Denaturing High-Performance Liquid Chromatography
2006; Elsevier BV; Volume: 8; Issue: 2 Linguagem: Inglês
10.2353/jmoldx.2006.050098
ISSN1943-7811
AutoresGiovanni Roti, Roberto Rosati, R Bonasso, Paolo Gorello, Daniela Diverio, Massimo Fabrizio Martelli, Brunangelo Falini, Cristina Mecucci,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoNPM1 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 chro-matograms, with mutations B and D differing for one nucleotide. Elution profiles and sequencing also identified four new variants. Our results suggest that DHPLC detects NPM1 mutations as well as direct sequencing and immunohistochemistry, providing a helpful approach in the diagnosis of NPMc+ AML. 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 chro-matograms, with mutations B and D differing for one nucleotide. Elution profiles and sequencing also identified four new variants. Our results suggest that DHPLC detects NPM1 mutations as well as direct sequencing and immunohistochemistry, providing a helpful approach in the diagnosis of NPMc+ AML. Acute myeloid leukemia (AML) with normal karyotype is the single largest cytogenetic subgroup, affecting approximately half of adult patients with primary AML.1Grimwade D Walker H Oliver F Wheatley K Harrison C Harrison G Rees J Hann I Stevens R Burnett A Goldstone A The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial.Blood. 1998; 92: 2322-2333Crossref PubMed Google Scholar2Mrozek K Heerema NA Bloomfield CD Cytogenetics in acute leukemia.Blood Rev. 2004; 2: 115-136Abstract Full Text Full Text PDF Scopus (540) Google Scholar Although the genetic mechanisms underlying this heterogeneous subgroup are primarily unknown, several gene mutations have been characterized. These involve the RAS gene, FLT3, and KIT (tyrosine kinase genes), and AML1, GATA1, and CEBPA transcription factors involved in normal hematopoiesis.3Marcucci G Mrozek K Bloomfield CD Molecular heterogeneity and prognostic biomarkers in adults with acute myeloid leukemia and normal cytogenetics.Curr Opin Hematol. 2005; 1: 68-75Crossref Scopus (107) Google Scholar The NPM1 gene is the translocation partner of MLF1 (myeloid leukemia factor 1) in AML with t(3;5), RARA (retinoic acid receptor α) in acute promyelocytic leukemia with t(5;17), and ALK (anaplastic lymphoma kinase) in large anaplastic cell lymphoma with t(2;5).4Yoneda-Kato N Look AT Kirstein MN Valentine MB Raimondi SC Cohen KJ Carroll AJ Morris SW The t(3;5)(q25.1;q34) of myelodys-plastic syndrome and acute myeloid leukemia produces a novel fusion gene, NPM-MLF1.Oncogene. 1996; 2: 265-275Google Scholar, 5Redner RL Rush EA Faas S Rudert WA Corey SJ The t(5;17) variant of acute promyelocytic leukemia expresses a nucleophosmin-retinoic acid receptor fusion.Blood. 1996; 87: 882-886Crossref PubMed Google Scholar, 6Morris SW Kirstein MN Valentine MB Dittmer K Shapiro DN Look AT Saltman DL Fusion of a kinase gene ALK, to a nucleolar protein gene NPM, in non-Hodgkin's lymphoma.Science. 1995; 267: 316-317Crossref PubMed Scopus (176) Google Scholar We recently identified NPM1 (nucleophosmin, B23, numatrin) mutations7Falini B Mecucci C Tiacci E Alcalay M Rosati R Pasqualucci L La Starza R Diverio D Colombo E Santucci A Bigerna B Pacini R Pucciarini A Liso A Vignetti M Fazi P Meani N Pettirossi V Saglio G Mandelli F Lo-Coco F Pelicci PG Martelli MF for the GIMEMA Acute Leukemia Working Party Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.N Engl J Med. 2005; 352: 254-266Crossref PubMed Scopus (1424) Google Scholar as the most frequent lesions, at ∼60% of adult AMLs with normal karyotype and none of the major fusion genes, ie, BCR/ABL1, PML/RARA, AML1/ETO, CBFB/ MYH11, and DEK/CAN. All NPM1 gene mutations occur at exon 12 and affect the C-terminus of the encoded nucleolar phosphoprotein, which is abnormally delocalized to the cytoplasm as shown by anti-NPM monoclonal antibodies (so called NPMc+ AML). In these patients NPM1 mutations are a good