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JMD 2006, Vol. 8, No. 2
Copyright © 2006 American Society for Investigative Pathology & Association for Molecular Pathology

Denaturing High-Performance Liquid Chromatography

A Valid Approach for Identifying NPM1 Mutations in Acute Myeloid Leukemia

Giovanni Roti^*, Roberto Rosati^*, Rossella Bonasso, Paolo Gorello^*, Daniela Diverio, Massimo Fabrizio Martelli, Brunangelo Falini, Cristina Mecucci^* for the Gruppo Italiano Malattie Ematologiche dell’ Adulto Working Party

From the Laboratories of Cytogenetics and Molecular Genetics * and Immunohistochemistry, and the Hematology Unit, University of Perugia, Perugia; and the Department of Cellular Biotechnologies and Hematology, La Sapienza University, Rome, Italy

	Abstract

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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 chromatograms, with mutations B and D differing for one nucleotide. Elution profiles and sequencing also identified four new variants. Our results suggest that DHPLC detects NPM1mutations as well as direct sequencing and immunohistochemistry, providing a helpful approach in the diagnosis of NPMc+ AML.

	Introduction

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Acute myeloid leukemia (AML) with normal karyotype is the single largest cytogenetic subgroup, affecting approximately half of adult patients with primary AML.^{1, 2} 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.³

The NPM1 gene is the translocation partner of MLF1 (myeloid leukemia factor 1) in AML with t(3;5), RARA (retinoic acid receptor ) in acute promyelocytic leukemia with t(5;17), and ALK(anaplastic lymphoma kinase) in large anaplastic cell lymphoma with t(2;5).^{4, 5, 6} We recently identified NPM1(nucleophosmin, B23, numatrin) mutations⁷ 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.^{7, 8, 9, 10} 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%.^{11, 12, 13}

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.

	Materials and Methods

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Patients
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).

Antibodies and Immunohistochemistry
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.^{7, 14} 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 and Molecular Analyses
Cytogenetic investigations were performed after short-term culture. Karyotypes were described according to the International System for Human Cytogenetic Nomenclature.¹⁵ Fluorescence in situ hybridization¹⁶ 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.¹⁷

Direct Sequencing
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'-CCTGGACAACATTTATCAAACACGGTA-3').

DHPLC
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').⁷ 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 (poly-styrene-divinylbenzene) particles (DNA-Sep; Transgenomic, 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 WaveMaker 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.

	Results

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NPMc+ AML
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 and Molecular Analyses
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.

DHPLC and Sequencing
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.

View larger version (22K): [in this window] [in a new window]   Figure 1. Chromatograms obtained with DHPLC technology. A: Colored lines show wild-type AML NPMc–. B: Reproducibility of the mutation A chromatograms in nine different cases. C: Mutations B and D with one different nucleotide. Violet, mutation B; green, mutation D. AML, acute myeloid leukemia; NPMc–, no nucleophosmin cytoplasmic delocalization/no NPM1 gene mutations; NPMc+, nucleophosmin cytoplasmic delocalization/NPM1 gene mutations.

View larger version (37K): [in this window] [in a new window]   Figure 2. Nucleotide and protein sequence mutations of nucleophosmin (NPM1). Top rows indicate amino acids in single-letter codes, and bottom rows represent the corresponding codons. The most common site of insertion mutations (position 959) is indicated. Bold red letters are inserted nucleotides; yellow boxes are conserved tryptophan residues; green boxes are nucleolar exporting signal (NES). *New NPM mutations unraveled by DHPLC.

View larger version (30K): [in this window] [in a new window]   Figure 3. New mutations detected by DHPLC technology. Chromatograms were compared to wild-type (broken line) in all cases (A–D) and to known mutations in three of four cases (A–C). A: Dark green line: mutation B; light green line: [ins964_965(CTCT); 965G>T;966G>T; 967A>C; 968G>T; 969G>A]. B: Dark green line: mutation D; black line: [ins963-964(AGGA)]. C: Dark green line: mutation D; light green line: mutation [ins964_965(GCTT); 965G>C; 967>A>C; 968>G>C; 969G>C]. D: Brown line: mutation [ins959_960(GCCA)].

	Discussion

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Only heterozygous NPM1 mutations have been found so far in more than 1839 cases of AML.^{8, 9, 10, 20, 21, 22} 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 reanneal, heteroduplex molecules are generated in heterozygous samples.¹¹ 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 chromatogram 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 colleagues²¹ 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.^{7, 8, 9} 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.^{7, 18, 19}

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.

	Acknowledgments

 
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.

	Footnotes

 
Address reprint requests to Cristina Mecucci, M.D., Ph.D., Laboratory of Cytogenetics and Molecular Genetics/Hematology, Policlinico Monteluce, 06123 Perugia, Italy. E-mail: crimecux{at}unipg.it

Supported in part by the Associazione Italiana per la Ricerca sul Cancro; the Associazione Umbra Leucemie e Linfomi; Associazione Sergio Luciani; Ministero Italiano Università e Ricerca; and the Fondazione Cassa di Risparmio, Perugia, Italy.

Accepted for publication November 7, 2005.

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