Nucleophosmin mutation in Southeast Asian acute myeloid leukemia: eight novel variants, FLT3 coexistence and prognostic impact of NPM1/FLT3 mutations

Abstract

NPM1 mutations were investigated in 400 Southeast Asian leukemia patients and were detectable in 105 cases (26.25%) of acute myeloid leukemia but in no cases of acute lymphoid leukemia or chronic myeloid leukemia. Eight novel and 5 known mutations were identified. All predicted novel proteins shared the last five amino acids VSLRK with the similar gain of nuclear exporting signal motif as known variants. Older age, high white blood cell and platelet counts, normal cytogenetics, and CD34-negativity were associated with NPM1 mutation. FLT3 mutation was more frequent in mutant NPM1 than wild-type cases (56.8% vs. 25.6%) whereas RAS and AML1 mutations were rarely found. Overall survival analysis based on the NPM1/FLT3 mutational status revealed a better outcome for the NPM1-positive/FLT3–negative subgroup. We conclude that: i) NPM1 mutation represents a common genetic hallmark in Southeast Asian acute myeloid leukemia with a normal karyotype; ii) NPM1 mutants coexisted mainly with FLT3 mutants, but not RAS or AML1; iii) FLT3 mutation had a negative prognostic impact on patients with mutant NPM1.

Introduction

Acute myeloid leukemia (AML) is a heterogeneous disease with distinct biological and prognostic characteristics.1 Various clinical subsets associated with unique chromosomal translocations have been identified in AML and appear to be associated with specific prognostic outcome depending on the underlying chromosome abnormalities. Three cytogenetic risk groups, i.e. favorable, intermediate and unfavorable, are well recognized. The intermediate risk group is a heterogeneous group of patients lacking any specific karyotypic abnormalities the majority of whom have a normal karyotype. Nucleophosmin is a member of the nucleophosmin/nucleoplasmin (NPM) family of nuclear chaperones whose members have been found throughout the animal kingdom.2 The NPM family has a diverse function in cellular processes such as genome stability, ribosome biogenesis, DNA duplication and transcriptional regulation.3 NPM1 is the most studied member of the NPM family as it plays an important role as a versatile partner in many chromosomal translocations.4 Frameshift mutation at exon 12 of the NPM1 gene is an alternative leukemogenetic mechanism rather than chromosomal translocations that was recently discovered in 2005.5–7 NPM1 mutation is now recognized as one of the most frequent mutations in AML patients with a normal karyotype in European countries.8–10 The aim of this study was to evaluate the prevalence and type of NPM1 gene mutations in Southeast Asian adult AML patients and to investigate the associated biological and clinical characteristics.

Design and Methods

Leukemia samples

Consecutive leukemia samples were obtained from 400 de novo AML, 30 acute lymphoid leukemia (ALL), and 30 chronic myeloid leukemia (CML) patients between 2000 and 2005. This study was part of a large leukemia project previously approved by the Ethical Committee for Human Research, Faculty of Medicine, Siriraj Hospital, Mahidol University. Morphological, immunophenotypic and chromosome analyses were performed according to the standard methods and karyotypes described according to the International System for Cytogenetic Nomenclature.11,12 FLT3 internal tandem duplication (ITD), RAS and AML1 mutations were analyzed according to our previously described methods.13–15

Table 1.Chromatograms of NPM1 wild type and NPM1 mutants.

Identification of NPM1 mutation

Mononuclear cells were isolated from the leukemia samples by Ficoll-Hypaque density-gradient centrifugation and genomic DNA extracted by the standard phenol-chloroform method. The polymerase chain reaction (PCR) amplification of the NPM1 gene exon 12 fragment was performed with the following oligonucleotide primers: forward (NPM11-F) (5′-ACCA-CATTTCTTTTTTTTTTTTTCCAGGCT-3′) and reverse (NPM12-R) (5′-CCTGGACAACATTTAT-CAAACACGGTA-3′). The total reaction volume of 25 μL contained 50 ng of genomic DNA, 2.5 mmol/L MgCl2, 0.4 mM dNTPs, 0.4 μM of each primers, 1 U of Taq DNA polymerase, and supplied buffer (Platinum Taq DNA Polymerase, Invitrogen, Brazil).

PCR amplification was performed under the following steps: initial denaturation at 95°C for 2 mins., 35 cycles at 94°C for one min., 61°C for 30 secs. and 72°C for 30 secs. A final elongation proceeded at 72°C for 2 mins. Denaturing high performance liquid chromatography (DHPLC) analysis was carried out using the WAVE Nucleic Acid Fragment Analysis System 3500HT with DNASep HT cartridge technology (Transgenomic Inc, Omaha, NE, USA).