prognostic index for response to induction therapy and overall survival.7Falini B Mecucci C Tiacci E Alcalay M Rosati R Pasqualucci L La Starza R Diverio D Colombo E Santucci A Bigerna B Pacini R Pucciarini A Liso A Vignetti M Fazi P Meani N Pettirossi V Saglio G Mandelli F Lo-Coco F Pelicci PG Martelli MF for the GIMEMA Acute Leukemia Working Party Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.N Engl J Med. 2005; 352: 254-266Crossref PubMed Scopus (1424) Google Scholar, 8Schnittger S Schoch C Kern W Mecucci C Tschulik C Martelli MF Haferlach T Hiddemann W Falini B Nucleophosmin gene mutations are predictors of favourable prognosis in acute myelogenous leukaemia with normal karyotype.Blood. 2005; 106: 3733-3739Crossref PubMed Scopus (592) Google Scholar, 9Suzuki T Kiyoi H Ozeki K Tomita A Yamaji S Suzuki R Kodera Y Miyawaki S Asou N Kuriyama K Yagasaki F Shimazaki C Akiyama H Nishimura M Motoji T Shinagawa K Takeshita A Ueda R Kinoshita T Emi N Naoe T Clinical characteristics and prognostic implications of NPM1 mutations in acute myeloid leukemia.Blood. 2005; 106: 2854-2861Crossref PubMed Scopus (223) Google Scholar, 10Dohner K Schlenk RF Habdank M Scholl C Rucker FG Corbacioglu A Bullinger L Frohling S Dohner H for the AML Study Group (AMLSG) Mutant nucleophosmin (NPM1) predicts favorable prognosis in younger adults with acute myeloid leukemia and normal cytogenetics-interactions with other gene mutations.Blood. 2005; 106: 3740-3746Crossref PubMed Scopus (671) Google Scholar Thus, molecular screening may improve risk assessment and therapeutic strategies after remission. A powerful technique for detecting mutations, such as single base substitutions as well as small insertions or deletions, is denaturing high-performance liquid chromatography (DHPLC), which has sensitivity and specificity ranging from 96 to 100%.11Xiao W Oefner PJ Denaturing high-performance liquid chromatography: a review.Hum Mutat. 2001; 6: 439-474Crossref Scopus (625) Google Scholar, 12Harvey JF Sampson JR Mutation scanning for the clinical laboratory: DHPLC.Methods Mol Med. 2004; 92: 45-66PubMed Google Scholar, 13O'Donovan MC Oefner PJ Roberts SC Austin J Hoogendoorn B Guy C Speight G Upadhyaya M Sommer SS McGuffin P Blind analysis of denaturing high-performance liquid chromatography as a tool for mutation detection.Genomics. 1998; 52: 44-49Crossref PubMed Scopus (290) Google Scholar In this study, we designed a DHPLC procedure to analyze NPM1 gene mutations in AML samples. Direct sequencing and/or immunohistochemistry with anti-NPM monoclonal antibodies were used to validate results. We show that DHPLC is a rapid, reliable, and straightforward approach for analyzing NPM1 gene mutations. A total of 120 adult AML blood and/or bone marrow samples were obtained at diagnosis (with leukemic blasts ranging from 40 to 90%) from the GIMEMA Cooperative Group. All patients provided informed consent to genetic analysis of their biological samples, and the study was approved by the Perugia University Institutional Review Board (FWA number 00005268). In all cases immunohistochemical studies were performed on bone marrow biopsies at diagnosis with the use of monoclonal antibodies against NPM. Immunostaining was performed with the monoclonal anti-alkaline phosphatase technique.7Falini B Mecucci C Tiacci E Alcalay M Rosati R Pasqualucci L La Starza R Diverio D Colombo E Santucci A Bigerna B Pacini R Pucciarini A Liso A Vignetti M Fazi P Meani N Pettirossi V Saglio G Mandelli F Lo-Coco F Pelicci PG Martelli MF for the GIMEMA Acute Leukemia Working Party Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.N Engl J Med. 2005; 352: 254-266Crossref PubMed Scopus (1424) Google Scholar14Cordell JL Pulford KAF Bigerna B Roncador G Banham A Colombo E Pelicci PG Mason DY Falini B Detection of normal and chimeric nucleophosmin in human cell.Blood. 