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Simultaneous occurrence of acute myeloid leukaemia with mutated nucleophosmin (NPM1) in the same family

Leukemia volume 23, pages 199203 (2009) | Download Citation

Acute myeloid leukaemia (AML) with mutated nucleophosmin (NPM1), accounting for about 30% of adult de novo AML1 and 7% of childhood AML,2 displays distinctive biological and clinical characteristics.3 We report for the first time on the clinical, pathological and molecular features of NPM1-mutated AML which developed almost simultaneously (at an interval of only 2 months) in a father and his daughter, and discuss the biological significance of this finding.

The father (SG) presented in April 2006, at the age of 33 years, because of fever. Laboratory examination revealed anaemia (8.5 g per 100 ml), moderate thrombocytopenia (74 000 per mm3), a white blood cell (WBC) count of 9820 per mm3 and a few blast cells in the peripheral blood smear. Bone marrow aspirate and trephine showed diffuse infiltration by blast cells that accounted for about 80% of the bone marrow population. Morphology (Figures 1a and b), immunophenotype of leukaemic cells, that is expression of myeloid (myeloperoxidase, CD33) and monocyte markers (macrophage-restricted CD68, CD14), were diagnostic of acute myelomonocytic leukaemia, according to the WHO (World Health Organization) classification. Leukaemic cells were CD34-negative (Figure 1c). Cytogenetics showed a normal karyotype (46, XY). Initially, no molecular studies for mutations of FLT3, NPM1 and CEBPA genes were performed. The patient was treated according to the GIMEMA (Gruppo Italiano Malattie Ematologiche dell’ Adulto) LAM 99-P protocol (Supplementary Materials). Bone marrow examinations carried out after induction and consolidation therapy showed complete remission (CR). The patient underwent allogeneic peripheral blood stem cell (PBSC) transplantation from his 40-year-old HLA-identical brother (Supplementary Materials). Currently (June 2008), the patient is in CR.

Figure 1
Figure 1

Bone marrow at initial diagnosis of AML (acute myeloid leukaemia, father). (a) Blasts with myelomonocytic appearance in bone marrow smear (May–Grunwald–Giemsa; × 1000). (b) The myelomonocytic blasts show irregular nuclei in a paraffin section from a trephine biopsy (hematoxylin–eosin; × 800). (c) Leukaemic cells are CD34 negative; the arrow indicates a CD34-positive vessel. (d) Immunostaining with anti-NPM monoclonal antibody reveals aberrant expression of nucleophosmin in the cytoplasm of the leukaemic cells (single arrow); residual normal hemopoietic cells show nucleus-restricted NPM positivity. (c and d) Alkaline phosphatase anti-alkaline phosphatase (APAAP) technique; paraffin-embedded sections from bone marrow trephine; hematoxylin counterstaining ( × 800).

