Nucleophosmin ( NPM1) Mutations in Acute Myeloid Leukemia: An Ongoing (Cytoplasmic) Tale of Dueling Mutations and Duality of Molecular Genetic Testing Methodologies

Abstract

This Commentary highlights two articles that focus on molecular techniques to identify mutations in nucleophosmin (NPM1), which is the most frequently mutated gene in cytogenetically normal acute myeloid leukemia (CN-AML) This Commentary highlights two articles that focus on molecular techniques to identify mutations in nucleophosmin (NPM1), which is the most frequently mutated gene in cytogenetically normal acute myeloid leukemia (CN-AML) Contemporary laboratory evaluation and classification of acute myeloid leukemia (AML) is multimodal, using a diverse array of tools such as morphology, cytochemistry, flow cytometric immunophenotyping, and genetic testing. Genetic testing incorporates both conventional cytogenetic karyotyping and molecular analysis, with the latter including fluorescence in situ hybridization- and polymerase chain reaction (PCR)-based approaches. Of all of these parameters, genetic features are the most pertinent with regard to the biology, classification, prognosis and, ultimately, therapy of AML.1Byrd JC Mrozek K Dodge RK Carroll AJ Edwards CG Arthur DC Pettenati MJ Patil SR Rao KW Watson MS Koduru PR Moore JO Stone RM Mayer RJ Feldman EJ Davey FR Schiffer CA Larson RA Bloomfield CD Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461).Blood. 2002; 100: 4325-4336Crossref PubMed Scopus (1371) Google Scholar2Grimwade 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. The Medical Research Council Adult and Children's Leukaemia Working Parties.Blood. 1998; 92: 2322-2333Crossref PubMed Google Scholar3Slovak ML Kopecky KJ Cassileth PA Harrington DH Theil KS Mohamed A Paietta E Willman CL Head DR Rowe JM Forman SJ Appelbaum FR Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia: a Southwest Oncology Group/Eastern Cooperative Oncology Group Study.Blood. 2000; 96: 4075-4083PubMed Google Scholar4Wheatley K Burnett AK Goldstone AH Gray RG Hann IM Harrison CJ Rees JK Stevens RF Walker H A simple, robust, validated and highly predictive index for the determination of risk-directed therapy in acute myeloid leukaemia derived from the MRC AML 10 trial. United Kingdom Medical Research Council's Adult and Childhood Leukaemia Working Parties.Br J Haematol. 1999; 107: 69-79Crossref PubMed Scopus (365) Google Scholar The importance of genetic changes was, for the first time, codified in the 2001 World Health Organization classification of AML and will assume an even greater role in the update, slated for publication later this year. In the 2001 classification, four recurrent cytogenetic abnormalities have been selected to define specific diagnostic entities. Those AMLs that harbor gene fusions resulting from t(8;21), t(15;17), or inv(16) have a favorable prognosis, whereas tumors with 11q23 translocations tend to be more aggressive, with a poor or occasionally intermediate prognosis, depending on the chromosomal partner. Not only does the karyotype define prognosis, but it determines specific treatment options as well.5Estey E Dohner H Acute myeloid leukaemia.Lancet. 2006; 368: 1894-1907Abstract Full Text Full Text PDF PubMed Scopus (1006) Google Scholar Thus, the decision to use bone marrow transplant as a first line treatment following induction of remission is guided, in large part, by the karyotype of the AML, and the use of targeted therapies, such as all-trans retinoic acid, is essentially restricted to those AMLs with t(15;17). Together, the aforementioned karyotypically detectable lesions are seen in approximately 30% of adult AMLs.2Grimwade 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. The Medical Research Council Adult and Children's Leukaemia Working Parties.Blood. 1998; 92: 2322-2333Crossref PubMed Google Scholar Approximately 30% of AMLs may contain other extremely diverse, but far less frequent, non-random chromosomal abnormalities.2Grimwade 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. The Medical Research Council Adult and Children's Leukaemia Working Parties.Blood. 1998; 92: 2322-2333Crossref PubMed Google Scholar Indeed, over 150 recurrent, and sometimes prognostically relevant, lesions have been described in AML, yet most have not currently been formally incorporated into classification schemes.6Kelly L Clark J Gilliland DG Comprehensive genotypic analysis of leukemia: clinical and therapeutic implications.Curr Opin Oncol. 