1999; 93: 632-642Crossref PubMed Google Scholar NPM subcellular distribution, ie, restriction to the nucleus (NPMc−) or presence in the cytoplasm (NPMc+), was assessed under an Olympus Provis microscope (Milano, Italy). Cytogenetic investigations were performed after short-term culture. Karyotypes were described according to the International System for Human Cytogenetic Nomenclature.15Mitelman F An International System for Human Cytogenetic Nomenclature. Karger, Basel1995Google Scholar Fluorescence in situ hybridization16Crescenzi B Fizzotti M Piattoni S La Starza R Matteucci C Carotti A Avcrsa F Martelli MF Mecucci C Interphase FISH for Y chromosome, VNTR polymorphisms, and RT-PCR for BCR-ABL in the monitoring of HLA-matched and mismatched transplants.Cancer Genet Cytogenet. 2000; 120: 25-29Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar and reverse transcriptase-polymerase chain reaction (RT-PCR) or Southern blotting were used to analyze PML/RARA, AML1/ETO, CBFB/MYH11, BCR/ABL, and DEK/CAN fusions and MLL rearrangements.17van Dongen JJ Macintyre EA Gabert JA Delabesse E Rossi V Saglio G Gottardi E Rambaldi A Dotti G Griesinger F Parreira A Gameiro P Diaz MG Malec M Langerak AW San Miguel JF Biondi A Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease: report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia.Leukemia. 1999; 13: 1901-1928Crossref PubMed Scopus (1006) Google Scholar RNA from NPMc+ patients was extracted by TRIzol (Invitrogen Life Technologies, Inc., Paisley, UK) and reverse transcribed with use of the Thermoscript RT-PCR System (Invitrogen, Carlsbad, CA). Sequencing was done using primers NPM1_25F (5′-GGTTGTTCTCTGGAGCAGCGT-TC-3′) and NPM1_1112R (5′-CCTGGACAACATTTAT-CAAACACGGTA-3 '). Genomic DNA of all patients was extracted from blood and/or bone marrow aspirate using standard procedures. PCR fragments of NPM1 exon 12 were amplified using the following oligonucleotide primers: NPM1-F (5′-TTAACTCTCTGGTGGTAGAATGAA-3′), NPM1-R (5′-CAAGACTATTTGCCATTCCTAAC-3′).7Falini B Mecucci C Tiacci E Alcalay M Rosati R Pasqualucci L La Starza R Diverio D Colombo E Santucci A Bigerna B Pacini R Pucciarini A Liso A Vignetti M Fazi P Meani N Pettirossi V Saglio G Mandelli F Lo-Coco F Pelicci PG Martelli MF for the GIMEMA Acute Leukemia Working Party Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.N Engl J Med. 2005; 352: 254-266Crossref PubMed Scopus (1424) Google Scholar PCR assays were performed in a volume of 25 μl containing 100 ng of genomic DNA, 6 pmol of forward and reverse primers, 200 μmol/L dNTPs, 0.3 U of Expand High Fidelity Plus Taq (Roche Diagnostics, Monza, Italy) and buffer 2 (1.75 mmol/L MgCl2). PCR assays were initiated by a denaturation step at 94°C for 5 minutes, followed by 33 cycles at 94°C for 45 seconds, 60°C for 30 seconds, and 72°C for 45 seconds; a final extension was performed at 72°C for 5 minutes. Exon 12 with flanking intron sequences of the NPM1 gene was screened for mutations by DHPLC (Wave System; MD Transgenomic Inc., Omaha, NE). PCR products were denatured at 95°C for 5 minutes and cooled via a temperature ramp of 1°C/minute to 65°C; the last step consisted of 1 minute at 65°C. Samples were kept cool until 10 μl were automatically inserted into a preheated reversed phase column based on nonporous (polystyrene-divinylbenzene) particles (DNA-Sep; Trans-genomic, San Jose, CA). DNA was eluted on a linear acetonitrile gradient consisting of buffer A (0.1 mol/L triethylammonium acetate; TEAA)/buffer B (0.1 mol/L TEAA, 25% acetonitrile). Gradient elution and melting temperature conditions were determined using Wave-Maker Navigator version 1.5.4 software (Transgenomic) and the DHPLC Melt Program (http://insertion.stanford.edu/melt.html). On the basis of the PCR product (553 bp) nucleotide sequence, both analysis programs calculate the melting temperature of the domains contained in the sequences of interest. Start elution gradient ranges between 40.8% buffer A and 54.2% buffer B, stop gradient at 5-minute intervals between 31.8% buffer A and 68.2% buffer B. Experimental