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[Cancer Research 66, 3310-3316, March 15, 2006]
© 2006 American Association for Cancer Research

Clinical Research

Nucleophosmin Mutations in De novo Acute Myeloid Leukemia: The Age-Dependent Incidences and the Stability during Disease Evolution

Wen-Chien Chou1,2, Jih-Luh Tang1, Liang-In Lin4, Ming Yao1, Woei Tsay1, Chien-Yuan Chen1, Shang-Ju Wu1, Chi-Fei Huang1, Rong-Jing Chiou1, Mei-Hsuan Tseng1, Dong-Tsamn Lin2,3, Kai-Hsin Lin3, Yao-Chang Chen1,2 and Hwei-Fang Tien1

Departments of 1 Internal Medicine, 2 Laboratory Medicine, and 3 Pediatrics, National Taiwan University Hospital; and 4 Graduate Institute of Medical Technology, National Taiwan University, Taipei, Taiwan

Requests for reprints: Hwei-Fang Tien, Department of Internal Medicine, National Taiwan University Hospital, 100 Taipei, Taiwan. Phone: 886-2-23123456, ext. 3955; Fax: 886-2-23959583; E-mail: hftd{at}ha.mc.ntu.edu.tw.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nucleophosmin (NPM) mutations have been found in a significant proportion of adults with de novo acute myeloid leukemia (AML), especially in those of a normal karyotype. These results provide a basis for studies of the pathogenesis in this specific subgroup of AML. In this study, NPM mutations were analyzed in 173 Chinese patients of de novo AML, including adults and children. We found that NPM mutations were present in 19.1% of the overall population and 40.3% of those with a normal karyotype. Adults had a significantly higher incidence of NPM mutations than children [32 of 126 (25.4%) versus 1 of 47 (2.1%), P < 0.001]. NPM mutations were closely associated with normal karyotype (P < 0.001) and internal tandem duplication of FLT3 (P = 0.002), but negatively associated with CEBPA mutations (P = 0.032) and expression of CD34 (P < 0.001) and HLA-DR (P = 0.003). Serial analyses of NPM mutations showed the mutation disappeared at complete remission, but the same mutation reappeared at relapse, except for one who lost the mutation at the second relapse, when new cytogenetic abnormalities emerged. None acquired novel mutations during the follow-up period. In conclusion, NPM mutations occur in an age-dependent fashion. Moreover, the findings that NPM mutations are stable during disease evolution and closely associated with disease status make it a potential marker for monitoring minimal residual disease. (Cancer Res 2006; 66(6): 3310-6)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Acute myeloid leukemia (AML) is a heterogeneous group of hematologic malignancy. Despite that recurrent chromosomal abnormalities are present in a significant proportion of AML patients, leukemia cells from ~40% to 50% of the patients have a normal karyotype and lack a reliable biological marker, thus making difficult the investigation of the etiologies and monitoring of residual disease in some of the de novo AML patients (1).

Recently, nucleophosmin (NPM) exon 12 mutations, which resulted in shift of the NPM protein from the nucleus to cytoplasm, were found in ~30% of de novo AML patients (2–8). The mutations were even more prominent in those with a normal karyotype and were frequently associated with FLT3 mutation. NPM protein shuttles between the nuclei and cytoplasm and is involved in ribosomal biogenesis (9, 10), centrosome duplication (11), and regulation of the functions of tumor suppressor proteins, such as p53 and alternative reading frame (12, 13). Although it is frequently a partner of translocations in various hematologic malignancies, such as anaplastic large cell lymphoma (NPM-ALK; ref. 14), acute promyelocytic leukemia (NPM-RAR{alpha}; ref. 15), myelodysplastic syndrome, and AML (NPM-MLF-1; ref. 16), the contribution of the mutated NPM protein to the leukemogenesis remains undetermined. The frequent mutation of NPM in de novo AML might provide a basis for investigation of the pathogenesis and monitoring of residual disease of AML with a normal karyotype.

Most reports of NPM mutations came from Europe and focused mainly on adults (2–7). In the current study, we investigated the mutation in 173 de novo AML patients, a population of mixed Chinese adults and children, and correlated the results with clinical features, cytogenetics, immunophenotyping, and other genetic alterations. We also did sequential analyses on some patient samples during the clinical course to investigate the stability and pathogenetic role of NPM mutation in AML.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients and sample collection. The NPM mutation was analyzed on the bone marrow cells from 156 consecutive patients with de novo AML diagnosed based on the French-American-British Cooperative Group criteria (17) at the National Taiwan University Hospital from 1995 to 2000. We subsequently recruited another 17 children with AML diagnosed outside this period to increase the number of pediatric patients. The bone marrow mononuclear cells were collected by heparinization followed by Ficoll-Hypaque gradient centrifugation. The isolated cells were frozen and stored at –80°C.

