Nucleophosmin (NPM) is an ubiquitously expressed multifunctional protein.1 Although NPM is known to shuttle back and forth between the nucleus and the cytoplasm,2 only a minimum amount of the protein is present at any time in the cytoplasm; this explains why with immunohistochemistry NPM is only detectable at the nucleolar level.3 We previously showed that NPM is aberrantly expressed in the cytoplasm of anaplastic large cell lymphoma cells carrying t(2;5), due to the presence of the NPM-ALK fusion protein.3 Ectopic NPM expression parallels that of ALK protein and serves as a highly specific diagnostic test on routine biopsies.3
The t(3;5)(q25;q35) is a rare chromosomal translocation which is detected in myelodysplastic syndromes and acute myeloid leukaemia (AML),4 more frequently of FAB-M6 type. Similarly to the t(2;5) in lymphomas, it generates a chimeric gene named NPM-myelodysplasia/myeloid leukaemia factor 1 (NPM-MLF1)5 which encodes for a fusion protein containing the N-terminal portion of NPM (amino acids 1–175) linked to almost the entire MLF1 protein as only the initial 16 amino acids at the N-terminus are excluded.
We used specific antibodies against NPM3 and a newly generated specific monoclonal antibody against MLF1 (Figure 1a) (Supplementary Information), an evolutionary conserved protein whose functions and expression are poorly understood,5, 6 to study the subcellular expression of wild-type NPM and MLF1, and the chimeric NPM-MLF1 proteins in four AML patients carrying the t(3;5)/NPM-MLF1 rearrangement.
In all cases, positivity for MLF1 was found predominantly in the nucleus (mainly the nucleoli) (Figure 1b, top panel, right) or in the nucleus and the cytoplasm (Figure 1b, middle panels), confirming previous report in a single pediatric patient.5 Notably, ectopic nuclear MLF1 positivity was specific of AML carrying the t(3;5), since it was not detected in normal haemopoietic cells (Figure 1b, top and middle panels) and in acute myeloid (n=90) and lymphoid (n=30) leukaemias without t(3;5) (Supplementary Information) used as control (not shown). MLF1 nuclear positivity reflects the presence of the NPM-MLF1 protein in the nucleus.5 The NPM-MLF1 fusion protein probably manages to enter the nucleus because the portion of NPM it retains (amino acids 1–175) contains one of the two NPM nuclear localization signals that target the chimera to the nucleus.5 Hetero-dimerization of NPM-MLF1 with the wild-type NPM also may contribute to import the NPM-MLF1 fusion protein to the nucleus, as occurs with NPM-ALK in lymphomas with t(2;5).3 The cytoplasmic labelling seen with the anti-MLF1 antibody is entirely due to the NPM-MLF1 protein, since the wild-type MLF1 is not expressed in leukaemic cells with t(3;5)5 (Figure 1a).
When primary leukaemic cells with t(3;5) were labelled with the antibody against the N-terminus of NPM (retained in the NPM-MLF1 fusion protein), cytoplasmic (in addition to nuclear) positivity of NPM was revealed (Figure 1b, bottom panel, left). In contrast, expression of nucleolin/C23 (the most abundant nucleolar protein beside NPM) was nucleus-restricted (Figure 1b, bottom panel, right). These findings are consistent with the presence of the NPM-MLF1 fusion protein in the cytoplasm of leukaemic cells.
However, these experiments do not provide any information whether the NPM wild-type is also present in the cytoplasm since the anti-NPM N-terminus antibodies cannot discriminate between the NPM-MLF1 and wild-type NPM proteins.3 To address this issue, we immunostained marrow paraffin sections from our cases with two antibodies against the NPM C-terminus: one generated in our laboratory3 and the other (directed against an epitope lying within the 68 amino acids at the C-terminus of NPM) obtained from Sigma-Aldrich (Milan, Italy), both recognizing wild-type NPM but not NPM-MLF1 protein. Both antibodies detected the NPM C-terminus in the cytoplasm of AML cells with t(3;5) (Figure 2, middle panels). This staining pattern clearly differed from the nucleus-restricted NPM C-terminus expression which is found in NPM-ALK positive lymphomas (Figure 2, top panels). However, it was identical to cytoplasmic NPM C-terminus expression in AML carrying a mutated NPM protein7 (Figure 2, bottom panels). These findings could indicate that the cytoplasm of AML cells with t(3;5) contains a mutated NPM protein, or a reciprocal MLF1-NPM fusion protein or even the wild-type NPM. In the two cases, we investigated (Supplementary Information), lack of mutations in the exon-12 of the NPM gene coding region precludes leukaemic cells from carrying a mutated NPM protein. It is unlikely that MLF1-NPM reciprocal protein accounts for cytoplasmic NPM positivity since MLF1-NPM transcripts are not detectable in leukaemic cells with t(3;5)5 (Figure 1a, lane 7). However, due to the lack of fresh material for Western blotting studies, we cannot exclude with certainty that our cases contained in the cytoplasm a MLF1-NPM reciprocal fusion protein.
