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Technical Briefs |
1 Dipartimento di Biopatologia e Diagnostica per Immagini and 3 Dipartimento di Medicina Interna, University Tor Vergata, Rome, Italy; 2 Laboratorio di Ricerca IRCCS, Ospedale Pediatrico Bambino Gesù, Rome, Italy; 4 Dipartimento di Medicina di Laboratorio, Policlinico Tor Vergata, Rome, Italy;
aaddress correspondence to this author at: Dipartimento di Biopatologia e Diagnostica per Immagini, Università Tor Vergata, via Montpellier, 1-00133 Rome, Italy; fax 39-0672596281, e-mail francesco.lo.coco{at}uniroma2.it
Nucleophosmin (NPM1) is a multifunctional, highly conserved protein found most frequently in nucleoli. NPM1 acts as a molecular chaperone (1) and is thought to participate in preribosome maturation and centrosome duplication(2); in addition, it has been implicated in the regulation of the ArF-p53 tumor suppressor pathway(3)(4).
NPM1 mutations have recently been reported to occur at high frequency in acute myeloid leukemia (AML), for which they currently represent the most common detectable genetic lesion (
35% of cases). This abnormality is strongly associated with normal-karyotype AML and has never been detected in AMLs bearing major cytogenetic abnormalities. It has not been observed in other hematopoietic tumors (5). Two main types of mutations have been described to date. The first and most frequent consists of a 4-nucleotide (nt) insertion (YWTG; YUPAC code) downstream from nucleotide 959; the second is deletion of a GGAGG sequence at positions 965 through 969 and substitution with 9 extra nt (GenBank accession no. NM_002520). Both mutations lead to aberrant cytoplasmic localization of NPM1 as shown after immunostaining with anti-NPM1 monoclonal antibodies. In addition to their high frequency and clustering with normal karyotype, NPM1 mutations may identify a subset of AMLs with distinct response to therapy(5). Together, these findings may have repercussions in AML classification and suggest that analysis of NPM1 mutational status should integrate modern genetic characterization of AML. We describe here a rapid and reproducible method for screening NPM1 mutations by reverse transcription-PCR followed by denaturing HPLC (DHPLC).
Bone marrow samples showing at least 70% bone marrow infiltration by leukemic cells were collected at diagnosis from 56 patients with newly diagnosed AML observed at the Department of Biopathology at the University Tor Vergata (Rome). According to the French–American–British classification (6)(7), the following subtypes were included in the study population: 6 M0, 9 M1, 14 M2, 15 M4, 6 M5a, 4 M5b, and 2 M6. Fifty-three of 56 patients were evaluable for karyotype. Of these, 10 patients had favorable karyotypes [5 with t(8;21) and 5 with inv(16)], 30 patients had intermediate karyotypes [20 with normal karyotype (6 + 8), and 4 cases with other intermediate lesions], and 13 patients had unfavorable karyotypes (7 with deletion of chromosomes 5 and/or 7, and 6 cases with complex karyotypes).
Written informed consent was obtained from all patients. Total RNA was extracted from Ficoll-Hypaque–isolated leukemic blasts by the method of Chomczynski and Sacchi (8). RNA (1 µg) was reverse-transcribed to cDNA by use of random examer primers as described previously in the BIOMED-1 Concerted Action protocol(9).
cDNA (2 µL) was amplified in a total volume of 50 µL. The reaction mixture contained 2.0 mM MgCl2, 200 µM of each deoxynucleotide triphosphate, 1x PCR buffer, 1.5 U of Taq-Gold DNA polymerase (Perkin-Elmer Cetus), and 10 pmol each of the forward (5'-CTCTTCCCAAAGTGGAAGGCAAA-3') and reverse (5'-ACCATTTCCATGTCTGAGCACC-3') primers. A G-to-C mutation was introduced in the reverse primer to reduce amplification of NPM1 pseudogenes. After the mixture was preheated at 94 °C for 7 min, it was subjected to 30 cycles of 45 s at 55 °C, 30 s at 72 °C, and 30 s at 94 °C. A final extension of 5 min was carried out at 72 °C on a GeneAmp PCR System 2400 (Perkin-Elmer). DHPLC was carried out on a WAVE DNA fragment analysis system (TransgenomicTM) equipped with a DNASep® column (Transgenomic). DNA was eluted from the column by a linear acetonitrile gradient in 0.1 mol/L triethylamine acetate buffer (TEAA; Transgenomic) at a constant flow rate. The reversed-phase gradient was formed by mixing buffer A (0.1 mol/L TEAA, pH 7.0) and buffer B (250 mL/L acetonitrile in 0.1 mol/L TEAA, pH 7.0). Oven temperature for optimum separation of heteroduplex molecules was deduced from the Transgenomic software (NavigatorTM software, Ver. 1.6.0). For detection of NPM1, 8 µL of PCR product was loaded in partially denaturing conditions (55.3 °C), and eluted DNA was detected by the absorbance at 260 nm. Samples were amplified with the NPM1_25F/NPM1_1112R primer pair (5). PCR products, purified by standard methods, were sequenced from both strands with NPM1 Dir3 or NPM1 Rev3 internal primers on a CEQ 8000 Genetic Analysis system (Beckman Coulter). The primer sequences are shown in Table 1
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The study recently reported by Falini et al. (5) showed that NPM1, already described to be involved in rearrangements in leukemia(10) and lymphomas(11), is mutated in the leukemic cells of 35% of primary adult AML. This finding was based on direct sequence analysis of leukemic DNA derived from patients with aberrant dislocation of the NPM1 protein as revealed by immunohistochemistry. In this study we established a DHPLC-based assay to routinely detect NPM1 mutations in AML and suggest its inclusion in the genetic diagnostic work-up of this disease. With the melting temperature predicted by the Navigator software, we were able to distinguish heteroduplex from homoduplex peaks. The chromatogram from each tested sample was overlaid with the wild-type profile, and samples with an extra peak were scored as mutated: the first peak corresponding to the heteroduplex was detected at 4.3 min, and the second, corresponding to the homoduplex product, was detected at 5 min.