Mutation analysis. Analysis of NPM exon 12 mutation was done as described by Falini et al. (2). The final volume for PCR reaction was 35 µL containing 200 ng DNA, 200 nmol/L deoxynucleotide triphosphate, 2 mmol/L MgSO4, 140 nmol/L of each primer, and 1 unit of AmpliTaq Gold polymerase (Applied Biosystems, Foster City, CA). PCR was done by heating at 95°C for 10 minutes, followed by 35 cycles of 95°C for 45 seconds, 49°C for 1 minute, and 72°C for 1 minute, with a final step for 10 minutes at 72°C. PCR products were electrophoresed on 2% agarose gels, purified and sequenced using the BigDye Terminator v3.1 Cycle Sequencing kit, which contained AmpliTaq DNA polymerase FS (Applied Biosystems), on an automated ABI-3100 Genetic Analyzer (Applied Biosystems). Abnormal sequencing results were confirmed by at least two repeated analyses. The DNA extracted from the peripheral blood mononuclear cells of 13 healthy persons was used as normal controls.

Point mutations at codons 12, 13 (exon 1), and 61 (exon 2) of the N-RAS and K-RAS genes were analyzed by PCR on genomic DNA and direct sequencing (18). Methylation status of SOCS1 (suppressor of cytokine signaling-1) and SHP1 (protein-tyrosine phosphatase containing 2 Src homology domains), both encoding proteins functioning as negative regulators of signal transduction, was analyzed by methylation-specific PCR as described (19–24). Analyses of mutations of CEBPA and internal tandem duplication of FLT3 (FLT3/ITD) were done according to previous studies (18, 25).

TA cloning. We chose the PCR products of all types of detected mutation and cloned with pGEM-T Easy TA cloning kit (Promega, Madison, WI), followed by sequencing to identify the specific mutation in a single allele.

Immunophenotyping. A panel of monoclonal antibodies to myeloid-associated antigens, including CD13, CD33, CD11b, CD15, CD14, and CD41a, as well as lymphoid-associated antigens, including CD2, CD5, CD7, CD19, CD10, and CD20, and lineage nonspecific antigens HLA-DR, CD34, and CD56 were used to characterize the phenotypes of the leukemia cells. Expression of surface antigens on the leukemia cells was shown by an indirect immunoalkaline phosphatase method (26) before 1998 and by flow cytometry thereafter.

Cytogenetic study. Bone marrow samples were aspirated into heparinized syringes and chromosomal analyses by G-banding method was done on bone marrow cells after 1 to 3 days of unstimulated culture as described previously (26).

Statistics. {chi}2 Test was used to calculate the significance of association between NPM mutation and other discrete variables, such as expression of antigens, cytogenetics, mutation, or methylation of a specific gene, etc., and Mann-Whitney tests were used to compare continuous variables. Kaplan-Meier curve was calculated by SPSS software (Chicago, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NPM exon 12 mutations. A total of 173 de novo AML patients, including 105 males and 68 females, with a median age of 41 years (range, 0-85 years), were analyzed. There were 126 adults and 47 pediatric patients (≤18 years old). Overall, the NPM mutation occurred in 33 (19.1%) of all de novo AML patients. There were four types of mutations, all involving the COOH-terminal portion of the transcript of NPM with a four-nucleotide insertion between positions 960 and 961. The frequencies and resulting changes of amino acid sequences are shown in Fig. 1A to E . All cases with the mutation were heterozygous and retained a wild-type allele. We also detected a polymorphism of nucleotide T deletion at position 1,146 in the 3' untranslated region (Fig. 1F). The polymorphism was detected in 123 patients (71.1%), homozygous in 27 and heterozygous in 96, and in 11 of the 13 healthy persons.


Figure 1
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  		Figure 1. The NPM mutation patterns. The sequences of (A-E) were derived from single clones by cloning with pGEM-T easy kit. A, wild-type NPM sequence. B to E, mutation of NPM. Boxed tetranucleotides, insertion sequences between nucleotides 960 and 961 from the transcription start site. The underlying denotes the corresponding alteration of encoded amino acid sequences and incidences. F, polymorphism with deletion of T nucleotide at position 1,146. The sequences were read forward in (A-E), but backward in F.