conditions (melting temperature or elution time shift) were optimized by studying alterations in the sample elution profiles. Electropherograms from patients were compared with normal sequenced controls. Immunohistochemistry showed cytoplasmic NPM in 26 of 120 leukemic samples. RT-PCR and direct sequencing of the NPM1 coding region revealed mutations affecting exon 12 in all NPMc+ AML. Cytogenetic data were available for 93 of 120 patients with AML (26 NPMc+, 67 NPMc−). Of 67 NPMc− patients, 21 were positive for t(8;21)(q22;q22), 22 for inv(16)(p13q22), 10 for MLL rearrangement, and 14 showed normal or complex karyotype. Karyotype was normal in the 26 NPMc+ patients. Screening analysis with DHPLC was performed in all 120 patients. Wild-type chromatogram with one single peak (Figure 1A), indicative of no mutations, was obtained in 92 of 94 NPMc- AMLs. In the other two patients, a double-peak elution profile was observed, and subsequent sequencing detected a del(T) at position 1146 of the NM_002520 sequence of the noncoding region. All electropherogram profiles in the 26 NPMc+ leukemias were different from the wild type, indicating 100% sensitivity of the DHPLC to detect NPM1 mutations. Elution profiles varied in the specific time of retention between homo- and heteroduplex, shape, and number of peaks. Sequencing categorized mutations A, B, and D, as previously named,4Yoneda-Kato N Look AT Kirstein MN Valentine MB Raimondi SC Cohen KJ Carroll AJ Morris SW The t(3;5)(q25.1;q34) of myelodys-plastic syndrome and acute myeloid leukemia produces a novel fusion gene, NPM-MLF1.Oncogene. 1996; 2: 265-275Google Scholar in 9 of 26, 5 of 26, and 5 of 26 patients, respectively (Figure 2). All nine cases of mutation A gave identical elution profiles (Figure 1B). The other groups showed a typical chromatogram, with mutations B and D differing for one nucleotide in the inserted sequence (Figure 1C). Mutations A, B, and D all led to a frameshift in the region encoding the NPM C-terminus. Mutation A, the most frequent, was an insertion of a TCTG tetranucleotide at position 959 of the reference sequence NM_002520 (GenBank). The resulting shift in the reading frame alters the NPM C-terminal portion by replacing the last seven amino acids (WQWRKSL) with 11 different residues (CLAVEEVSLRK). Mutations B and D included distinct 4-bp insertions at position 959, which resulted in the same frameshift and alterations in the NPM C-terminal portion as mutation A. In the other seven patients elution profiles and sequencing identified four new variants (Figures 2 and 3): [ins964_965(GCTT); 965G>C; 967>A>C; 968>G>C; 969G>C]; [ins963-964(AGGA)]; [ins964_965(CTCT); 965G>T; 966G>T; 967A>C; 968G>T; 969G>A]; and [ins959_960(GCCA)]. All four new NPM variant proteins maintained the same last five amino acid residues (VSLRK). All mutations involved one or both of the tryptophan residues at positions 288 and 290 and acquisition of a leucine-rich nuclear export signal motif (Figure 2).7Falini B Mecucci C Tiacci E Alcalay M Rosati R Pasqualucci L La Starza R Diverio D Colombo E Santucci A Bigerna B Pacini R Pucciarini A Liso A Vignetti M Fazi P Meani N Pettirossi V Saglio G Mandelli F Lo-Coco F Pelicci PG Martelli MF for the GIMEMA Acute Leukemia Working Party Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.N Engl J Med. 2005; 352: 254-266Crossref PubMed Scopus (1424) Google Scholar18Bogerd HP Fridell RA Benson RE Hua J Cullen BR Protein sequence requirements for function of the human T-cell leukaemia virus type 1 rex nuclear export signal delineated by a novel in vivo randomization-selection assay.Mol Cell Biol. 1996; 16: 4207-4214Crossref PubMed Scopus (321) Google Scholar19Nakagawa M Kameoka Y Suzuki R Falini B Mecucci C Martelli MF Nucleophosmin in acute myelogenous leukemia.N Engl J Med. 2005; 352: 1819-1820PubMed Google Scholar No changes in melting temperature (52.9°C) or elution gradient were needed to unravel these seven mutations. In