2002; 14: 10-18Crossref PubMed Scopus (43) Google Scholar However, three additional genetically defined AML categories to be included in the new (2008) World Health Organization classification are AMLs with t(6;9), inv(3) and t(1;22). Although these numerous recurrent karyotypic aberrations are both biologically and clinically significant, approximately 40% of AMLs in adults (and ∼25% of pediatric cases) do not harbor genetic alterations that can be detected by conventional cytogenetics.2Grimwade 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. The Medical Research Council Adult and Children's Leukaemia Working Parties.Blood. 1998; 92: 2322-2333Crossref PubMed Google Scholar Variability in the frequency of these so-called cytogenetically normal (CN) AMLs as reported in the literature is likely due to a number of factors. These include age (more CN AMLs are evident with increasing age), methodology (direct preparation versus 24- or 48-hour culture, with the latter sometimes providing a higher yield of clonal abnormalities), and specimen source (in ∼5% of cases, bone marrow may be superior to peripheral blood in detecting clonal abnormalities). A fourth factor to consider is biological, in that a minor proportion of cases harboring one of the four common translocations/genetic fusions alluded to above may be karyotypically cryptic. Either reverse transcription (RT)-PCR or fluorescence in situ hybridization can be used to uncover these infrequent events, and given the clinical importance of proper diagnosis, they should not be misclassified as CN-AML. In large clinical outcome analyses, CN-AMLs, collectively the most common group of AMLs, are assigned to an intermediate prognosis category.2Grimwade 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. The Medical Research Council Adult and Children's Leukaemia Working Parties.Blood. 1998; 92: 2322-2333Crossref PubMed Google Scholar However, CN-AMLs are clearly not homogeneous, and numerous insights into the diversity present in this group have been unraveled recently.7Mrozek K Bloomfield CD Chromosome aberrations, gene mutations and expression changes, and prognosis in adult acute myeloid leukemia.Hematology Am Soc Hematol Educ Program. 2006; : 169-177Crossref PubMed Scopus (110) Google Scholar Indeed, distinct subsets of CN-AMLs have been recognized to possess genetic or epigenetic alterations in specific genes, as well as (sometimes related) expression profiles, dictating their biological and clinical characteristics. While the entire spectrum of genetic alterations has certainly not been defined, many recurrent genetic abnormalities have been identified and have been demonstrated to be important in both tumorigenesis and the clinical outcome of CN-AMLs. For example, internal tandem duplication (ITD) within FLT3, which leads to ligand-independent activation of this receptor tyrosine kinase, has a significantly adverse effect on clinical outcome.8Small D FLT3 mutations: biology and treatment.Hematology Am Soc Hematol Educ Program. 2006; : 178-184Crossref PubMed Scopus (187) Google Scholar Similarly, partial tandem duplications of MLL (also disrupted in 11q23 translocations, noted above) are associated with a poor prognosis. Conversely, mutations in CEBPA correlate with favorable prognosis. Yet other biologically and clinically relevant genetic lesions are being uncovered, including mutations of RUNX1, KIT, N-RAS, and K-RAS, as well as overexpression of BAALC, ERG, MN1, and EVI1. Although enriched in the group of CN-AMLs, the above lesions are not found exclusively in these cases, and a number of genetic aberrations have been noted to occur simultaneously, with intriguing biological and clinical associations. In brief, most of the aforementioned translocations disrupt transcription factors (such as RUNX1 and RARA in the t(8;21) and t(15;17), respectively), in a dominant-negative fashion, and lead to a block in normal myeloid differentiation (so-called class 2 mutations).9Kelly LM Gilliland DG Genetics of myeloid leukemias.Annu Rev Genomics Hum Genet. 