Mutated nucleophosmin detects clonal multilineage involvement in acute myeloid leukemia: impact on WHO classification

Abstract

Because of a lack of specific clonality markers, information on lineage involvement and cell of origin of acute myeloid leukemia with normal karyotype (AML-NK), is missing. Because Nucleophosmin (NPM) gene is frequently mutated in AML-NK and causes aberrant NPM cytoplasmic localization (NPMc ؉ ), it was used as an AML lineage clonality marker. Clonal NPM exon 12 mutations were detected in myeloid, monocytic, erythroid, and megakaryocytic cells but not in fibroblasts or endothelia that were lasermicrodissected from 3 patients with NPMc ؉ AML. Aberrant cytoplasmic expression of mutated NPM proteins was identified with anti-NPM antibodies in 2 or more myeloid hemopoietic cell lineages in 99 (61.5%) of 161 of NPMc ؉ AML paraffin-embedded bone marrow biopsies; lymphoid involvement was excluded in 3 investigated cases. These findings suggest that NPMc ؉ AML derives from either a common myeloid or earlier progenitor. IntroductionClonal cell lineage involvement in myelodysplastic syndrome and acute myeloid leukemia (AML) carrying recurrent genetic abnormalities can be successfully investigated by a number of techniques, including cytogenetics, fluorescence in situ hybridization (FISH), FISH combined with immunophenotyping, and polymerase chain reaction (PCR) on purified cell populations, which are all able to detect leukemia-specific molecular alterations. 1-7 Unfortunately, no specific genetic markers are as yet available for a significant proportion of AML. Under these circumstances, cell lineage clonality can be investigated by analysis of X-chromosome inactivation patterns, 8 but this technique has not been applied widely to AML. Thus, no or scarce information is available on cell lineage involvement or cell of origin in most AMLs, especially those with normal karyotype (AML-NK), 9 which lack specific clonality markers and account for 40% to 50% of de novo adult AML. 10 All genetically poorly defined AMLs, including AML-NK, are now included into the category of "acute myeloid leukemia not otherwise characterized" of the World Health Organization (WHO) classification. 11 This large, poorly characterized subgroup of AML is currently defined according to criteria of the French-AmericanBritish (FAB) classification 12 (with some modification), which is based on morphologic and cytochemical features of the leukemic cells and degree of maturation. Given its frequency and extreme heterogeneity, AML not otherwise characterized clearly needs to be defined better.We recently identified mutations of the NPM gene exon 12 as the most common and specific genetic lesion associated with AML-NK, 13 being observed in 50% to 60% of cases, as confirmed to date in more than 3500 patients with AML. [14][15][16][17][18][19] At the NPM protein C-terminus, these mutations modify critical tryptophan(s) and create a new nuclear export signal (NES) motif, which act together to aberrantly localize NPM leukemic mutants in the cytoplasm 20,21 -hence the term NPMc ϩ (cytoplasmic-positive) AML. 13 As the NPM mutant protein dislocates the NPM wild-t...

Altered nucleophosmin transport in acute myeloid leukaemia with mutated NPM1: molecular basis and clinical implications

“…On the contrary, excess doses of NPM1wt relocated NPM1 leukaemic mutant from cytoplasm to the nucleoli of transfected cells, 27 a pattern never observed in NPM1-mutated AML patients in whom the mutant was always cytoplasmic restricted. 81 This evidence points at a dose-dependent relationship between wild-type and mutated NPM1 proteins, and supports the view that NPM1 leukaemic mutants are 'born to be exported'. 41 …”

“…When stained with these reagents, AML cells harbouring an NPM1 mutation appear positive in the nucleus (which contains a fraction of NPM1wt) and in cytoplasm that contains mutated NPM1 and NPM1wt (recruited by the mutant through heterodimer formation) 45 (Figures 3 and 4). Although polyclonal antibodies recognizing only mutant NPM1 protein 45,68 are very useful for investigating lineage involvement 81 and for diagnosing AML with mutated NPM1 by western blotting, 97 they are of limited value in immunohistochemical detection of cytoplasmic nucleophosmin on paraffin sections 81 because antigenic epitopes are frequently denaturated by fixation/decalcification procedures.…”

Growth and Differentiation Effects of Homer3 on a Leukemia Cell Line

“…Acute myeloid leukemia (AML), a deadly form of hematopoietic malignancies, is a group of heterogeneous diseases with considerable diversity in terms of clinical behavior and prognosis, it is the most common malignant cancer in children and young people (Weltermann et al, 2004;Pasqualucci et al, 2006;Falini et al, 2007;Mills et al, 2009;Burnett et al, 2011). Gene-expression detection technology in the hematopoietic cell of AML patients have been applied in scientific research and clinical testing, in hope of identifying candidate genes which may refer to the development and progression of AML and marker of diagnosis or prognosis (Delaunay et al, 2003;Weltermann et al, 2004;Gonzalez Garcia et al, 2006;Foran, 2010;Flach et al, 2011).…”

A western blot assay for detecting mutant nucleophosmin (NPM1) proteins in acute myeloid leukaemia

“…The present study assessed the value of western blot in identifying NPM1 mutants in cytological AML samples, using rabbit polyclonal antibodies that react with mutated, but not wild-type, NPM1 proteins. 8,9 We applied western blot analysis to leukaemic samples from 213 AML patients at first diagnosis (Table 1). A total of 135 consecutive patients were analyzed prospectively using freshly collected cells (see Supplementary Materials), whereas 78 patients were studied retrospectively using liquid nitrogen snap-frozen dry pellets of bone marrow or peripheral blood mononuclear cells that had been stored for the past 3 years.…”

“…The inclusion criterion was the availability of good quality protein extracts, as defined by the absence of protein degradation when membranes were probed with an anti-NPM wild-type specific antibody (anti-NPM, Clone FC-61991; Invitrogen, Carlsbad, CA, USA). Western blot analysis was performed according to the standard procedure on whole-cell lysate from bone marrow or peripheral blood samples (Supplementary Materials), using two rabbit polyclonal antibodies (Sil-A and Sil-C) 8,9 raised against synthetic peptides corresponding to the C-terminal portion of the NPM1 mutant A (Figure 1a and Supplementary Materials). The specific wild-type NPM monoclonal antibody was used as the positive control.…”