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.
Besides their role as pathogenetic events, genetic alterations of acute myeloid leukemia (AML) are known to be major determinants of patient response to therapy and outcome.1 As a consequence, detection of these abnormalities in conjunction with other laboratory characterization analysis is nowadays considered as an established practice in the diagnostic work-up of AML.1 In addition to aberrations that are revealed by conventional karyotyping, submicroscopic lesions undetectable at chromosome level have been described whose presence in AML blasts may confer distinct prognosis. These include Flt3 receptor gene mutations and, more recently, NPM1 gene mutations.2, 3, 4, 5, 6
Flt3 is mutated gene in approximately 20–30% of AML cases and has been implicated in its pathogenesis.3, 4, 5, 6, 7, 8 Such alterations consist most frequently of internal tandem duplications (ITD) in the juxtamembrane (JM) region of the receptor cytoplasmic domain and involve exon 14 and less frequently part of exon 15. Compared to patients with unmutated Flt3, AML patients carrying this aberration have been shown to have poorer outcome. The approach most commonly used for large-scale diagnostic screening of Flt3 ITD consists of RT-PCR followed by agarose gel electrophoresis.
We have recently shown that alterations in the NPM1 gene represent the most common genetic lesion currently detectable in AML, being found in nearly 35% of adult AML patients. Interestingly, such alterations are strongly associated with normal karyotype (NK-AML), being detectable in about 60% of AML without visible chromosomal aberrations. This lesion consists of small insertion/deletions in the NPM1 gene region coding for the nucleolar localization signal of the protein.2
Two kinds of NPM1 mutations have been described so far: (1) insertion of the four nucleotides (nt) repeat YWTG (YUPAC code) downstream nucleotide 959 (Gene Bank accession number NM_002520); and (2) deletion of a GGAGG sequence at position 965–969 and substitution with nine extra nt. All described mutations cause a frameshift that leads to the loss of one or two tryptophans that are essential for nucleolar localization of the protein.9
We describe here a method for simultaneous detection of the two most frequent genetic alterations associated with AML , that is, Flt3-ITD and NPM1 mutations .
Materials and methods
Patients, RNA extraction, cDNA synthesis, and PCR amplification
Bone marrow or peripheral blood leukemic cells were collected at presentation from 38 AML patients diagnosed at the Department of Hematology of the University Tor Vergata of Rome. Written informed consent was obtained from all patients. In all samples, the presence of Flt3-ITD was previously investigated by RT-PCR as reported elsewhere.8 Total RNA was extracted from Ficoll–Hypaque isolated mononuclear cells using the method of Chomczynsky and Sacchi.10 RNA was reverse-transcribed using random examers primers as previously described.11 With the aim of simultaneously analyzing both ITD and NPM1 mutations, we adopted a multiplex PCR strategy. In order to easily discriminate NPM1 and FLT3 PCR products regardless of size, we used 5′ end D4 and D3 WellRED dye labeled reverse primers (Beckman Coulter).
NPM1 mutations so far described are all located in a small region spanning from nt 959 (Gene Bank accession number NM_002520) to the 3′end of the locus. In this region NPM1 and its seven pseudogenes are highly homologous. Therefore, to rule out the amplification of pseudogenes, we introduced in forward and revers primers a C/A and C/G mutation, respectively.12 Primer sequences used for NPM1 and FLT3 amplification are shown in Table 1.
In all, 2 μl of cDNA were amplified in a total volume of 25 μl of the reaction mixture containing 0.2 mM of each GIBCO dNTP, 1 × GIBCO PCR buffer, 2 mM of GIBCO MgCl2, 1.6 U of GIBCO Platinum Taq polymerase, and 10 pmol of each primer for the amplification of Flt3-ITD and NPM1 exon 12.
Preheating of the mixture at 94°C for 5 min was followed by 30 cycles of 30 s at 94°C, 45 s at 56 °C, and 30 s at 72°C. A final extension of 7 min was carried out at 72°C on a Gene Amp PCR System 2400 (Perkin Elmer, Emeryville, CA, USA).
Sample preparation for loading into the CEQ-8000
Sample dilutions of 1:20 and 1:400 were prepared in a total volume of 20 μl SLS (CEQ™ SLS p/n 608082, Beckman Coulter) containing 0.25 μl of CEQ 600 size standard mixture (CEQ™ 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 NPM
In order to validate NPM1 results obtained from the electropherograms, 5 NPM1 mutated and 10 NPM1 wild-type (wt) samples were amplified with standard procedures with the NPM1_25F/ NPM1_1112R pair of primers.2 PCR products, purified by standard methods, were sequenced directly from both strands with NPM1_649F or NPM1_1105R internal primers. Primers used for sequence analysis are shown in Table 2.
Results and discussion
Capillary electrophoresis of fluorescently labeled multiplex PCR products was used to screen for the presence of both FLT3-ITD and NPM1 mutations simultaneously. The labeled fragment sizes corresponding to NPM1 and Flt3 wt genes were 349 bp (blue) and 366 bp (green), respectively (Figure 1a). Of the 38 AML samples screened,5 were NPM1 mutated (NPM1m) and 33 wt (NPM1wt). All NPM1m samples were heterozygous, showing a double blue peak at positions 349 (wt) and 353 (Figure 1b). In all cases, mutations were confirmed by RT-PCR and direct sequencing with an internal primer on both strands. Similarly, the wt sequence was confirmed by sequence analysis in 10 out of 33 randomly chosen NPM1wt cases. In four NPM1m cases, we found the most common NPM1 mutation previously described: a duplication of TCTG tetranucleotide at positions 956–959 of the reference sequence (NM_002520).2 In the fifth case, nt 965–969 (GGAGG) were substituted by the new 9-mer IndexTermIndexTermGCTTTAGTC, not described so far: the resulting frameshift leads to a five amino acid longer product with the new C-terminal CFSQVSLRK.
Of 38 AML samples analyzed three were Flt3-ITD positive and showed one peak at 366 corresponding to the wt and a second peak at 394, 419 or 443, respectively. All samples were previously analyzed by conventional RT-PCR for FLT3-ITD followed by agarose gel electrophoresis. The results showed 100% concordance with those obtained with the NFmPCR assay. In only one case both NPM1 and FLT3 mutations were detected, as shown in Figure 1b. To assess the sensitivity of the NFmPCR assay, we performed dilution experiments using RNA of wt and mutant for both NPM1 and Flt3 genes (ratio mutant/wt 0.01, 0.10, 0.25, 0.50, 0.75, 1.00). The results indicated that this assay could detect the NPM1 mutation present in concentrations as low as 10%.
In this report, we show that multiplex PCR followed by capillary electrophoresis can be used for a fast and reliable diagnosis of both NPM1 and FLT3-ITD mutations. Capillary electrophoresis of fluorescently labeled PCR products is a very sensitive method for the detection of DNA fragment size variation. Up to 40 samples can be screened in a single analytical run. Analysis is based on differences in fragment size for each fluorescent dye and discriminates up to 1 bp of difference, allowing the detection of subtle mutations undetectable through conventional agarose gel electrophoresis. We conclude that this assay, rapid, highly reproducible, nonisotopic, and amenable to automation, may be included in routine molecular diagnosis of AML.
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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.
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