Immunohistochemistry predicts nucleophosmin (NPM) mutations in acute myeloid leukemia

Abstract

IntroductionAcute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease. Cytogenetic and/or molecular studies are used to assign 30% to 40% of AML cases carrying specific genetic lesions (eg, t(15;17), t(8;21) or Inv(16)) to different prognostic subgroups in order to monitor minimal residual disease and to select patients who could benefit from targeted therapies. 1 However, they cannot be applied to 40% to 50% of patients with AML who at conventional cytogenetics exhibit a normal karyotype. 1,2 We recently found exon-12 nucleophosmin (NPM) gene mutations in approximately 60% of AMLs with normal karyotype (about one third of all adult AML) that are characterized by distinct morphologic, phenotypic, and molecular features, 3 as well as distinct gene expression profile signature. 4 NPM mutations predict good response to induction therapy and favorable prognosis, 5-8 and could serve to monitor minimal residual disease. 9 Thus, analysis of NPM mutations emerges as a major new step in the diagnostic/prognostic work-up of patients with AML with a normal karyotype.Reverse-transcriptase-polymerase chain reaction (RT-PCR), mutational analysis, or gene expression profiling is usually used for detecting genetic abnormalities or specifically overexpressed genes with diagnostic/prognostic significance. However, demand is growing for simple, inexpensive, and specific immunologic tests 10 that can serve as a surrogate to, or used in integration with, molecular studies. Examples include immunohistochemical detection of proteins such as anaplastic lymphoma kinase (ALK) in CD30 ϩ anaplastic large cell lymphoma, 10 Zap-70 in chronic lymphocytic leukemia, 11 annexin were involved in biochemical studies; I.N. and R.M. were involved in the confocal microscopic analysis of cells; R.P., A.T., and E.T. were involved in the immunohistochemical study of AML samples; C.M. was involved in the identification of NPM mutations; R.B., D.D., G.R., R.R., M.A., L.L., S.V., L.B., E.G., A.G., G.S., F.P., F.L.-C., and P.-G.P. were involved in the analysis of NPM gene mutations in the GIMEMA-EORTC trial; and F.M., S.A., G.S., and M.F.M. were involved in organizing the clinical trial.M.P.M. and N.B. contributed equally to this work.Reprints: Brunangelo Falini, Institute of Hematology, Policlinico Monteluce, 06122 Perugia, Italy; e-mail: faliniem@unipg.it.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 U.S.C. section 1734. For personal use only. on April 8, 2019. by guest www.bloodjournal.org From A1 in hairy cell leukemia, 12 myeloid leukemia factor-1 (MLF1) in myelodysplasia/AML with t(3;5), 13 and promyelocytic leukemia (PML) in acute promyelocytic leukemia carrying the t(15;17). 14 In a small group of patients with AML, we showed that aberrant cytoplasmic expression of NPM, a multifunctional 15-20 nucleocytoplasmic shuttling protein 21 with restricted nucleolar localization, 10...

Clinicopathologic analysis of acute myeloid leukemia arising from chronic myelomonocytic leukemia

“…34,35 Although there were too few cases to attempt formal statistical comparisons regarding the NPM1 or FLT3 gene mutations, survival of the acute myeloid leukemia ex chronic myelomonocytic leukemia cases containing the NPM1 mutation were generally favorable compared with the other patients. This finding, as well as the rapid progression of NPM1-mutated chronic myelomonocytic leukemia patients to acute myeloid leukemia in our series and in another published series, 24 suggests that cases diagnosed as chronic myelomonocytic leukemia with an NPM1 mutation may actually represent early de novo acute myeloid leukemia exhibiting dysplastic features and monocytosis mimicking chronic myelomonocytic leukemia. 36 It may thus be useful to screen chronic myelomonocytic leukemia cases for NPM1 mutation and carefully follow cases with NPM1 mutation for possible rapid transformation to 'bona fide' acute myeloid leukemia.…”

“…17 NPM1 and FLT3 Mutation Analysis NPM1 and FLT3 mutation status was obtained from available molecular diagnostic reports of acute myeloid leukemia, myelodysplastic syndrome, and chronic myelomonocytic leukemia cases. For the chronic myelomonocytic leukemia and acute myeloid leukemia ex chronic myelomonocytic leukemia cases with available paraffin-embedded material, NPM1 immunohistochemical staining was performed on deparaffinized bone marrow trephine sections 24 using the FLEX monoclonal mouse anti-human nucleophosmin antibody, Clone 376 (ready-to-use; Dako North America, Carpinteria, CA, USA) incubated overnight at 4 1C after antigen retrieval. The secondary antibody Mouse HRP (incubated for 15 min at room temperature) and the DAB Chromogen from Dako kit (K4006) were used.…”

“…The presence of cytoplasmic NPM1 staining was considered indicative of an NPM1 mutation. 24 Nuclear C23 expression (C23 mouse monoclonal antibody; 1:60 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as a control for all cases showing cytoplasmic NPM1 expression.…”

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

“…For example, we have found that in human bone marrow, early progenitors, such as proerythroblasts or promyelocytes, express much more nuclear nucleophosmin than terminally differentiated cells, such as normoblasts or neutrophils. The usual positivity of NPM1wt in paraffin-embedded samples is 'nucleus-restricted, diffuse' rather than nucleolar, 26,45 reflecting the limits of immunohistochemical staining of paraffin sections in detecting subnuclear structures, such as nucleoli or nuclear bodies. 46 In normal human tissues, immunohistochemistry also identifies a small percentage of cells that express nucleophosmin in cytoplasm in the absence of NPM1 mutations.…”

Nucleophosmin mutations in acute myeloid leukemia: A tale of protein unfolding and mislocalization

“…29 This is the hallmark of this kind of leukemia that can be detected by immunohistochemistry. 30 Because of its distinctive features, AML with NPM1 mutations has been included as a provisional distinct entity in the 2008 WHO classification of myeloid neoplasms. 31 The observation that AML-associated NPM1 mutations lead to the replacement of one or both of Trp288 and Trp290, and consequently alter the hydrophobic core of the C-terminal domain, led investigators to try to correlate the folding mechanism of the protein (wild-type versus the mutated one) with the altered cellular localization.…”