2002; 3: 179-198Crossref PubMed Scopus (418) Google Scholar Experimentally, and also likely clinically, such defects are necessary but not sufficient for the AML phenotype. Only when combined with an additional mutation, typically one having a positive effect on proliferation (a so-called class 1 mutation), does the AML manifest, at least experimentally. In patients, the combination of the two types of mutations typically has prognostically adverse connotations. Thus, prognosis for patients with t(8;21) and an associated KIT mutation, or with t(15;17) and a FLT3-ITD mutation, is typically worse than for patients with the translocations alone.10Callens C Chevret S Cayuela JM Cassinat B Raffoux E de Botton S Thomas X Guerci A Fegueux N Pigneux A Stoppa AM Lamy T Rigal-Huguet F Vekhoff A Meyer-Monard S Ferrand A Sanz M Chomienne C Fenaux P Dombret H Prognostic implication of FLT3 and Ras gene mutations in patients with acute promyelocytic leukemia (APL): a retrospective study from the European APL Group.Leukemia. 2005; 19: 1153-1160Crossref PubMed Scopus (115) Google Scholar,11Lasa A Carricondo MT Carnicer MJ Perea G Aventin A Nomdedeu JF A new D816 c-KIT gene mutation in refractory AML1-ETO leukemia.Haematologica. 2006; 91: 1283-1284PubMed Google Scholar To date, the most frequently identified mutated gene in CN-AML is nucleophosmin (NPM1).12Falini 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 Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.N Engl J Med. 2005; 352: 254-266Crossref PubMed Scopus (1386) Google Scholar Mutations in this gene, which are typically small insertions (usually of 4 bp, sometimes up to 11 bp) in the coding region of the terminal exon (exon 12), occur in ∼50 to 60% of CN-AMLs, equivalent to ∼20 to 25% of all AMLs.12Falini 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 Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.N Engl J Med. 2005; 352: 254-266Crossref PubMed Scopus (1386) Google Scholar The nucleophosmin gene product is an ∼37-kDa protein that actively shuttles between the nucleolus, nucleoplasm, and cytoplasm; however, as its name suggests, it is predominantly found in the nucleolus. The nucleophosmin protein was initially suspected to be involved in promoting cell growth, in part through its mediation of ribosomal biogenesis. Consistent with this hypothesis, NPM1 is overexpressed in a variety of human tumors. Studies have demonstrated that NPM1 might also promote growth inhibition by its functional interactions with the tumor suppressors p14ARF and p53.13Cheng K Grisendi S Clohessy JG Majid S Bernardi R Sportoletti P Pandolfi PP The leukemia-associated cytoplasmic nucleophosmin mutant is an oncogene with paradoxical functions: arf inactivation and induction of cellular senescence.Oncogene. 2007; 26: 7391-7400Crossref PubMed Scopus (44) Google Scholar Mutations in NPM1 found in CN-AML alter tryptophan residues required for proper nucleolar localization and create a putative nuclear export signal at the C terminus of the protein. Consequently, the mutant nucleophosmin protein is predominantly localized to the cytoplasm and, through dimerization, causes the mislocalization of the wild-type protein as well.14Falini B Martelli MP Bolli N Bonasso R Ghia E Pallotta MT Diverio D Nicoletti I Pacini R Tabarrini A Galletti BV Mannucci R Roti G Rosati R Specchia G Liso A Tiacci E Alcalay M Luzi L Volorio S Bernard L Guarini A Amadori S Mandelli F Pane F Lo-Coco F Saglio G Pelicci PG Martelli MF Mecucci C Immunohistochemistry predicts nucleophosmin (NPM) mutations in acute myeloid leukemia.Blood. 2006; 108: 1999-2005Crossref PubMed Scopus (146) Google Scholar This leads to the mislocalization and destabilization of p14ARF and to the inhibition of p53 activity.13Cheng K Grisendi S Clohessy JG Majid S Bernardi R Sportoletti P Pandolfi PP The leukemia-associated cytoplasmic nucleophosmin mutant is an oncogene with paradoxical functions: arf inactivation and induction of cellular senescence.Oncogene. 2007; 26: 7391-7400Crossref PubMed Scopus (44) Google Scholar Thus cytoplasmic nucleophosmin (NPMc) may be oncogenic, a hypothesis that is supported by tissue culture studies.13Cheng K Grisendi S Clohessy JG Majid S Bernardi R Sportoletti P Pandolfi PP The leukemia-associated cytoplasmic nucleophosmin mutant is an oncogene with paradoxical functions: arf inactivation and induction of cellular senescence.Oncogene. 