The optimized condition and temperature was predicted by the Navigator™ software. Approximately 5–10 μL of the crude PCR products were injected onto the DHPLC system and eluted under an optimized temperature at 55.5°C. DNA fragments were analyzed for changes in the chromatographic peak pattern. PCR products showing a change in the DHPLC pattern were identified as containing potential mutations and reamplified for sequencing analysis. The sequences were compared to the wildtype NPM1 cDNA (GenBank Accession number, NM_002520).

Results and Discussion

Characterization of NPM1 mutations by DHPLC and sequencing

Wildtype chromatograms performed in one single peak were detected in 295 cases (73.75%) by DHPLC. Fourteen patterns of chromatograms that differed from the wildtype are shown in Table 1, thirteen of which were confirmed as NPM1 mutations by sequencing while one pattern was found to represent a single nucleotide polymorphism (SNP). The SNP was detected in 9 patients (2.3%). None of the patterns of 30 ALL and 30 CML cases was found to be aberrant.

All mutated cases were heterozygous and retained a wild type allele. Similar to other studies from the western countries,6–10 the most frequent mutation variant in the Southeast Asian population (81 cases, 20.3%) was a type A mutation which was a 4-bp TCTG insertion between the position 956 and 959, followed by type D (CCTG) in 7 cases (1.8%), type B (CATG) in 5 cases (1.3%), type J (TATG) in 2 cases (0.5%), and type DD-4 (TGTG) in one case (0.3%). Eight variants, Thai (TH)1 to TH8, were identified as novel mutations and each novel mutation was detected in a separate patient, including a 4-bp insertion between the position 960 and 961 (type TH1, and TH2), between the position 964 and 965 (type TH3), and between the position 958 and 959 (type TH4). Mutation type TH5 and TH6 were a 6-bp insertion associated with a 2-bp deletion (ins6del2) at the position 960. Mutation type TH7 was a 9-bp insertion associated with a 5-bp deletion (ins9del5) at the position 965 and mutation type TH8 was a 10-bp insertion associated with a 6-bp deletion (ins10del6) at the position 958. Noticeably, all mutations resulted in a net of 4-bp addition either from insertion alone or associated with additional deletion. It is interesting that all eight novel NPM1 variants from this study appeared to maintain the last five amino acid residues, VSLRK, and gain the nuclear exporting signal (NES) motif in much the same way as other previously reported variants.6–9 Our findings indicated some common features of NPM1 mutations among Southeast Asian patients and worldwide with respect to nucleotide base changes and their mutant proteins. The disruption of NPM1 nucleolar localization signal caused accumulation of NPM1 protein in the cytoplasm, possibly a critical step in malignant transformation.16

Associated genetic abnormalities and clinical parameters of patients with NPM1 mutations

Chromosomal aberrations were rarely seen in patients with NPM1 mutations in this study (4.2%) and other studies.7–10 Only 4 NPM1 mutated cases had abnormal cytogenetics, i.e. del(9), inv(9), del(10), and inv(10). Chromosomal abnormalities are probably secondary events in the molecular pathogenesis of AML as cells with an abnormal karyotype could represent a subclone within the leukemic population with a normal karyotype. It is intriguing, however, that among the 4 AML cases with abnormal cytogenetics in our series, the abnormal chromosomes mainly involved chromosomes 9 and 10. The association of del(9q) with NPM1 mutations has recently been recognized by Corbacioglu et al.17 who reported the incidence of the NPM1 mutation of 29% in 35 AML patients with del(9q). Although del(9q) was found in only 9 out of 400 AML cases in this study, we were able to identify one patient with del(9q) who also had NPM1 mutation (11%). Because of the mutually exclusive nature of NPM1 mutations and recurrent chromosomal abnormalities, Falini et al. recently proposed that AML patients with NPM1 mutations or cytoplasmic NPM should be categorized as a distinct entity in the upcoming World Health Organization classification.10

With respect to additional genetic aberrations, three different types of genes were studied including FLT3, AML1 and RAS (Table 2). FLT3-ITD mutation was detected in 56.8% of mutated NPM1 cases compared to 25.6% of wild-type cases (p value <0.0001). Only one case in the mutated NPM1 group and 25 cases in the wild-type NPM1 group had RAS mutation. No AML1 mutation was found among the mutated NPM1 group whereas 11 cases of AML1 mutation were found in the wild-type NPM1 group. The higher frequency of FLT3 mutations in cases with mutated NPM1 suggests a possible pathogenic link between these two gene mutations. Mutated FLT3 could induce chronic myeloid disorders in the murine model, but it alone is not sufficient to induce AML.1 Additional mutation may be involved or needed in the pathogenesis of AML with FLT3 mutation. Although the role of NPM1 mutation in AML is still not clear as there is currently no established animal model of AML with mutated NPM1, the coexistence of NPM1 and FLT3 mutations could suggest that the mutant NPM protein may serve to impair differentiation of hematopoietic cells in the same way as AML1 and CEBPA. It is, therefore, possible that NPM1 could co-operate with FLT3, but not RAS or AML1, in the multi-step pathogenetic model of AML.1

Table 2.Clinical parameters of acute myeloid leukemia patients carrying NPM1 mutant (MT) and NPM1 wild-type (WT).