the present study we developed a DHPLC methodology to identify NPM1 mutations typically occurring at exon 12 in AML with normal karyotype. These heterozygous NPM1 mutations are the most frequently acquired genetic event identified to date in adult AML with normal karyotype.7Falini B Mecucci C Tiacci E Alcalay M Rosati R Pasqualucci L La Starza R Diverio D Colombo E Santucci A Bigerna B Pacini R Pucciarini A Liso A Vignetti M Fazi P Meani N Pettirossi V Saglio G Mandelli F Lo-Coco F Pelicci PG Martelli MF for the GIMEMA Acute Leukemia Working Party Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.N Engl J Med. 2005; 352: 254-266Crossref PubMed Scopus (1424) Google Scholar The C-terminus of the nucleolar nucleophosmin encoded by NPM1 is affected, and NPM is delocalized to the cytoplasm as shown by specific anti-NPM monoclonal antibodies (NPMc+ AML).7Falini B Mecucci C Tiacci E Alcalay M Rosati R Pasqualucci L La Starza R Diverio D Colombo E Santucci A Bigerna B Pacini R Pucciarini A Liso A Vignetti M Fazi P Meani N Pettirossi V Saglio G Mandelli F Lo-Coco F Pelicci PG Martelli MF for the GIMEMA Acute Leukemia Working Party Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.N Engl J Med. 2005; 352: 254-266Crossref PubMed Scopus (1424) Google Scholar In NPMc+ AML without FLT3 mutations, the NPM1 mutation is associated with good response to induction treatment and overall survival,7Falini B Mecucci C Tiacci E Alcalay M Rosati R Pasqualucci L La Starza R Diverio D Colombo E Santucci A Bigerna B Pacini R Pucciarini A Liso A Vignetti M Fazi P Meani N Pettirossi V Saglio G Mandelli F Lo-Coco F Pelicci PG Martelli MF for the GIMEMA Acute Leukemia Working Party Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.N Engl J Med. 2005; 352: 254-266Crossref PubMed Scopus (1424) Google Scholar, 8Schnittger S Schoch C Kern W Mecucci C Tschulik C Martelli MF Haferlach T Hiddemann W Falini B Nucleophosmin gene mutations are predictors of favourable prognosis in acute myelogenous leukaemia with normal karyotype.Blood. 2005; 106: 3733-3739Crossref PubMed Scopus (592) Google Scholar, 9Suzuki T Kiyoi H Ozeki K Tomita A Yamaji S Suzuki R Kodera Y Miyawaki S Asou N Kuriyama K Yagasaki F Shimazaki C Akiyama H Nishimura M Motoji T Shinagawa K Takeshita A Ueda R Kinoshita T Emi N Naoe T Clinical characteristics and prognostic implications of NPM1 mutations in acute myeloid leukemia.Blood. 2005; 106: 2854-2861Crossref PubMed Scopus (223) Google Scholar, 10Dohner K Schlenk RF Habdank M Scholl C Rucker FG Corbacioglu A Bullinger L Frohling S Dohner H for the AML Study Group (AMLSG) Mutant nucleophosmin (NPM1) predicts favorable prognosis in younger adults with acute myeloid leukemia and normal cytogenetics-interactions with other gene mutations.Blood. 2005; 106: 3740-3746Crossref PubMed Scopus (671) Google Scholar making mutational analysis of the NPM1 gene at diagnosis an important step in the prognostic assessment of adult AML. Only heterozygous NPM1 mutations have been found so far in more than 1839 cases of AML.8Schnittger S Schoch C Kern W Mecucci C Tschulik C Martelli MF Haferlach T Hiddemann W Falini B Nucleophosmin gene mutations are predictors of favourable prognosis in acute myelogenous leukaemia with normal karyotype.Blood. 2005; 106: 3733-3739Crossref PubMed Scopus (592) Google Scholar, 9Suzuki T Kiyoi H Ozeki K Tomita A Yamaji S Suzuki R Kodera Y Miyawaki S Asou N Kuriyama K Yagasaki F Shimazaki C Akiyama H Nishimura M Motoji T Shinagawa K Takeshita A Ueda R Kinoshita T Emi N Naoe T Clinical characteristics and prognostic implications of NPM1 mutations in acute myeloid leukemia.Blood. 2005; 106: 2854-2861Crossref PubMed Scopus (223) Google Scholar, 10Dohner K Schlenk RF Habdank M Scholl C Rucker FG Corbacioglu A Bullinger L Frohling S Dohner H for the AML Study Group (AMLSG) Mutant nucleophosmin (NPM1) predicts favorable prognosis in younger adults with acute myeloid leukemia and normal cytogenetics-interactions with other gene mutations.Blood. 