Eight of the 56 analyzed samples were scored as NPMmut and 48 as NPMwt. The NPM1 mutational status detected in 2 representative cases is shown in Fig. 1
. All NPMmut samples and 10 NPMwt samples were also analyzed by reverse transcription-PCR and direct sequencing, which confirmed in all cases the results obtained by DHPLC. The French–American–British subtypes of NPMmut cases were as follows: M0, 2 cases; M1, 1 case, M4, 2 cases, M5a, 1 case; M5b, 2 cases. With respect to cytogenetic characterization, 7 of the NMPmut patients had normal karyotypes, and 1 had deletion of both chromosomes 5q and 7q. As to the type of mutation detected, in 7 NPMmut cases we found the most frequently reported mutation type: a duplication of TCTG tetranucleotide at positions 956–959 of the reference sequence (NM_002520). In the fifth case, nucleotides 965–969 (GGAGG) were substituted by the 9mer GCTTTAGTC. In all mutated cases, the resulting frameshift led to a product 5 amino acids longer with the new C-terminal tail CFSQVSLRK, peculiar to the NPM1-mutated product. Because NPM1 mutations currently are the most frequently reported genetic aberration in AML and in light of their potential prognostic significance, we believe that inclusion of this DHPLC assay in diagnostic evaluations may improve genetic characterization of AML and allow assignment of patients to better-defined risk categories. Compared with immunohistochemical analysis to detect aberrant cytoplasmic NMP1 localization, this assay overcomes the need to obtain a bone marrow biopsy at the time of AML diagnosis, which is not an established procedure in the routine diagnostic work-up of acute leukemia. Moreover, the DHPLC approach is less time-consuming and less expensive than direct NPM1 sequencing and could therefore represent a suitable technique for rapid screening of AML.
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Acknowledgments
This work was supported by Ministero dell’Istruzione dell’Università e della Ricerca Cofin 2003, and by Associazione Italiana per la Ricerca Cancro. At the time of the study, N.I.N. was on a leave of absence from Department of Chemical Biochemistry (Hematology), Universidad Nacional de Rosario (Argentina), and E.A. was on a leave of absence from the Division of Hematology of the University of Palermo (Italy). N.I.N. is a member of Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET), Argentina.
References
The following articles in journals at HighWire Press have cited this article:
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  T. Ottone, E. Ammatuna, S. Lavorgna, N. I. Noguera, F. Buccisano, A. Venditti, L. Gianni, M. Postorino, G. Federici, S. Amadori, et al. An Allele-Specific RT-PCR Assay to Detect Type A Mutation of the Nucleophosmin-1 Gene in Acute Myeloid Leukemia J. Mol. Diagn., May 1, 2008; 10(3): 212 - 216. [Abstract] [Full Text] [PDF]
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 B. Falini, I. Nicoletti, N. Bolli, M. P. Martelli, A. Liso, P. Gorello, F. Mandelli, C. Mecucci, and M. F. Martelli Translocations and mutations involving the nucleophosmin (NPM1) gene in lymphomas and leukemias Haematologica, April 1, 2007; 92(4): 519 - 532. [Abstract] [Full Text] [PDF]
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 B. Falini, I. Nicoletti, M. F. Martelli, and C. Mecucci Acute myeloid leukemia carrying cytoplasmic/mutated nucleophosmin (NPMc+ AML): biologic and clinical features Blood, February 1, 2007; 109(3): 874 - 885. [Abstract] [Full Text] [PDF]
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