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Case Reports
. 2021 Nov;35(11):3113-3126.
doi: 10.1038/s41375-021-01222-4. Epub 2021 Apr 20.

Diagnostic and therapeutic pitfalls in NPM1-mutated AML: notes from the field

Brunangelo Falini  1 , Sofia Sciabolacci  2 , Lorenza Falini  2 , Lorenzo Brunetti  2 , Maria Paola Martelli  2
Affiliations
Case Reports

Diagnostic and therapeutic pitfalls in NPM1-mutated AML: notes from the field

Brunangelo Falini et al. Leukemia. 2021 Nov.

Abstract

Mutations of Nucleophosmin (NPM1) are the most common genetic abnormalities in adult acute myeloid leukaemia (AML), accounting for about 30% of cases. NPM1-mutated AML has been recognized as distinct entity in the 2017 World Health Organization (WHO) classification of lympho-haematopoietic neoplasms. WHO criteria allow recognition of this leukaemia entity and its distinction from AML with myelodysplasia-related changes, AML with BCR-ABL1 rearrangement and AML with RUNX1 mutations. Nevertheless, controversial issues include the percentage of blasts required for the diagnosis of NPM1-mutated AML and whether cases of NPM1-mutated myelodysplasia and chronic myelomonocytic leukaemia do exist. Evaluation of NPM1 and FLT3 status represents a major pillar of the European LeukemiaNet (ELN) genetic-based risk stratification model. Moreover, NPM1 mutations are particularly suitable for assessing measurable residual disease (MRD) since they are frequent, stable at relapse and do not drive clonal haematopoiesis. Ideally, combining monitoring of MRD with the ELN prognostication model can help to guide therapeutic decisions. Here, we provide examples of instructive cases of NPM1-mutated AML, in order to provide criteria for the appropriate diagnosis and therapy of this frequent leukaemia entity.

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Conflict of interest statement

BF licensed a patent on NPM1 mutants (n. 102004901256449). BF and MPM declare honoraria from Rasna Therapeutics, Inc for scientific advisor activities. MPM also declares consultancy at scientific advisory board for Abbvie, Amgen, Celgene, Janssen, Novartis, Pfizer, Jazz Pharmaceuticals, and honoraria from Amgen, Celgene, Janssen, Novartis. LB declares consultancy at scientific advisory boards for Abbvie.

Figures

Fig. 1
Fig. 1. NPM1 MRD monitoring by RT-qPCR in case 1.
Monitoring of NPM1mut transcripts during therapy and follow-up (see text). 0.0001%NPM1mut/ABL is equivalent to MRD negativity. 1st CHT, first line chemotherapy; 2nd CHT, salvage chemotherapy; allo-HSCT, allogeneic haematopoietic stem cell transplant.
Fig. 2
Fig. 2. AML with multilineage dysplasia and cell of origin of NPM1-mutated AML.
A Multilineage involvement is documented by the presence of myeloid blasts (single arrow) expressing cytoplasmic NPM1 (blue) and immature erythroid cells (double arrows) expressing cytoplasmic NPM1 (blue) and surface glycophorin (brown) (double staining for NPM1/immune-alkaline phosphatase APAAP technique and glycophorin/ immunoperoxidase, x400). B Another area of BM biopsy showing cytoplasmic NPM1 in dysplastic megakaryocytes (arrow) and myeloid cells (APAAP immunostaining; x400) C Cartoon depicting the putative cell of origin of NPM1-mutated AML. Lighting bolts indicate the suggested putative cells of origin.
Fig. 3
Fig. 3. NPM1/FLT3-D835/DNMT3A triple-mutated AML with skin involvement.
A Skin biopsy showing marked dermal infiltration by leukaemic cells. E indicates the overlying epidermis (Haematoxylin-Eosin, x400). B Leukaemic cells express cytoplasmic NPM1 (single arrow) whilst the cells of the overlying epidermis show nucleus-restricted NPM1 positivity (double arrows) (APAAP immunostaining, x400). E indicates the overlying epidermis. C MRD monitoring by RT-qPCR. 0.0001%NPM1mut/ABL is equivalent to MRD negativity. CHT + FLT3i, chemotherapy plus midostaurin; AZA + VEN, azacytidine plus venetoclax. Arrows indicate the beginning of each cycle of azacytidine plus venetoclax.
Fig. 4
Fig. 4. AML with mutation of NPM1, exon 11.
A Diffuse BM infiltration by leukaemic cells. Dysplastic megakaryocytes are also present (arrow). B Mononucleated blasts and dysplastic megakaryocytes (arrow) express cytoplasmic NPM1 (APAAP immunostaining, x400). C Sanger sequencing of exon 11 showing a heterozygous 8 nucleotides insertion leading to a stop codon at amino acid 275. D Schematic representation of the new mutant protein (Mut) compared with the wild-type (WT). Analysis of the new protein sequence predicted a truncated protein (274 aa length), with a newly acquired NES motif (VxxxFxxLxIx). Both proteins are recognized by the anti-NPM1, Clone 376 mAb: monoclonal antibody.
Fig. 5
Fig. 5. AML with NPM1 mutation and BCR-ABL1 rearrangement.
A G-banding showing a subclone with the following karyotype: 47,XY,t(9;22)(q34;q11),+8 (see the text for the full karyotype). B Sanger trace of NPM1 exon 12, demonstrating mutation B. C Massive BM infiltration by leukaemic cells. T indicates bone trabecula (BM biopsy, haematoxylin-eosin, x400). D Leukaemic cells exhibit aberrant cytoplasmic expression of NPM1 (BM biopsy, APAAP staining, x400). E BM re-evaluation after the first cycle of CHT. Good recovery of normal haematopoietic cells showing nucleus-restricted NPM1 positivity (indicative of NPM1 wild-type). A small cluster of residual leukaemic cells with cytoplasmic NPM1 is seen (arrow) (BM biopsy, APAAP immunostaining, x100). F Higher magnification of the small cluster of NPM1 cytoplasmic leukaemic cells (arrow) shown in Fig. 4E (BM biopsy, APAAP immunostaining, x400).
Fig. 6
Fig. 6. Differential diagnosis of NPM1-mutated AML.
Decisional algorithm for distinguishing NPM1-mutated AML from AML with BCR-ABL1 and AML with RUNX1 mutations (two provisional entities of the 2017 WHO classification of haematopoietic tumors).
Fig. 7
Fig. 7. Decisional algorithm for the molecular diagnosis of NPM1-mutated AML and monitoring of MRD.
Immunohistochemical analysis (IHC) on BM trephine discriminates between AML with nuclear localization of nuclephosmin (predictive of wild-type NPM1 gene), and AML with cytoplasmic staining for NPM1 (predictive of NPM1 mutations). Standard Sanger sequencing of NPM1 exon 12 (involved in almost all cases) allows identification of the specific NPM1 mutations. The discrepancy between IHC (cytoplasmic NPM1) and conventional molecular analysis of exon 12 (absence of NPM1 mutation) should prompt to study the entire NPM1 coding sequence to exclude mutations in other exons. Application of RT-qPCR to monitor MRD to be performed during CHT and at interval of 3 months for at least 2 years after the end of CHT + /− allo-HSCT. NGS has the potential to identify all NPM1 mutations but the commercially available panels should be implemented to include, together with exon 12, at least exon 11, 9 and 5. *Should the entire coding sequence be wild-type, FISH to exclude very rare NPM1 fusions should be considered. RNA sequencing should be also performed in these cases to identify novel NPM1 translocations. NGS can be used also for MRD monitoring. Relapse with no detectable NPM1 mutation points towards a diagnosis of second AML.
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