Can cytoplasmic nucleophosmin be detected by immunocytochemical staining of cell smears in acute myeloid leukemia?

Mattsson, Göran; Turner, Sue; Cordell, Jacqueline; Ferguson, David J. P.; Schuh, Anna; Grimwade, Lizz F.; Bench, Anthony J.; Weinberg, Olga K.; Marafioti, Teresa; George, Tracy I.; Arber, Daniel A.; Erber, Wendy N.; Mason, David Y.

doi:10.3324/haematol.2009.011817

Cited by 14 publications

(13 citation statements)

References 24 publications

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“…This is conducted by direct microscopic examination of AML cytospins immunostained with NPM. Under microscopy, the morphological appearance of cells will help track NPM during mitosis [5]. Immunoblotting is another laboratory technique that allows studying NPM [6], it is a procedure that helps not only to identify an antigen but also to determine its MW, quantity and possible protein interactions.…”

Section: Introductionmentioning

confidence: 99%

Role of Nucleophosmin Gene Mutation in Leukemogenesis of Acute Myeloid Leukemia

et al. 2018

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Background: Acute myeloid leukemia (AML) is a hematopoietic stem cell disorder that carries very poor prognosis. Understanding molecular basis of AML leukemogenesis could lead to the emergence of effective targeted therapies for AML. AML bearing nucleophosmin (NPM) gene mutation has distinct features. This study was conducted to investigate the role of mutated (m) NPM in pathogenesis of de novo AML through studying its contribution in proliferation of AML cell line cells.

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Section: Introductionmentioning

confidence: 99%

Role of Nucleophosmin Gene Mutation in Leukemogenesis of Acute Myeloid Leukemia

et al. 2018

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“…17 As bone marrow trephines are not routinely performed in AML patients in all hematologic centers, the ability to predict NPM1 mutations from cytoplasmic expression of nucleophosmin in smears or cytospins would be particularly useful. In the present issue of Haematologica, Mattsson et al 43 found no significant correlation between sub-cellular expression of nucleophosmin and NPM1 gene status in their immunocytochemical study of smears and cytospins from 60 AMLs (31 NPM1-mutated; 29 NPM1 wild-type). Why cytoplasmic nucleophosmin is detected in fixed paraffin-embedded material but not cytological samples remains unclear.…”

Section: © F E R R a T A S T O R T I F O U N D A T I O Nmentioning

confidence: 99%

“…Mattsson et al's 43 claim that cytoplasmic nucleophosmin in paraffin sections may represent non-specific staining appears unfounded because there is much strong evidence indicating that in NPM1-mutated AML immunohistochemistry on paraffin sections depicts the real status of nucleophosmin sub-cellular distribution. In fact, aberrant cytoplasmic expression of nucleophosmin in B5 fixed/EDTA decalcified bone marrow trephines is fully predictive of NPM1 mutations.…”

Section: © F E R R a T A S T O R T I F O U N D A T I O Nmentioning

confidence: 99%

“…30 Finally, laser confocal microscopy of transfected cells clearly showed that fluorescent-tagged wild-type and mutated NPM1 proteins localized respectively in the nucleolar and cytoplasmic cell compartments. 4,38 Whatever the reason for the failure to detect cytoplasmic nucleophosmin in smears or cytospins, the comprehensive study by Mattsson et al 43 clearly indicates this method cannot be at present recommended as surrogate marker for NPM1 mutations. One promising approach based on the use of intracellular flow cytometry for rapid, specific detection of nucleophosmin in the cytoplasm of leukemic cells has been recently proposed as an alternative to bone marrow trephines in the diagnosis of NPM1-mutated AML.…”

Section: T a S T O R T I F O U N D A T I O Nmentioning

confidence: 99%

“…In the present issue of Haematologica, Mattsson et al 43 found no significant correlation between sub-cellular expression of nucleophosmin and NPM1 gene status in their immunocytochemical study of smears and cytospins from 60 AMLs (31 NPM1-mutated; 29 NPM1 wild-type). Why cytoplasmic nucleophosmin is detected in fixed paraffin-embedded material but not cytological samples remains unclear.…”

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Molecular and alternative methods for diagnosis of acute myeloid leukemia with mutated NPM1: flexibility may help

