The discovery of NPM1 mutations in acute myeloid leukemia

TITLE Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.

AUTHORS Falini B, Mecucci C, Tiacci E, et al.

JOURNAL The New England Journal of Medicine. 2005;352(3):254-266. doi: 10.1056/NEJMoa041974.

In 2005 a paper appeared in The New England Journal of Medicine describing cytoplasmic nucleophosmin (NPM1) and NPM1 gene mutations in acute myeloid leukemia (AML) with a normal karyotype.¹ These findings were going to change the way we manage patients with AML. Falini and his colleagues discovered NPM1 mutations in the era before whole sequencing, stemming from immunohistochemical studies with an anti-NPM1 monoclonal antibody on anaplastic large cell lymphoma with t(2;5) that revealed the NPM1-ALK fusion protein to be aberrantly dislocated in the cytoplasm of lymphoma cells.² Further immunohistochemical studies of fixed, paraffin-embedded biopsies from other human tumors showed the expected nuclear expression of NPM1, with the exception of bone marrow biopsies from AML patients who aberrantly expressed NPM1 (a nucleolar located protein) in the cytoplasm of leukemic cells.¹ The authors named these cases NPMc⁺ (cytoplasmic positive) to distinguish them from the NPMc^– (cytoplasmic negative) ones.¹

Notably, ectopic cytoplasmic expression of NPM1 was mainly associated with a de novo origin of the leukemia, normal karyotype (85% of cases), negativity for CD34 and high sensitivity to chemotherapy.¹ Moreover, it was mutually exclusive with the known recurrent translocations defining AML entities, such as PML-RARA, inv(16), t8(;21) and MLL rearrangements.¹ The authors also found that about 30% of NPMc⁺ AML cases carried FLT3-internal tandem duplication, establishing, for the first time, a mechanistic link between the two mutations in promoting AML¹ that has been further confirmed in mouse models.³ These immunohistochemical findings prompted sequencing of the NPM1 gene which led to mutations being found in exon 12. Finally, the authors used transfection techniques to demonstrate a causal relationship between NPM1 mutations, which were predicted to alter the protein at its C-terminal, and the cytoplasmic dislocation of NPM1.¹ This landmark study contributed to unravel the molecular abnormalities underlying AML with a normal karyotype, since NPM1 mutations accounted for 60% of cases with these characteristics. Moreover, the authors predicted that NPM1-mutated AML may represent a new leukemia entity and suggested that “Immunohistochemistry plus mutational analysis of NPM may assist in the monitoring of minimal residual disease in a setting (normal karyotype and CD34 negativity) in which no molecular or immunophenotypic markers are available” and that “Understanding the mechanisms in NPMc+ AML may lead to more specific anti-leukemic therapies”.¹

Remarkably, all the authors’ predictions came true.⁴ Because of its distinctive clinico-pathological features, NPM1-mutated AML is now recognized as a distinct leukemia entity both in the 2022 International Consensus Classification and in the fifth edition of the World Health Organization’s classification of myeloid neoplasms. A search for NPM1 mutations (in combination with other mutations) represents a critical step in the genetic-based risk stratification of AML patients according to the European LeukemiaNet. Evaluation of measurable residual disease by quantitative polymerase chain reaction or next-generation sequencing can serve as a tool to guide therapeutic decisions. Finally, better knowledge of the molecular basis of aberrant transport of mutant NPM1, association of NPMc⁺ with a unique gene expression profile characterized by upregulation of HOX genes, and the activity of NPM1 mutant at the chromatin level have resulted in the development of new promising therapies for NPM1-mutated AML based on XPO1 (CRM1) and menin inhibitors.

Figure 1.Mutations in exon 12 of the nucleophosmin (NPM) gene and in the encoded protein. (A) A schematic representation of the NPM gene as deduced from GenBank sequences NM_002520, NM_199185, and AB042278. Green indicates coding sequences, and yellow 3’ and 5’ untranslated regions. MB: metal-binding domain; Ac: acidic domain; NLS: nuclear localization signal; NAB: nucleic acid-binding domain. Primers for amplification of genomic DNA (NPM1-F and NPM1-R [blue arrowheads]) and complementary DNA (NPM1_25F and NPM1_1112R [red arrowheads]) are shown in their approximate positions above the map. (B) The wild-type NPM sequence (nucleotides 952 through 989) is aligned with six mutant variants, called A to F. Red type indicates nucleotide insertions. The predicted protein is also shown, with boxed areas indicating the positions of the two C-terminal tryptophan (W) residues; the wild-type tryptophan residue is shown in yellow, and the mutated residues are shown in gray. The new amino-acid sequence common to all the mutated proteins is shown in green. For each variant, the number and percentage of affected cases, of the 52 total NPMc⁺ cases, are given. (C) Sequencing results from one patient bearing mutation A, as obtained by direct sequencing (top diagram) and after cloning and sequencing of the two individual alleles (middle [wild-type] and bottom [mutated allele] diagrams). The arrow and the dashed line indicate the position where the two alleles diverge, and the box indicates the most frequently mutated nucleotides. (D) As shown in the images, the mutated NPM protein is dislocated in the cytoplasm. The images are tridimensional reconstructions of confocal micrographs of NIH-3T3 cells transfected with plasmids encoding wild-type and mutant NPM alleles tagged with enhanced green fluorescent protein; the nuclei were counterstained with propidium iodide (which appears as red). The wild-type protein is located in the nucleoli and nuclear membrane, whereas the mutated NPM shows aberrant cytoplasmic localization. Reproduced, with permission, from the paper by Falini et al. in The New England Journal of Medicine.¹

Footnotes

Correspondence

paolo.sportoletti@unipg.it

Disclosures

No conflicts of interest to disclose.