His daughter (SV), aged 7 years, presented in June 2006, because routine blood tests showed Hb 8.9 g per 100 ml, platelets 195 000 per mm3, and a WBC count of 46 920 per mm3 with 90% blasts. The morphological features (Figure 2a) and positivity for myeloperoxidase of a fraction of blast cells (not shown) indicated an AML without maturation, according to the WHO classification. Leukaemic cells were CD34 negative (Figure 2b). Cytogenetics showed a normal karyotype (46, XX). Molecular studies revealed an NPM1 gene mutation of type B (insertion of 4 bp CATG after nucleotide 1018 of the reference sequence, GenBank accession number NM_002520), which is one of the most common NPM1 mutation variants in childhood AML.4 Further studies also revealed an internal tandem duplication of the FLT3 gene (FLT3-ITD). The patient was treated according to the high-risk arm of the Associazione Italiana Ematologia Oncologia Pediatrica protocol LAM 2002/01 (Supplementary Materials). Complete haematological remission was followed by autologous PBSC transplantation (December 2006; Supplementary Materials). Bone marrow biopsy (February 2007) showed about 15% leukaemic cells. Rescue therapy was immediately started according to the I-BFM-SG protocol but only the first FLAG (fludarabine, cytarabine, granulocyte-colony stimulating factor) cycle was administered because of severe neutropenia. In April 2007, bone marrow biopsy showed massive infiltration by leukaemic cells exhibiting the original morphological and phenotypic characteristics (myeloperoxidase positivity, CD34 negativity). In May 2007, the child underwent a haploidentical PBSC transplant from her mother at the Division of Pediatric Oncology and Haematology, and Haematopoietic Stem Cell transplantation Unit of the University of Perugia, Italy (Supplementary Materials). A CR was sustained from June to November 2007. As bone marrow biopsy in December 2007 indicated AML had relapsed, the patient was treated with a donor lymphocyte infusion (5 × 105 per kg). In February 2008, bone marrow biopsy showed massive leukaemic infiltration. Molecular analyses confirmed the presence of NPM1 and FLT3-ITD mutations. In March 2008, the child received unmanipulated bone marrow stem cells from the same donor after conditioning with treosulfan, thiotepa and clofarabine. She developed grade II acute graft-versus-host disease and achieved a CR with a full-donor type chimerism.

Finding that the daughter's leukaemic cells harboured a NPM1 mutation and both father's and daughter's showed normal karyotype and CD34 negativity (which are distinguishing features of AML with mutated NPM11), prompted us to retrospectively investigate the father's leukaemic cells, which had been frozen at diagnosis of AML in April 2006. NPM1 gene sequencing revealed a 4 bp (TCTG) insertion after nucleotide 1018 of the reference sequence (GenBank accession number NM_002520). This corresponds to NPM1 mutation A,1 the most common type of NPM1 mutation in adult AML, which is found in 75–80% of cases.3 The father's leukaemic cells, unlike his daughter's, carried no FLT3-ITD mutation. In conclusion, the daughter's AML cells were NPM1 mutated (type B) and FLT3-ITD positive whereas her father's were NPM1 mutated (type A) and harboured two FLT3 gene alleles in germline configuration. These different molecular profiles of leukaemic cells in father and daughter suggested that NPM1 mutations were not inherited.

Immunostaining with a mouse monoclonal anti-NPM antibody of bone marrow at diagnosis (in father and daughter) and relapse (daughter) showed aberrant nucleophosmin expression in the cytoplasm of leukaemic cells (Figures 1d and 2c). This expression pattern is predictive of a mutated NPM1 gene.5 In contrast, normal residual cells (haemopoietic precursors, vessel endothelial cells, fibroblasts and osteoblasts) showed a nucleus-restricted nucleophosmin expression (Figures 1d, 2c and d) which is predictive of NPM1 gene in germline configuration.5 Nuclear NPM positivity was also found in all normal cells during CR (not shown). Immunohistochemical results were confirmed by molecular studies that detected an NPM1 mutation in leukaemic cells but not in bone marrow mononuclear cells taken during CR (Figure 3). None of the other family members we studied (I,1—I,2—II,1—III,2) showed NPM1 or FLT3-ITD mutations (Figure 3), proving the NPM1 mutation was somatic in origin in father and daughter. Thus, the mutation pattern in our patients is quite different from what is observed in familial leukaemia associated with heterozygous mutations of genes encoding the runt-related transcription factor 1 (RUNX1) or CEBPA, both of which are of germline type and linked to familial susceptibility to AML.6, 7

Figure 2
Figure 2

Bone marrow at first relapse of AML (acute myeloid leukaemia, daughter). (a) Diffuse marrow infiltration by myeloid blasts without maturation (paraffin section from bone marrow trephine; hematoxylin–eosin; × 800). (b) Leukaemic cells are CD34 negative; the arrow indicates a CD34-positive vessel. (c and d) Immunostaining with anti-NPM monoclonal antibody reveals aberrant expression of nucleophosmin in the cytoplasm of leukaemic cells (single arrows); normal endothelial cells in a vessel (c; double arrows) and residual normal haemopoietic cells, including macrophage (d; double arrows) show nucleus-restricted NPM positivity. (c and d) Alkaline phosphatase anti-alkaline phosphatase (APAAP) technique; paraffin-embedded sections from bone marrow trephine; hematoxylin counterstaining ( × 800).