Falini¹,

Martelli²,

Pileri³

et al. 2010

Haematologica

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© F e r r a t a S t o r t i F o u n d a t i o nseparate provisional entity in the 2008 World Health Organization (WHO) classification of myeloid neoplasms, under the heading of "AML with recurrent genetic abnormalities". 7 In the 2001 WHO classification, this category included only AML with t(15;17), t(8;21), inv(16), and MLL rearrangements and in 2008 it was expanded to include AMLs carrying t(6;9), inv(3) or t(3;3), AML (megakaryoblastic) with t(1;22) and two provisional entities, i.e. AML with mutated NPM1 and AML with mutated CEBPA. Thus, in the 2008 WHO classification, the category of "AML with recurrent genetic abnormalities" covers about 60% of AML.The identification of the specific genetic alteration underlying each of the AML subtypes listed in the category of "AML with recurrent genetic abnormalities" is of critical importance since it helps to assign patients to different prognostic groups, thus influencing therapy. For example, in AML patients under 60 years of age, NPM1 mutations consistently predict favorable prognosis, 6 when no concomitant FLT3-ITD mutation is present, whilst in AML patients 70 years old or over NPM1 mutations appear to be the only factor influencing prognosis in multivariate analysis. 8 The search for genetic alterations using molecular methods would ideally be the gold standard for diagnosis. Unfortunately, not all centers, especially in developing countries, are equipped for molecular studies and this may hamper the worldwide use of the WHO classification. These problems could potentially be solved by developing simple surrogates for molecular studies. Here, we briefly review the molecular methods and their alternatives that are currently available for diagnosing AML with mutated NPM1. Detection of NPM1 mutations by molecular techniquesThe normal NPM1 gene configuration and the first six NPM1 mutations we identified in AML, 2 which lead to common structural changes of the NPM1 protein C-terminus, are shown in Figure 1. Over the past five years, several qualitative and quantitative molecular assays for identifying NPM1 mutations have been developed and tested in a large number of AML patients.(i) Qualitative assays for NPM1 mutations: these highly sensitive and specific assays for detecting NPM1 mutations 9-15 are best applied to RNA or DNA extracted from fresh bone marrow or peripheral blood leukemic cells 13 but plasma 16 and paraffin-embedded samples 17 are also suitable. More than 50 molecular variants of NPM1 mutations have been identified to date, 18 mostly involving exon-12, and occasionally other exons.19 NPM1 mutations occur in about 30% of adult AML 4 (50-60% of AML with normal karyotype). NPM1 mutation A (a duplication of TCTG at position 956 to 959 of the reference sequence) accounts for 75-80% of cases.2 Mutation B and D account for about 10% and 5% of cases, respectively; other mutations are very rare. NPM1 mutations are less frequent in childhood (about 8% of pediatric AML) 20,21 and have been never found in children under three years of age. 21Pediatric an...

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Detection of cytoplasmic nucleophosmin expression by imaging flow cytometry

et al. 2012

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Mutations within the nucleophosmin NPM1 gene occur in approximately one-third of cases of acute myeloid leukemia (AML). These mutations result in cytoplasmic accumulation of the mutant NPM protein. NPM1 mutations are currently detected by molecular methods. Using samples from 37 AML patients, we investigated whether imaging flow cytometry could be a viable alternative to this current technique. Bone marrow/peripheral blood cells were stained with anti-NPM antibody and DRAQ5 nuclear stain, and data were acquired on an ImageStream imaging flow cytometer (Amnis Corp., Seattle, USA). Using the similarity feature for data analysis, we demonstrated that this technique could successfully identify cases of AML with a NPM1 mutation based on cytoplasmic NPM protein staining (at similarity threshold of 1.1 sensitivity 88% and specificity 90%). Combining data of mean fluorescence intensity and % dissimilar staining in a 0-2 scoring system further improved the sensitivity (100%). Imaging flow cytometry has the potential to be included as part of a standard flow cytometry antibody panel to identify potential NPM1 mutations as part of diagnosis and minimal residual disease monitoring. Imaging flow cytometry is an exciting technology that has many possible applications in the diagnosis of hematological malignancies, including the potential to integrate modalities. ' 2012 International Society for Advancement of Cytometry Key termsimaging flow cytometry; acute leukemia; nucleophosmin; hematology NUCLEOPHOSMIN (NPM) is both a nuclear (predominantly nucleolar) and cytoplasmic protein that functions as a molecular chaperone (1). It has many functions including the prevention of protein aggregation in the nucleolus and regulation of p53 levels (2). Mutations within the C-terminal region (exon 12) of the NPM1 gene occur in approximately one-third of cases of de novo acute myeloid leukemia (AML) (3,4) leading to increased nuclear export and aberrant cytoplasmic accumulation of the mutant NPM protein (5). A number of methods have been assessed to detect cytoplasmic NPM (cNPM) in clinical specimens. Immunohistochemistry has been reported to be reliable, especially on B5-fixed bone marrow trephines, with close correlation between cNPM and the presence of mutated NPM1 (6). Immunocytochemistry of air-dried blood and bone marrow smears can also detect cNPM in myeloblasts but is insufficiently reliable for diagnostic use (7). Fluorescent confocal microscopy and standard flow cytometry have both been evaluated for cNPM detection (8,9). Standard flow cytometry cannot be used to assess the localization of molecules within specific cellular compartments, but two alternative approaches have been assessed. The first, using antibodies to mutant NPM1, was able to identify NPM1-mutated AML cases exclusively with or without the addition of a de novo NPM ''control'' antibody (8,9). The second method specifically addressed mean fluorescence intensity (MFI) and showed higher MFI in NPM1 mutated than wild-type AML cases (10). However, discrepancie...

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Can cytoplasmic nucleophosmin be detected by immunocytochemical staining of cell smears in acute myeloid leukemia?

Cited by 14 publications

References 24 publications

Role of Nucleophosmin Gene Mutation in Leukemogenesis of Acute Myeloid Leukemia

Role of Nucleophosmin Gene Mutation in Leukemogenesis of Acute Myeloid Leukemia

Molecular and alternative methods for diagnosis of acute myeloid leukemia with mutated NPM1: flexibility may help

Detection of cytoplasmic nucleophosmin expression by imaging flow cytometry

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