2007; 26: 7391-7400Crossref PubMed Scopus (44) Google Scholar In addition to this interesting pathobiology, the protein mislocalization may be exploited diagnostically since cytoplasmic nucleophosmin can be detected immunohistochemically, and some reports suggest an essentially 100% correlation between immunohistochemical (NPMc) and molecular (NPM1 mutations) findings. Despite this concordance, the terms NPMc and NPM1 mutation signify fundamentally distinct phenomena—with distinct methods of detection—and should probably not, therefore, be used interchangeably. Given the high frequency of NPM1 mutations in CN-AMLs, multiple studies assessing the relationship between these mutations and survival have been performed. One study showed a weak, but statistically significant increase in disease-free and overall survival in patients with an AML that possessed an NPM1 mutation.15Thiede C Koch S Creutzig E Steudel C Illmer T Schaich M Ehninger G Prevalence and prognostic impact of NPM1 mutations in 1485 adult patients with acute myeloid leukemia (AML).Blood. 2006; 107: 4011-4020Crossref PubMed Scopus (572) Google Scholar This benefit, however, is affected by the FLT3 status. FLT3 ITDs are enriched in AMLs with NPM1 mutations, in that they are seen twice as often in this group as compared with AMLs with wild-type NPM1.16Falini B Nicoletti I Martelli MF Mecucci C Acute myeloid leukemia carrying cytoplasmic/mutated nucleophosmin (NPMc+ AML): biologic and clinical features.Blood. 2007; 109: 874-885Crossref PubMed Scopus (424) Google Scholar Numerous studies have demonstrated that patients who lack an NPM1 mutation and possess an FLT3-ITD have a much worse prognosis, as gauged by both disease-free survival and overall survival, than do patients who possess a NPM1 mutation without a concomitant FLT3-ITD.15Thiede C Koch S Creutzig E Steudel C Illmer T Schaich M Ehninger G Prevalence and prognostic impact of NPM1 mutations in 1485 adult patients with acute myeloid leukemia (AML).Blood. 2006; 107: 4011-4020Crossref PubMed Scopus (572) Google Scholar,17Dohner K Schlenk RF Habdank M Scholl C Rucker FG Corbacioglu A Bullinger L Frohling S Dohner H Mutant nucleophosmin (NPM1) predicts favorable prognosis in younger adults with acute myeloid leukemia and normal cytogenetics: interaction with other gene mutations.Blood. 2005; 106: 3740-3746Crossref PubMed Scopus (665) Google Scholar,18Gale RE Green C Allen C Mead AJ Burnett AK Hills RK Linch DC Medical Research Council Adult Leukaemia Working Party The impact of FLT3 internal tandem duplication mutant level, number, size and interaction with NPM1 mutations in a large cohort of young adult patients with acute myeloid leukemia.Blood. 2008; 111: 2776-2784Crossref PubMed Scopus (557) Google Scholar Overall survival of these latter patients approaches that of patients with AMLs that harbor karyotypes correlated with a favorable prognosis, such as t(8;21), t(15;17), or inv(16) and for whom bone marrow transplant may not have survival benefit. In fact, CN-AML patients with mutant NPM1 and wild-type FLT3 may not benefit from bone marrow transplant. It has been proposed that combining the status of these two “dueling” mutations allows for stratification into three prognostic groups.18Gale RE Green C Allen C Mead AJ Burnett AK Hills RK Linch DC Medical Research Council Adult Leukaemia Working Party The impact of FLT3 internal tandem duplication mutant level, number, size and interaction with NPM1 mutations in a large cohort of young adult patients with acute myeloid leukemia.Blood. 2008; 111: 2776-2784Crossref PubMed Scopus (557) Google Scholar,19Gallagher RE Dueling mutations in normal karyotype AML.Blood. 2005; 106: 3681-3682Crossref Scopus (10) Google Scholar Accordingly, patients may be assigned to good (FLT3-ITD−/NPM1+), intermediate (FLT3-ITD−/NPM1− or FLT3-ITD+/NPM1+), and poor (FLT3-ITD+/NPM1−) categories. Detection of a mutant NPM1 allele may also be of clinical benefit in following the treatment course of patients with CN-AML. Longitudinal studies have demonstrated that NPM1 mutations are stable throughout the course of the disease, presumably because they often occur as an early or initiating event in the genesis of the AML and are required for tumor maintenance.15Thiede C Koch S Creutzig E Steudel C Illmer T Schaich M Ehninger G Prevalence and prognostic impact of NPM1 mutations in 1485 adult patients with acute myeloid leukemia (AML).Blood. 2006; 107: 4011-4020Crossref PubMed Scopus (572) Google Scholar,20Palmisano M Grafone T Ottaviani E Testoni N Baccarani M Martinelli G NPM1 mutations are more stable than FLT3 mutations during the course of disease in patients with acute myeloid leukemia.Haematologica. 