Figure 3
Figure 3

NPM1 mutation detection in family members. Exon 12 and flanking intron sequences of the NPM1 gene were screened for mutations by DHPLC (Wave System; MD Transgenomic Inc., Omaha, NE, USA) and direct sequencing (data not shown) in all family members, including diagnostic and remission samples of the affected patients, II,2 and III,1. Electropherograms from patients’ samples were compared with normal sequenced controls. Chromatograms obtained with DHPLC technology are shown beside each family member. Sequencing results are indicated on the bottom of each chromatogram. Nucleotide numbers are referred to the GenBank NPM1 wild-type sequence NM_002520.

To further clarify the significance of our findings, we used the Affymetrix GeneChip 250K SNP (single-nucleotide polymorphism) mapping array technology and CNAG (v3) software8 to perform an high-throughput and genome-wide profiling of chromosomal alterations and loss of heterozygosity (LOH) events in several family members. In the father's cells we observed an altered status of chromosome 3p (II,2), which had not been detected by routine cytogenetics. A hemizygous deletion from 3p14.1 to 3p12.3 (from 67 525 645 to 79 347 258 bp of Human May 2004 Assembly, 11.8 Mb in length) was found at diagnosis but not at remission (Figure 4), which suggested it was a tumour-specific alteration. Moreover, a large LOH region, spanning from 3p23 to 3p22.1 (from 32 328 557 to 43 502 256 bp, 11 Mb in length), was present at diagnosis and maintained during remission (Figure 4). This germline LOH event was associated with combined deletion of one allele and duplication of the other, thus preserving a diploid copy number status (defined as copy number neutral LOH). Stretches of homozygosity have been found through the genome of normal individuals.9 These traits of homozygosity can originate from uniparental dysomy, but most probably they represent autozygosity traits found in region of broad linkage disequilibrium and low frequency of recombination, due to presence of a common ancestor and consanguinity. These regions might include genes, which could be affected by mutations, or they could contain loci related to disease predisposing events. None of the other family members presented this peculiar combination of chromosome 3 alterations. No recurrent-specific DNA aberrations that might suggest familial predisposing genetic factors were identified. Moreover, there was no history of leukaemias or other malignant diseases in the family tree.

Figure 4
Figure 4

Map of DNA alterations on chromosome 3 in father's cells at diagnosis and remission. DNA from father's cells at diagnosis (II,2 diagnosis) and remission (II,2 remission) were analysed on Affymetrix GeneChip 250K Nsp SNP Mapping Arrays. Whole-genome DNA profiles were assembled using Copy Number Analyzer for Affymetrix GeneChip (CNAG, v3) software and comparing each sample to a pool of normal controls (48 HapMap samples available on Affymetrix website). Chromosome 3 map is shown from p to q end (from right to left) in both samples. The upper two graphs represent single SNP copy number signals on log scale (red dots) and total copy number values averaged on adjacent 10 SNPs (blue lines), whereas copy number values for each allele (red and green lines) are shown below. Green and pink bars in the middle represent heterozygous genotype calls and homozygous calls between each father's sample and normal controls, respectively. The two bars at the bottom represent the colour-coded visualisation of total copy number status (yellow, diploidy; pink, amplification) and LOH (loss of heterozygosity; blue, significant LOH; yellow, no LOH). Deletion at 3p14.1–12.3 (from 67 525 645 to 79 347 258 bp, according to UCSC Genome Browser, Human Assembly May 2004) observed in II,2-diagnostic sample and CNN-LOH at 3p23–22.1 (from 32 328 557 to 43 502 256 bp) observed both in II,2-diagnosis and II,2-remission samples are shown.

