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

Abstract

Mutations of nucleophosmin 1 are frequently found in acute myeloid leukemia and lead to aberrant cytoplasmic accumulation of nucleophosmin protein. Immunohistochemical staining is therefore recommended as the technique of choice in front-line screening. 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 nucleophosmin 1 mutation status in 119 patients with acute myeloid leukemia. Discrepant cases were further characterized by gene expression analyses and fluorescence in situ hybridization. A large overlap between both methods was observed. Nevertheless, nine patients demonstrated discordant results at initial screening. Five cases demonstrated nuclear staining of nucleophosmin 1 by immunohistochemistry, but a nucleophosmin 1 mutation by molecular analysis. In two cases this could be attributed to technical issues and in three cases minor subpopulations of myeloblasts had not been discovered initially. All tested cases exhibited the characteristic nucleophosmin-mutated gene expression pattern. Four cases had cytoplasmic nucleophosmin 1 staining and a nucleophosmin-mutated gene expression pattern without a detectable nucleophosmin 1 mutation. In two of these cases we found the chromosomal translocation t(3;5)(q25;q35) encoding the NPM-MLF1 fusion protein. In the other discrepant cases the aberrant cytoplasmic nucleophosmin staining and gene expression could not be explained. In total six patients (5%) had true discordant results between immunohistochemistry and mutation analysis. We conclude that cytoplasmic nucleophosmin localization is not always caused by a conventional nucleophosmin 1 mutation and that in the screening for nucleophosmin 1 abnormalities, most information will be obtained by combining immunohistochemistry with molecular analysis.A single center analysis of nucleophosmin in acute myeloid leukemia: value of combining immunohistochemistry with molecular mutation analysis

Nucleophosmin mutation analysis in acute myeloid leukaemia

“…However, this assay detects only the NPM1A mutation10, which although is most common (75 to 80% of cases) but is not seen in approximately 20 per cent of the cases10. NPMc+ by IHC besides being caused by the NPM1 A mutation can also be caused by other variants of NPM1 mutations1213. In this patient, more extended exon screening test was required but due to cost constraints this could not be done.…”

“…Two patients positive by PCR assay were not detected by IHC. This could be because mutated NPM1 does not always reveal overt cytoplasmic staining of NPM1 on formalin fixed bone marrow biopsies12. The NPM1c+ detection can be improved by analyzing thin 2-3 μm sections and using B5 fixative instead of formalin12.…”

“…This could be because mutated NPM1 does not always reveal overt cytoplasmic staining of NPM1 on formalin fixed bone marrow biopsies12. The NPM1c+ detection can be improved by analyzing thin 2-3 μm sections and using B5 fixative instead of formalin12. However, the potential toxicity of mercuric chloride containing B5 fixative and waste disposal hazards are major disadvantages.…”

A novel mechanism of NPM1 cytoplasmic localization in acute myeloid leukemia: the recurrent gene fusion NPM1-HAUS1

“…Paulo Vidal Campregher, 1 Welbert de Oliveira Pereira, 2 Bianca Lisboa, 2 Renato Puga, 2 Elvira Deolinda Rodrigues Pereira Velloso, 3 Ricardo Helman, 4 Luciana Cavalheiro Marti, 2 João Carlos Campos Guerra, 5 Kalliopi N. Manola, 6 Roberta Cardoso Petroni, 5 Alanna Mara Pinheiro Sobreira Bezerra, 5 Fernando Ferreira Costa, 7 Nelson Hamerschlak, 4 and Figure 2. NPM1-HAUS1 localizes both in the nucleus and cytoplasm through a Crm1-dependent transport.…”

“…6 While a fraction of these patients harbor the t(3;5)(q25;q35) (NPM1-MLF1), 6,7 the remaining patients have, thus far, unknown operating genomic mechanisms. The identification of such patients and mechanisms is important since this group could clinically and biologically overlap with the entity "AML with mutated NPM1".…”