2005; 106: 3740-3746Crossref PubMed Scopus (671) Google Scholar20Boissel N Renneville A Biggio V Philippe N Thomas X Cayuela JM Terre C Tigaud I Castaigne S Raffoux E de Botton S Fenaux P Dombret H Preudomme C Prevalence, clinical profile and prognosis of NPM mutations in AML with normal karyotype.Blood. 2005; 106: 3618-3620Crossref PubMed Scopus (192) Google Scholar, 21Verhaak RG Goudswaard CS van Putten W Bijl MA Sanders MA Hugens W Uitterlinden AG Erpelinck CA Delwel R Lowenberg B Valk PJ Mutations in nucleophosmin NPM1 in acute myeloid leukemia (AML): association with other gene abnormalities and previously established gene expression signatures and their favorable prognostic significance.Blood. 2005; 106: 3747-3754Crossref PubMed Scopus (479) Google Scholar, 22Cazzaniga G Dell'Oro MG Mecucci C Giarin E Masetti R Rossi V Locatelli F Martelli MF Basso G Pession A Biondi A Falini B Nucleophosmin mutations in childhood acute myelogenous leukemia with normal karyotype.Blood. 2005; 106: 1419-1422Crossref PubMed Scopus (139) Google Scholar The DHPLC technology is a powerful approach for detecting these mutations because it is based on the differential retention of homo-and hetero-duplex DNA molecules by ion-pair chromatography under conditions of partial heat denaturation. When homologous DNA single strands carrying point mutations, small insertions or deletions rean-neal, heteroduplex molecules are generated in heterozygous samples.11Xiao W Oefner PJ Denaturing high-performance liquid chromatography: a review.Hum Mutat. 2001; 6: 439-474Crossref Scopus (625) Google Scholar Heteroduplex spikes are generally eluted ahead of the homoduplexes, producing at least one additional peak. Because all known NPM1 mutations derive from insertions, DHPLC may be applied in full denaturing conditions to discriminate the wild-type and mutant alleles according to sequence length. In partial denaturation, the type of nucleotide sequence, besides sequence length, determines the chromatograms. Thus given the heterogeneity of the NPM1 mutations, we opted for this approach. In all 26 NPMc+ AMLs, one or more peaks in addition to the standard wild-type spike were observed. At a constant melting temperature of 52.9°C, we obtained seven different chromatograms, each one corresponding to a different mutation. DHPLC missed no case with NPM delocalization and/or mutations after sequencing. Moreover, a single nucleotide difference as in mutations B and D (Figure 1C) was visualized as two distinct chromatograms. These results highlight the sensitivity of the methodology, which emerges as a reliable diagnostic test with the potential for clinical application. Regarding specificity, each of the seven mutations corresponded to a distinct elution profile, indicating that the nature of the sequence variations can be predicted independently of sequencing. Indeed, in this study we defined the chro-matogram of mutations A, B, and D, which together cover 92 to 96% of all variants in adult AML. Reproducibility was excellent because chromatograms were always identical in each mutation (Figure 1B). Recently Verhaak and col-leagues21Verhaak RG Goudswaard CS van Putten W Bijl MA Sanders MA Hugens W Uitterlinden AG Erpelinck CA Delwel R Lowenberg B Valk PJ Mutations in nucleophosmin NPM1 in acute myeloid leukemia (AML): association with other gene abnormalities and previously established gene expression signatures and their favorable prognostic significance.Blood. 