The apparent absence of genetic predisposing factors concurs with the observation that the NPM1 mutation seems to be a founder genetic lesion rather than a secondary event, as it is specific for AML,1, 10 does not usually associate with secondary AML,1, 11 is mutually exclusive of other recurrent genetic abnormalities (except for rare cases bearing NPM1 and CEPBA mutations),12 is stable over the course of the disease13 and is associated with distinctive gene expression profile3 and micro-RNA signature.14 However, although the SNP array did not identify genetic predisposing factors, we cannot exclude that our patients carried leukaemia susceptibility genes whose alterations could not be detected by SNP arrays (as in case of balanced translocations, inversions or point mutations) or that might have been affected by epigenetic changes.

An alternative hypothesis of the almost simultaneous occurrence of AML in father and daughter is exposure to a common environmental agent. Investigation into family occupations and workplace revealed that father and mother managed a cafeteria within a gasoline station for a period of about 10 years, where their daughter used to spend time almost every day after school. As exposure to benzene is related to higher risk of leukaemia,15 it could be speculated that low but continuous exposure to benzene from gasoline might have played a role in the development of AML in our patients. On the other hand, although extremely improbable, disease onset in father and daughter might just have been coincidental. Thus, despite extensive investigation, the almost simultaneous occurrence of AML with mutated NPM1 in the two family members remains unexplained.

Father and daughter both underwent allogeneic PBSC transplantation but with different outcomes. The father's favourable outcome was expected as leukaemic cells carried an NPM1 mutation in the absence of FLT3-ITD.3 Assessment of minimal residual disease by quantitative PCR after allogeneic transplant revealed no NPM1 mutant copies, which is indicative of complete molecular remission and may be predictive of long-term survival.16 In contrast, AML in the daughter was characterized by an initial response to therapy followed by early relapses, even after haploidentical PBSC transplantation. Her unfavourable prognosis was probably due to co-existence of an FLT3-ITD (documented at diagnosis and during relapses), which is known to counteract the favourable prognostic impact of NPM1 mutations.3

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Acknowledgements

Supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC), MIUR PRIN and Fondazione Cariplo (to GC and AB). LLN was supported by IBISCUS. This work was supported by MIUR (Ministero Università e Ricerca) through a FIRB grant RBLA03ER38 and funds to Interdisciplinary Center for Biomolecular Studies and Industrial Applications (CISI) and Department of Biomedical Sciences and Technologies, University of Milan, Italy. We thank Dr GA Boyd for editorial assistance. B Falini has applied for a patent on the clinical use of NPM mutants.

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Author notes

    • G Cazzaniga
    •  & L Lo Nigro

    These authors contributed equally to this work.

Affiliations

  1. Centro Ricerca Tettamanti, Clinica Pediatrica, Ospedale San Gerardo, Università di Milano-Bicocca, Monza, Italy

    • G Cazzaniga
    •  & A Biondi
  2. Center of Pediatric Haematology Oncology, University of Catania, Catania, Italy

    • L Lo Nigro
    •  & E Mirabile
  3. Institute of Biomedical Technologies (ITB), National Research Council (CNR), Milan, Italy

    • I Cifola
  4. Institute of Haematology, Ospedale Ferrarotto, University of Catania, Catania, Italy

    • G Milone
    •  & F Di Raimondo
  5. MLL Munich Leukemia Laboratory, Munich, Germany

    • S Schnittger
    •  & T Haferlach
  6. Ospedale Cannizzaro, Catania, Italy

    • F Costantino
  7. Section of Haematology and Immunology, IBit Foundation, University of Perugia, Perugia, Italy

    • M P Martelli
    • , E Mastrodicasa
    • , F Aversa
    •  & B Falini

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Correspondence to B Falini.

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https://doi.org/10.1038/leu.2008.170

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

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