2007; 92: 1268-1269Crossref PubMed Scopus (47) Google Scholar Thus, an NPM1 mutation, if present, represents a marker that can be used to monitor for minimal residual disease (MRD). It has further been demonstrated that quantitative assessment of these mutations correlates well with responsiveness to initial induction chemotherapy.21Chou WC Tang JL Wu SJ Tsay W Yao M Huang SY Huang KC Chen CY Huang CF Tien HF Clinical implications of minimal residual disease monitoring by quantitative polymerase chain reaction in acute myeloid leukemia patients bearing nucleophosmin (NPM1) mutations.Leukemia. 2007; 21: 998-1004PubMed Google Scholar Monitoring patients by quantitative assessment of NPM1 mutations can predict overt clinical relapse. Accordingly, these data suggest that NPM1 mutation detection may have clinical and treatment ramifications not only at initial diagnosis but also subsequently for monitoring remission and predicting relapse. Nascent molecular discoveries and their demonstrated associated clinical relevance are characteristically and predictably followed by a flurry of publications detailing methodologies that might be used in a molecular diagnostic laboratory. These laboratories are then charged with determining the most appropriate assay to adopt. Such is the case for NPM1, and in this regard, two papers in this issue of The Journal of Molecular Diagnostics are of note. The study by Szankasi and colleagues22Szankasi P Jama M Bahler DW A new DNA-based test for detection of nucleophosmin exon 12 mutations by capillary electrophoresis.J Mol Diagn. 2008; 10: 236-243Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar details a non-quantitative, genomic DNA-based PCR assay for detecting NPM1 mutations. This assay can be used on paraffin-embedded tissue and can detect mutations when the leukemic cells represent 5% of the population. Since the assay is performed using genomic DNA as a template, intronic primers are used to avoid the amplification of the known pseudogenes. Moreover, since small-length nucleotide insertions distinguish wild-type and mutant alleles, a polymerase with editing capabilities is used. By contrast, the methodology described by Ottone et al23Ottone T Ammatuna E Lavorgna S Noguera NI Buccisano F Venditti A Gianni L Postorino M Federici G Amadori S Lo Coco F An allele-specific RT-PCR assay to detect type A mutation of the nucleophosmin-1 gene in acute myeloid leukemia.J Mol Diagn. 2008; 10: 212-216Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar begins with an RNA template, employs an RT-PCR protocol with and without semi-nesting, and uses allele-specific oligonucleotide (ASO) primers. The assay only detects the most common NPM1 A mutation, which represents 75 to 80% of mutated cases and consists of a tetranucleotide TCTG insertion in exon 12. Although the latter protocol does not detect the full range of NPM1 mutations, it can detect mutant clones that represent as little as 0.001% of the population. The greater lability of the RNA template in this assay is a potential shortcoming; however, since the molecular assays to detect translocations in AML require RNA, this ought not to be a major issue for molecular diagnostic laboratories. Although both articles in this issue present viable methods for detection of NPM1 mutations, a comparison of the two methodologies highlights recurring issues surrounding both mutational testing in general and NPM1 in particular. The genomic DNA-based method detects all known NPM1 mutations, yet the diagnostic utility of the test is limited to cases in which leukemic cell proportion represents at least 5% of the assayed population. While this relatively low analytic sensitivity is not a limitation at diagnosis, since by definition there will be >20% blasts, it diminishes the utility of the assay for MRD detection. The RNA-ASO-based method has superior analytic sensitivity. However, this advantage comes at a price, in that diagnostic sensitivity is restricted to detection of one specific mutation and bypasses other NPM1 mutations that are seen in 20 to 25% of positive cases. The trade-off evident in these two different methodologies is thus quite striking and is practically inherent in the detection of multiple mutant alleles: a gain in the range of mutation detection is associated with a sacrifice in the sensitivity of detecting a specific allele. Such factors are clearly germane to deciding which assay to institute in a molecular diagnostic laboratory, which in turn is dictated by the specific clinical questions that are being asked. If the primary goal of NPM1 testing is to stratify patients at diagnosis, a qualitative PCR of NPM1 exon 12 from genomic DNA followed by separation of products by capillary electrophoresis (similar to the assay described by Szankasi et al22Szankasi P Jama M Bahler DW A new DNA-based test for detection of nucleophosmin exon 12 mutations by capillary electrophoresis.J Mol Diagn. 