Cytoplasmic localization of nucleophosmin in bone marrow blasts of acute myeloid leukemia patients is not completely concordant with NPM1 mutation and is not predictive of prognosis

Abstract

Background NPM1 mutations are reported to predict a favorable prognosis in acute myeloid leukemia (AML) patients. Aberrant cytoplasmic localization of NPM protein is reported be a surrogate for NPM1 gene mutation. Methods Using immunohistochemical analysis (IHC), we assessed for NPM (clone 376) expression in formalin-fixed, formic acid-decalcified bone marrow biopsy specimens. DNA sequencing of the exon 12 of NPM1 gene was performed in 104 patients. Results The study included 252 AML patients: 192 de novo AML, 33 AML following a myelodysplastic syndrome or chronic myelomonocytic leukemia, and 27 therapy-related AML. The median age was 62 years and 115 patients were ≤ 60 years old. All patients received intensive chemotherapy. Cytoplasmic NPM was detected in 59 of 252 (23%) patients, including 48 of 192 (25%) de novo AML and 33 of 94 (35%) with a normal karyotype. DNA sequencing identified NPM1 mutations in 30 of 38 cases with cytoplasmic NPM and 10 of 66 cases with nuclear NPM. Cytoplasmic NPM was associated with young patient age (p=0.024), FLT3/ITD (p=0.005), CD34- negative blasts (p<0.001), high peripheral blood blast counts (p=0.041), and high serum albumin level (p=0.028). No statistical differences in overall or event-free survival were found on the basis of NPM localization. Similar results were obtained in patients ≤ 60 years old with normal karyotype and wild-type FLT3 (p=0.768). Conclusions Immunohistochemical assessment for NPM localization did not predict prognosis in this patient cohort. The discordance between immunohistochemistry and DNA sequencing results indicates that DNA sequencing cannot be replaced by IHC assessment.

The concurrent use of N- and C-terminal antibodies anti-nucleophosmin 1 in immunofluorescence experiments allows for precise assessment of its subcellular localisation in acute myeloid leukaemia patients

“…The pattern of staining was consistent with a prevalently cytoplasmic localisation of the mutant-specific T26 antibody in the OCI-AML3 cells and in bone marrow smears of primary patients' material, whereas the signal of the 23H antibody had a dual nucleolar and cytoplasmic pattern. Moreover, the nucleolar staining of the 23H antibody seems more robust than the staining obtained with previously published murine antibodies as we have never seen any cytoplasmic diffusion in patients without a mutation, formerly reported by Konoplev et al 12 and Mattson et al 13 To this point, the false cytoplasmic localisation of the murine 322 in the OCI-AML2 cells and of the 376 antibody's signal in the lower panel of the two wt NPM1 AML patients has been otherwise not seen with the 23H antibody. Finally, the novel antibody reported here may be of use as a research tool on samples deriving from transgenic mice generated in order to study the NPMc þ driven processes of leukaemogenesis.…”

“…Notably, this distribution pattern is extremely rare in patients' samples, although it has been described in small percent of cells in some of the cases tested with the use of the T26 antibody by immunofluorescence. 9 The reliability of detecting the cytoplasmic localisation of NPM1 as a surrogate marker for the presence of the mutations in diagnostic settings has recently been questioned by Konoplev et al 12 and Mattsson et al 13 As these groups have used 376 and NA24 antibodies previously validated and successfully used by other researchers, 5,6 it appears that the results may be strongly influenced by technicalities related to sample preparation and fixation. Furthermore, data published to date originate from single-colour staining experiments as all of the antibodies available that work well in immunofluorescence are of murine origin, thus making it difficult to design co-staining experiments, necessary for obtaining contemporaneously a precise picture of wt NPM1 and NPMc þ distribution.…”

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

“…7,8 Less reliable results were reported in bone marrow biopsies fixed in formalin and decalcified in formic acid. 71 Preliminary findings from our laboratory suggest discrepancies may result from the decalcifying agent (formic acid) rather than to formalin fixation (B.F., unpublished results, February 2010). 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.…”

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

“…On the other hand, compared to molecular analyses, immunohistochemistry is more prone to inter-observer variability and variability due to technical issues. Indeed, in the study by Konoplev et al 9 immunohistochemical staining was not completely predictive for NPM1 mutations. 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.…”