We favour the hypothesis that leukaemic cells with the t(3;5) may express ectopic cytoplasmic wild-type NPM. To determine whether the NPM-MLF1 fusion protein caused localization of wild-type NPM in the cytoplasm, we tranfected COS cells with pMT2-NPM-MLF1 and wild-type NPM-green fluorescent protein (pEGFP) constructs7 and immunostained them with the anti-MLF1 antibody (Supplementary Information). No perturbation in the subcellular expression of wild-type NPM was observed in transfected cells (not shown). Thus, the mechanism underlying the putative cytoplasmic dislocation of wild-type NPM remains to be clarified.
Trilineage displasia has been previously described in AML with t(3;5).4 To investigate whether the NPM-MLF1 fusion protein was present in different haemopoietic cell lineages, we immunostained two t(3;5)-AMLs with FAB-M6 morphology (Figure 3a). The nuclear plus cytoplasmic MLF1 staining which is typical of AML with t(3;5) was observed both in myeloid blasts and erythroid precursors (Figure 3b). Double immunoenzymatic staining for MLF1 and glycophorin confirmed this finding (Figure 3c). Both erythoid and myeloid populations also showed aberrant cytoplasmic expression of wild-type NPM (not shown). Among erythoid precursors, proerythroblasts were the cells showing the strongest aberrant expression of MLF1 and NPM. Both erythroid and myeloid populations were CD34-negative (not shown). Our immunohistochemical findings suggest that pathogenesis of AML with t(3;5) may involve at least a committed myeloid stem cell. Interestingly, HLS7, the murine gene counterpart to human MLF1, influences erythroid/myeloid lineage switching and development of normal haemopoietic cells.8
Aberrant cytoplasmic expression of NPM and involvement of several haemopoietic cell lineages are also distinctive features of NPMc+ AML which is associated with normal karyotype.7 Despite these similarities, AML with t(3;5) and NPMc+ AML have several different features. Sequencing of NPM in leukaemic cells with t(3;5) (Supplementary Information) did not show mutations at exon-12 of NPM (or in any other part of its coding sequence). Clinically, NPMc+ AML accounts for about one-third of adult AML with the highest peak in the fifth decade7 and is usually associated with a de novo origin and favourable prognosis.9 In contrast, AML bearing t(3;5) (0.25% of all adult AML, aged 15–60 years, in the ongoing GIMEMA LAM99P/AML12 EORT trial), mainly occurs in young adults as a rapidly evolving myelodysplastic syndrome or as a de novo AML with dysplasia; it is usually associated with a poor prognosis.4 In fact, three of the four patients in our series died of their disease. The other, has been in complete clinical remission for more than three years after HLA-identical allogeneic peripheral blood stem cell transplantation.
The exact mechanism(s) through which the NPM-MLF1 protein induces malignant transformation remain unknown. Since MLF1 is not usually expressed in normal haemopoietic tissues,5, 6 NPM-MLF1 is hypothesized to contribute, at least in part, to the genesis of myelodysplasia/AML by promoting ectopic expression of MLF1 in haemopoietic cells.5, 6 Interestingly, MLF1 prevents erythroleukaemic cells from undergoing biological and morphological maturation in response to erythropoietin,10 possibly through interaction with the recently identified MLF1IP protein.11 This may also explain why AML with t(3;5) is most frequently of the FAB-M6 type. Besides, the oncogenic potential of NPM-MLF1, NPM haploinsufficiency caused by only one allele coding for wild-type NPM could play a role in leukaemogenesis. Notably, in a knockout mouse model,1 NPM is a key regulator of haematopoiesis (especially erythropoiesis). Moreover, NPM+/- heterozygous mice develop a haematological disorder with features resembling human myelodysplastic syndromes.1
In conclusion, antibodies against MLF1 and NPM are valuable tools for the biological study of AML with t(3;5). Moreover, the immunohistochemical test is rapid and inexpensive and could serve as a diagnostic surrogate for molecular studies, especially in less privileged countries.
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This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC) and MURST. Supported in part by NCI Grant CA 76301 and CA 69179 (SWM), NCI Cancer Center CORE grant CA 21765 and by the American Lebanese Syrian Associated Charities (ALSAC), S. Jude Children's Research Hospital. We thank Dr Xiaoli Cui for the expert technical assistance.
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