2005; 106: 3747-3754Crossref PubMed Scopus (479) Google Scholar applied a DHPLC protocol to a large series of 275 AMLs. This approach used cDNA instead of genomic DNA, and two melting temperatures were needed to unravel new mutations. However, given the methodological differences between this approach and ours in the present study, it is not possible to compare both sets of results. Four new NPM1 gene mutations were identified (Figure 3, A to D) with features similar to those that have already been described.7Falini B Mecucci C Tiacci E Alcalay M Rosati R Pasqualucci L La Starza R Diverio D Colombo E Santucci A Bigerna B Pacini R Pucciarini A Liso A Vignetti M Fazi P Meani N Pettirossi V Saglio G Mandelli F Lo-Coco F Pelicci PG Martelli MF for the GIMEMA Acute Leukemia Working Party Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.N Engl J Med. 2005; 352: 254-266Crossref PubMed Scopus (1424) Google Scholar, 8Schnittger S Schoch C Kern W Mecucci C Tschulik C Martelli MF Haferlach T Hiddemann W Falini B Nucleophosmin gene mutations are predictors of favourable prognosis in acute myelogenous leukaemia with normal karyotype.Blood. 2005; 106: 3733-3739Crossref PubMed Scopus (592) Google Scholar, 9Suzuki T Kiyoi H Ozeki K Tomita A Yamaji S Suzuki R Kodera Y Miyawaki S Asou N Kuriyama K Yagasaki F Shimazaki C Akiyama H Nishimura M Motoji T Shinagawa K Takeshita A Ueda R Kinoshita T Emi N Naoe T Clinical characteristics and prognostic implications of NPM1 mutations in acute myeloid leukemia.Blood. 2005; 106: 2854-2861Crossref PubMed Scopus (223) Google Scholar Independent of the number of inserted nucleotides, all four new mutant proteins showed a frameshift leading to the same last five amino acid residues (VLSRK). At least one of the tryptophan residues at positions 288 and 290 was involved, and a leucine-rich nuclear export signal was acquired (Figure 2). The last two features probably underlie cytoplasmic localization of the NPM mutant proteins.7Falini B Mecucci C Tiacci E Alcalay M Rosati R Pasqualucci L La Starza R Diverio D Colombo E Santucci A Bigerna B Pacini R Pucciarini A Liso A Vignetti M Fazi P Meani N Pettirossi V Saglio G Mandelli F Lo-Coco F Pelicci PG Martelli MF for the GIMEMA Acute Leukemia Working Party Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.N Engl J Med. 2005; 352: 254-266Crossref PubMed Scopus (1424) Google Scholar'18Bogerd HP Fridell RA Benson RE Hua J Cullen BR Protein sequence requirements for function of the human T-cell leukaemia virus type 1 rex nuclear export signal delineated by a novel in vivo randomization-selection assay.Mol Cell Biol. 1996; 16: 4207-4214Crossref PubMed Scopus (321) Google Scholar'19Nakagawa M Kameoka Y Suzuki R Falini B Mecucci C Martelli MF Nucleophosmin in acute myelogenous leukemia.N Engl J Med. 2005; 352: 1819-1820PubMed Google Scholar Interestingly, in 2 of the 96 cases of NPMc- AMLs, DHPLC detected a different profile to the wild-type. Sequencing showed that the noncoding region of the NPM1 gene was involved in both cases, confirming immunohistochemical results. We are not able to classify these DHPLC profiles, both of which corresponded to a thymidine deletion at position 1146 (NM_002520) of the noncoding sequence. No polymorphisms are known in this region (NCBI 135:5:170746125: 170771092:1), and no material was available in our case for further molecular investigation. Therefore, we consider these cases as false-positive in the detection of leukemogenic mutations. Consequently, DHPLC accuracy in the identification of new leukemic mutations emerges at 98.4%. Further developments with alternative primers will indicate whether this drawback can be eliminated. In conclusion, our results indicate that DHPLC is a highly sensitive, reliable, and rapid diagnostic test that detects NPM1 mutations as well as direct sequencing or immunohistochemistry with an anti-NPM monoclonal antibody. It served to identify four new NPM mutants that add new insights into the heterogeneity of genomic insertions at exon 12. As expected, DHPLC may need to be performed in concert with sequencing to unravel the significance of abnormal chromatograms that certainly indicate nucleotide variations and possibly sporadic new mutations. We thank Dr. Geraldine Boyd for assistance in the preparation of the manuscript. We also thank the Gruppo Italiano Malattie Ematologiche dell' Adulto working party for providing AML samples.
Referência(s)