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

Abstract

© 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...

Molecular Study of NPM1-A (Nucleophosmin1-A) Mutation in Iraqi Adult Acute Myeloid Leukemia Patients; Its Correlation with Clinicopathological Parameters

“…Which was differing from that had been stated by several clinical studies. [12,16,17,18] Population genetics, environmental factors, smaller sample number of enrolled patients and mean age of female was lower than that of males (38.95 vs 47.65 years), or a combination of all might account for the lower frequency of NPM1-A mutation in females.…”

A single center analysis of nucleophosmin in acute myeloid leukemia: value of combining immunohistochemistry with molecular mutation analysis

“…Differences in fixation, decalcification and staining procedures, leading to difficulties in microscopic evaluation, have also been addressed in previously published studies. 1,[7][8][9]11 However, additional studies directly comparing different fixation techniques are necessary to assess the relevance of the fixation technique.…”

“…Still, immunohistochemical staining is recommended as the technique of choice in simple front-line screening, with a reported sensitivity and specificity of 100% on B5-fixed and EDTA-decalcified bone marrow biopsies, and for the diagnosis of AML patients presenting with a "dry tap" or myeloid sarcoma. 10,11 In this study, we assessed the sensitivity and specificity of immunohistochemistry on formalin-fixed bone marrow biopsies compared with gold standard molecular analysis to predict NPM1 mutation status in a large cohort of AML patients from our institution. The cases that were found to have a discrepancy between the two methods were extensively evaluated.…”

Acute myeloid leukemia with mutated nucleophosmin (NPM1): is it a distinct entity?

“…Expression of cytoplasmic NPM was difficult to assess by immunocytochemistry in smears, 72 probably because of artifact diffusion among cell compartments and even outside leukemic cells. 54 More recently, flow cytometry was successfully used to detect nucleophosmin accumulation in leukemic cell cytoplasm 73,74 (Figure 4). This assay could serve as a complementary or even as an alternative procedure to bone marrow biopsy immunohistochemistry, allowing rapid measurement of cytoplasmic NPM1 and correlations with other markers in routine immunophenotyping.…”