NPM1 mutations were particularly associated with specific clinical factors; for instance, a higher platelet count was one of the parameters. This was similarly observed by Thiede et al.9 who suggested that blasts with NPM1 mutation might retain a certain capacity for thrombocytic differentiation as demonstrated by in vitro experiments. Immunophenotypically, CD34-negativity and multi-lineage involvement, a common intrinsic feature of NPM1 mutation that has already been reported, were also observed in this study.7–10 Age-dependency was another NPM1 mutation characteristic due to the significantly higher incidence found in adults than in pediatric patients.18 Furthermore, among adult patients, NPM1 mutations were found more frequently in the older age group than the younger age group, as shown in Table 2, and this finding is supported by other studies.7–10

Table 3.Incidence of NPM1 mutations in acute myeloid leukemia patients from various countries.

The effect of NPM1 mutation in AML patients was found to be a favorable prognosis in most studies.7–9 We could not observe a major difference in the overall survival (OS) in the Thai patients with and without NPM1 mutation (p=0.376). Interestingly, statistical analyses according to the combined NPM1/FLT3 mutational status revealed a better outcome (p=0.036) for the NPM1-mutated/FLT3 ITD–negative than NPM1⁺/FLT3-ITD⁺ and NPM1-/FLT3-ITD⁺ subgroup, implicating the negative impact of FLT3 mutations regardless of the NPM1 mutational status. Nevertheless, because of the retrospective nature of this study, it might be useful to initiate a prospective clinical trial to confirm the true impact of NPM1/FLT3 mutation on the clinical outcome of Southeast Asian patients.

Despite the fact that the fusion between the NPM1 gene to its various partner genes had been known to be involved in hematologic malignancies for decades, the non-translocation mechanism of NPM1 gene in AML, i.e. NPM1 mutation, was only reported recently in 2005.5–7 We report the first series of NPM1 mutation from the ethnically distinct Southeast Asian region, representing the largest series in Asia (n=400). The incidence of NPM1 mutation in Asian AML populations was previously known only from reports from China (n=28, 156),19,20 Japan (n=257),21 and Taiwan (n=173),18 as summarized in Table 3. European reports were from Italy (n=107–2,562),5,10,22,23 Germany (300–1,485),7–9 and the Netherlands (n=275).6 No AML studies concerning the NPM1 mutations from other ethnic populations have been reported. Of all studies worldwide, NPM1 mutations were mainly detected in AML patients, especially in adult AML patients with a normal karyotype (ranging from 40–60%). Only 2 cases of myelodysplastic syndrome from China20 and 3 cases of chronic myelomonocytic leukemia from the United States were reported to have NPM1 mutation type A.24 The frequency of NPM1 mutation in Thailand of 26.2% as reported by this study was comparable to that of Japan (24.9%), Taiwan (19.1%), Italy (21.7–35.2%) and Germany (27.5%) despite geographical and ethnic differences.

To summarize, NPM1 mutations were most prevalent in Thai AML patients with a normal karyotype. Eight novel variants were identified and all the predicted mutated proteins gained the NES motif, supporting the aberrant process of NPM cytoplasmic localization associated with malignant transformation. Distinct prognostic subclasses of adult AML patients were identified based on the presence of NPM1 and FLT3 mutations. Targeting FLT3 with specific FLT3 inhibitors should provide benefits for the majority of NPM1-mutated AML patients who have coexistent unfavorable FLT3 mutations.

Footnotes

• Authorship and Disclosures CUA was responsible for the initiation and execution of the entire project and writing of the manuscript. WT supervised the molecular and data analysis and contributed to the revision of the manuscript. CB performed the experiments and data analysis and contributed to the drafting of the manuscript. We thank the staff of the Division of Hematology, Department of Medicine, Faculty of Medicine Siriraj Hospital for excellent care of the patients in this study. CUA was the recipient of an award from the Anandamahidol Foundation and is the current principal investigator of the leukemia project funded by Mahidol University. CB is a graduate student whose thesis is partially supported by the Mahidol University Graduate Student Alumni Association. The authors reported no potential conflicts of interest.

• Received February 22, 2008.
• Revision received May 15, 2008.
• Accepted June 5, 2008.