2008; 10: 236-243Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) may be instituted. Alternatively, if the major goal of testing is MRD detection in a subset of NPM1-mutated AMLs, an ASO-PCR or RT-RCR assay (similar to that described by Ottone et al23Ottone T Ammatuna E Lavorgna S Noguera NI Buccisano F Venditti A Gianni L Postorino M Federici G Amadori S Lo Coco F An allele-specific RT-PCR assay to detect type A mutation of the nucleophosmin-1 gene in acute myeloid leukemia.J Mol Diagn. 2008; 10: 212-216Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), is preferred. If both allele detection at diagnosis and MRD detection are desirable, a combination of methods is required. In this scenario, both assays could be performed at diagnosis to classify patients, and MRD testing would only be performed if the allele-specific assay were positive. Alternatively, individual patient/mutation-specific ASO assays could be developed, although this may be prohibitively expensive. The methods described in the reports in this issue of the JMD, and highlighted in this commentary, are among numerous other assays that have been reported for NPM1 mutation detection. Table 1 outlines some of the key features of various different assays, which may help determine which assay is most applicable to a particular laboratory. Ultimately, the decision as to which assay to use is likely predicated on a number of factors, including i) what is the clinical question and ii) what material is available.Table 1Reported Methodologies for Detecting NPM1 MutationsTechnologySubstrateDiagnostic sensitivity (%)Analytic sensitivity (%)ReferencesPCR and capillary electrophoresis*Assay may be performed on paraffin-embedded tissue.DNA∼100∼522Szankasi P Jama M Bahler DW A new DNA-based test for detection of nucleophosmin exon 12 mutations by capillary electrophoresis.J Mol Diagn. 2008; 10: 236-243Abstract Full Text Full Text PDF PubMed Scopus (34) Google ScholarASO-Nested PCR and capillary electrophoresisRNA∼75–80∼0.00123Ottone T Ammatuna E Lavorgna S Noguera NI Buccisano F Venditti A Gianni L Postorino M Federici G Amadori S Lo Coco F An allele-specific RT-PCR assay to detect type A mutation of the nucleophosmin-1 gene in acute myeloid leukemia.J Mol Diagn. 2008; 10: 212-216Abstract Full Text Full Text PDF PubMed Scopus (39) Google ScholarPCR and melting curve analysis/FRETDNA∼900.0124Scholl S Mugge LO Landt O Loncarevic IF Kunert C Clement JH Hoffken K Rapid screening and sensitive detection of NPM1 (nucleophosmin) exon 12 mutations in acute myeloid leukaemia.Leuk Res. 2007; 31: 1205-1211Abstract Full Text Full Text PDF PubMed Scopus (6) Google ScholarPCR/SequencingRNA∼98–100NR12Falini 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 Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.N Engl J Med. 2005; 352: 254-266Crossref PubMed Scopus (1386) Google Scholar25Roti G Rosati R Bonasso R Gorello P Diverio D Martelli MF Falini B Mecucci C Denaturing high-performance liquid chromatography: a valid approach for identifying NPM1 mutations in acute myeloid leukemia.J Mol Diagn. 2006; 8: 254-259Abstract Full Text Full Text PDF PubMed Scopus (32) Google ScholarPCR/SequencingDNA∼100NR15Thiede C Koch S Creutzig E Steudel C Illmer T Schaich M Ehninger G Prevalence and prognostic impact of NPM1 mutations in 1485 adult patients with acute myeloid leukemia (AML).Blood. 2006; 107: 4011-4020Crossref PubMed Scopus (572) Google Scholar17Dohner K Schlenk RF Habdank M Scholl C Rucker FG Corbacioglu A Bullinger L Frohling S Dohner H Mutant nucleophosmin (NPM1) predicts favorable prognosis in younger adults with acute myeloid leukemia and normal cytogenetics: interaction with other gene mutations.Blood. 2005; 106: 3740-3746Crossref PubMed Scopus (665) Google ScholarPCR/dHPLCDNA∼100NR25Roti G Rosati R Bonasso R Gorello P Diverio D Martelli MF Falini B Mecucci C Denaturing high-performance liquid chromatography: a valid approach for identifying NPM1 mutations in acute myeloid leukemia.J Mol Diagn. 2006; 8: 254-259Abstract Full Text Full Text PDF PubMed Scopus (32) Google ScholarLNA-mediated PCR clampingDNA∼1000.1–126Thiede C Creutzig E Illmer T Schaich M Heise V Ehninger G Landt O Rapid and sensitive typing of NPM1 mutations using LNA-mediated PCR clamping.Leukemia. 