assay, we analyzed in parallel all diagnostic samples by DNA sequencing (see below) as well as by a previously developed RT-PCR assay followed by capillary electrophoresis. The primers and PCR conditions to amplify NPM1 exon 12 have been described previously.8
For capillary electrophoresis, sample dilutions of 1:20 and 1:400 were prepared in a total volume of 20 µl of Sample Loading Solution (SLS) (CEQTM SLS p/n 608082; Beckman Coulter, Fullerton, CA) containing 0.25 µl of CEQ 600 size standard mixture (CEQTM DNA Size Standard Kit-600 p/n 608095; Beckman Coulter). Samples were loaded in 96-well plates and covered with mineral oil. The amplified products were separated with a capillary electrophoresis-based system (CEQ 8000 Genetic Analysis system; Beckman Coulter) using the Frag Test default run method.
Sequencing Analysis of NPM1 Exon12
All samples analyzed were sequenced to confirm the results obtained by ASO-RT-PCR. cDNAs were amplified using the primers NPM-REV-6 and NM-F2 (see Table 1
) and PCR conditions reported elsewhere.8
The amplified products were purified using a QIAquick PCR extraction kit (Qiagen Inc., Chatsworth, CA). PCR products were cloned into a plasmid vector pCR 2.1-TOPO and then transformed into chemically competent TOP10F' One Shot Escherichia coli (TOPO TA Cloning; Invitrogen, Carlsbad, CA). For each sample, at least five recombinant colonies were selected, and plasmid DNA was prepared using a QIAprep Spin Miniprep Kit (Qiagen Inc.) and sequenced (CEQ 8000 Genetic Analysis system; Beckman Coulter). Sequences were compared with the NPM1-w/t cDNA (GenBank accession number NM_002520). Numbering of nucleotide positions refers to the coding sequence. Primer sequences used for ASO-RT-PCR, semi-nested ASO-PCR, sequencing, and fragment analysis are shown in Table 1
.
Results and Discussion
Using our ASO-RT-PCR assay, a fragment of the expected size of 320 bp presumably containing the NPM1-mutA was amplified in 21 of the 52 patients, whereas no amplification bands were detected in the remaining 31 cases. The ABL fragment of 258 bp used as internal control was successfully amplified in all patients. The results of the ASO-RT-PCR assay in some representative cases are shown in Figure 1
.
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. This comparison showed concordance of results in 100% of cases by confirming in all instances NPM1-mutA and NPM1-w/t cases. The specificity of our assay test was finally confirmed by sequencing the PCR product in all 52 samples. The results of sequencing analysis in two representative samples with NPM1-mutA and NPM1-w/t are shown in Figure 3 A and B
.
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).
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. The prospective analysis of a larger number of cases is needed to assess the clinical relevance of sequential PCR monitoring using this test.
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Footnotes
Address reprint requests to Francesco Lo-Coco, M.D., Dept. of Biopathology, Via Montpellier 1, 00133 Roma, Italy. E-mail: francesco.lo.coco{at}uniroma2.it
See related Commentary on page 198
Supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC), and by a European Organization for Research and Treatment of Cancer (EORTC) Leukemia Group Translational Research grant (to F.L.C.).
Accepted for publication January 25, 2008.
References
This article has been cited by other articles:
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  G. Wertheim and A. Bagg Nucleophosmin (NPM1) Mutations in Acute Myeloid Leukemia: An Ongoing (Cytoplasmic) Tale of Dueling Mutations and Duality of Molecular Genetic Testing Methodologies J. Mol. Diagn., May 1, 2008; 10(3): 198 - 202. [Abstract] [Full Text] [PDF]
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) and NPM1-w/t (
) results of the semi-nested ASO-PCR assay.