2006; 20: 1897-1899Crossref PubMed Scopus (33) Google ScholarQuantitative PCRDNA>950.01–0.127Gorello P Cazzaniga G Alberti F Dell'Oro MG Gottardi E Specchia G Roti G Rosati R Martelli MF Diverio D Lo Coco F Biondi A Saglio G Mecucci C Falini B Quantitative assessment of minimal residual disease in acute myeloid leukemia carrying nucleophosmin (NPM1) gene mutations.Leukemia. 2006; 20: 1103-1108Crossref PubMed Scopus (227) Google ScholarQuantitative RT-PCRRNA∼900.0127Gorello P Cazzaniga G Alberti F Dell'Oro MG Gottardi E Specchia G Roti G Rosati R Martelli MF Diverio D Lo Coco F Biondi A Saglio G Mecucci C Falini B Quantitative assessment of minimal residual disease in acute myeloid leukemia carrying nucleophosmin (NPM1) gene mutations.Leukemia. 2006; 20: 1103-1108Crossref PubMed Scopus (227) Google ScholarImmunohistochemistry*Assay may be performed on paraffin-embedded tissue.Protein∼100NR14Falini B Martelli MP Bolli N Bonasso R Ghia E Pallotta MT Diverio D Nicoletti I Pacini R Tabarrini A Galletti BV Mannucci R Roti G Rosati R Specchia G Liso A Tiacci E Alcalay M Luzi L Volorio S Bernard L Guarini A Amadori S Mandelli F Pane F Lo-Coco F Saglio G Pelicci PG Martelli MF Mecucci C Immunohistochemistry predicts nucleophosmin (NPM) mutations in acute myeloid leukemia.Blood. 2006; 108: 1999-2005Crossref PubMed Scopus (146) Google ScholarFRET, fluorescence resonance energy transfer; dHPLC, denaturing high-performance liquid chromatography; LNA, locked nucleic acid; NR, not reported (for sequencing of other mutations, this is typically 15 to 25%).* Assay may be performed on paraffin-embedded tissue. Open table in a new tab FRET, fluorescence resonance energy transfer; dHPLC, denaturing high-performance liquid chromatography; LNA, locked nucleic acid; NR, not reported (for sequencing of other mutations, this is typically 15 to 25%). Real-time quantitative PCR provides a highly sensitive approach that is well suited for MRD detection, evaluation of response to therapy, and prediction of overt clinical relapse.21Chou WC Tang JL Wu SJ Tsay W Yao M Huang SY Huang KC Chen CY Huang CF Tien HF Clinical implications of minimal residual disease monitoring by quantitative polymerase chain reaction in acute myeloid leukemia patients bearing nucleophosmin (NPM1) mutations.Leukemia. 2007; 21: 998-1004PubMed Google Scholar Unfortunately, real-time quantitative PCR approaches may be limited by their requirement for allele-specific primers or probes, with an attendant increase in cost. Furthermore, given the remarkable sensitivity of real-time quantitative PCR, and the need to compare product levels from discrete time points (and possibly different laboratories), extensive standardization and quality control protocols will need to be established, analogous to the recent efforts to harmonize testing for BCR-ABL1 levels in patients with chronic myelogenous leukemia.28Hughes T Deininger M Hochhaus A Branford S Radich J Kaeda J Baccarani M Cortes J Cross NC Druker BJ Gabert J Grimwade D Hehlmann R Kamel-Reid S Lipton JH Longtine J Martinelli G Saglio G Soverini S Stock W Goldman JM Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results.Blood. 2006; 108: 28-37Crossref PubMed Scopus (994) Google Scholar As noted, the clinical impact of NPM1 mutations is affected by the mutational status of the FLT3 gene. Indeed, NPM1 mutation analysis is of little or no value in the absence of FLT3 testing and the two should always be performed together. Thus, clinical laboratories establishing an NPM1 assay may wish to attempt to multiplex this assay with an FLT3-ITD assay. Detection by capillary electrophoreses of FLT3-ITD mutations and NPM1 mutations in a single assay has been reported.29Noguera NI Ammatuna E Zangrilli D Lavorgna S Divona M Buccisano F Amadori S Mecucci C Falini B Lo-Coco F Simultaneous detection of NPM1 and FLT3-ITD mutations by capillary electrophoresis in acute myeloid leukemia.Leukemia. 2005; 19: 1479-1482Crossref PubMed Scopus (90) Google Scholar We have previously used a multiplex approach, with gel-based detection, to identify recurrent translocations in acute leukemia.30Salto-Tellez M Shelat SG Benoit B Rennert H Carroll M Leonard DG Nowell P Bagg A Multiplex RT-PCR for the detection of leukemia-associated translocations: validation and application to routine molecular diagnostic practice.J Mol Diagn. 2003; 5: 231-236Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar However, multiplex mutational testing may be particularly well suited to the use of a Luminex-based systems using X-MAP technology, as has been adopted at our institution to identify these recurrent translocations. Theoretically, if PCR and hybridization conditions were similar to those used for detection of recurrent translocations, identification of both recurrent karyotypic abnormalities and these intragenic mutations could be distilled into a single (RNA-based) assay. Additional clinically relevant mutations, such as those seen in CEBPA, KIT, and others, may also be included to increase significantly and comprehensively the prognostic yield. Indeed, using this bead-based technology, the number of discrete mutations that can be detected in a single assay is vast. Thus, the ultimate goal of detecting all clinically relevant mutations for AML—the “mutationome”—in a single reaction is theoretically feasible. Although the two articles in this issue of JMD, as well as multiple other studies, focusing on NPM1 have used molecular techniques to identify mutations, the cytoplasmic mislocalization of the mutant protein allows for the use of immunohistochemistry to determine NPM1 status.12Falini 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 Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.N Engl J Med. 2005; 352: 254-266Crossref PubMed Scopus (1386) Google Scholar Using immunohistochemistry to detect underlying mutations or dysregulated gene expression is certainly not without precedent in hematopathology, with the detection of BCL2 in follicular lymphoma (in contrast to follicular hyperplasia), CyclinD1 in most mantle cell lymphoma, and ALK in a subset of anaplastic large cell lymphoma being central to diagnosis of these lymphomas. Accordingly, in laboratories that lack molecular diagnostic capabilities but can perform adequate immunohistochemistry, this method may be a viable alternative to direct analysis of the NPM1 gene. However, interpretation of NPM1 localization may be difficult, especially in blasts that lack abundant cytoplasm. Moreover, NPM1 immunohistochemistry is unlikely to be useful for MRD detection, and NPM1 localization is only relevant with known FLT3 mutational status, which is only obtained through molecular characterization. Thus, it appears currently appropriate to recommend nucleic acid-based assays rather than immunohistochemical approaches for the detection of NPM1 mutations. Despite NPM1 being only one of many genes that are frequently mutated in AML, the high occurrence of mutation in CN-AML, the correlation with prognosis, the temporal stability of the mutation, and the possible impact the mutation may have on treatment options strongly argue that testing for NPM1 mutations should be routinely performed in molecular laboratories. However, the existence of multiple NPM1 mutant alleles complicates testing strategies, which currently ensures that a single testing methodology is not ideal for all clinical situations. As such, to institute the appropriate test or tests, laboratory directors must not only be familiar with the benefits and limitations of the various approaches (the duality of methodologies), but more importantly, must also be cognizant of specific and contextual (dueling mutations) clinical questions that are being asked, and what the clinical consequences of the answers are going to be. An Allele-Specific RT-PCR Assay to Detect Type A Mutation of the Nucleophosmin-1 Gene in Acute Myeloid LeukemiaThe Journal of Molecular DiagnosticsVol. 10Issue 3PreviewNucleophosmin-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. Full-Text PDF A New DNA-Based Test for Detection of Nucleophosmin Exon 12 Mutations by Capillary ElectrophoresisThe Journal of Molecular DiagnosticsVol. 10Issue 3PreviewMutations in nucleophosmin (NPM1) exon 12 are thought to be the most common genetic event in acute myelogenous leukemia (AML) and to confer favorable clinical prognoses. In this report, we describe a simple molecular test for the detection of NPM1 exon 12 mutations in patients with AML using polymerase chain reaction amplification of genomic DNA followed by the analysis of amplification products by capillary electrophoresis. Mutations were reproducibly detected when present in at least 5% of cells, and all NPM1 exon 12 mutations reported to date in AML could be identified using this method. Full-Text PDF