 
Sequential studies of NPM mutations. NPM exon 12 mutations were serially studied in 41 patients, including 13 patients with and 28 patients without NPM mutation at diagnosis; 20 of the latter patients had 1,146(–T) polymorphism at the NPM gene (Table 1 ; Supplementary Table). None of these 41 patients acquired a new mutation after a median follow-up time of 16 months (range, 1-60 months), although eight of them showed karyotypic evolution and two had different clonal abnormalities at relapse. The NPM mutations disappeared at complete remission in all patients who harbored the mutations at diagnosis and had available DNA samples for analysis (Table 1). The same mutations as those at diagnosis were detected at first relapse in all five patients analyzed (patients 22, 55, 63, 129, and 147). However, no mutation was detected at second relapse in patient 63; at that time, new clonal karyotypic abnormalities were present. In one patient (patient 32), the mutation remained detectable after chemotherapy when bone marrow blasts were 7.2%. In the 20 patients with 1,146(–T) polymorphism but without mutation at the NPM gene, the nucleotide T deletion persisted at complete remission and also at relapse with the exception of one patient (patient 69) in whom the homozygous polymorphism turned to heterozygous status after allogeneic bone marrow transplantation (Supplementary Table). In all patients without NPM mutation at diagnosis and having subsequent samples for analysis, none acquired mutation at relapse.


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  		Table 1. Results of sequential studies of NPM mutations and chromosomal changes in the patients with the mutation at diagnosis

 
Correlation of NPM mutations with clinical features and biological characteristics. One hundred and sixty-five patients had cytogenetic data. The NPM mutation was mainly seen in those of a normal karyotype (Table 2 ). Among the patients with a normal karyotype, 40.3% showed the NPM mutation, compared with 6.1% in those with chromosomal abnormalities. No NPM mutation was seen in patients with t(15;17), t(8;21), inv(16), or other recurrent chromosomal abnormalities, such as t(6;9), t(7;11), and deletions involving chromosome 5 or 7 (Table 2). The NPM mutation occurred much more frequently in adults than in pediatric patients [32 of 126 (25.4%) versus 1 of 47 (2.1%), P < 0.001]. It was infrequently detected in patients younger than 40 years [3 of 85 (3.5%); Fig. 2 ]. The lower incidence of the NPM mutation in children could not be explained by the lower rate of normal karyotype in pediatric patients than in adults [11 of 41 (26.8%) versus 56 of 124 (45.1%), P = 0.038] because the NPM mutation was still significantly less frequent in children than in adults when only the patients with a normal karyotype were considered [1 of 11 (9%) versus 26 of 56 (46.4%), P = 0.021]. Comparison of NPM mutations in different age groups revealed a gradient increase of mutation rate in the older patient groups (Fig. 2). Patients with AML M4 subtype had a higher NPM mutation rate than those with other subtypes, but the difference had not reached statistical significance yet (Table 2). NPM mutations were significantly associated with higher initial WBC counts, blast percentages, and platelet counts in the peripheral blood, but not sex, lactate dehydrogenase, and hemoglobin levels (Table 2).


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  		Table 2. Clinical data, French-American-British Cooperative Group types, and cytogenetic changes in AML patients

 

Figure 2
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  		Figure 2. Age-dependent incidences of NPM mutation. *, P < 0.001, compared with age groups 0 to 20 and 21 to 40. Percentages above the columns represent the incidences of the NPM mutation in that specific group of patients Figure 2, mutation; Figure 2, wild type.

 
We also analyzed mutations in CEBPA, N-RAS, and K-RAS, FLT3/ITD, and hypermethylation in SHP1 and SOCS1, which were frequently detected in AML (21, 27). The comparison of these genetic or epigenetic alterations between the patients with and without NPM mutation is summarized in Table 3 . FLT3/ITD and CEBPA mutations were positively and negatively associated with NPM mutations, respectively. None of other genetic changes was associated with NPM mutation.


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  		Table 3. Comparison of other genetic alterations between AML patients with and without NPM mutation

 
For immunophenotyping, the NPM mutation was associated with lack of expression of CD34 and HLA-DR on leukemia cells (P < 0.001 and P = 0.003, respectively; Table 4 ). Other markers, such as CD13, CD33, CD11b, CD14, CD15, CD19, CD10, CD7, CD56, and CD2, were not found to be significantly associated with NPM mutation status. We did not detect significant effects of the NPM mutation on the disease-free or overall survival whether FLT3 mutation status was considered or not (data not shown), probably because of the small sample size.


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  		Table 4. Comparison of immunophenotyping between AML patients with and without NPM mutation

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this comprehensive analysis of NPM mutations in de novo AML patients, we found a remarkable difference in the incidence of NPM mutation between adult and pediatric patients (25.4% versus 2.1%). A recent study has reported a low incidence of NPM mutation in pediatric patients (7%; ref. 28). Verhaak et al. (6) showed that NPM mutations were significantly less frequent in adult patients younger than 35 years. Our current report is the first to present a side-by-side comparison of the NPM mutation rate in Chinese children and adults with AML. The difference in the incidence of NPM mutation between these two age groups could not be explained by the lower incidence of normal karyotype in the pediatric patients than in adults because the same finding could be shown although only the patients with a normal karyotype were considered (a mutation rate of 46.4% in adults versus 9% in children, P = 0.021). This is another proof that childhood leukemia can be very different from that of adults in pathogenesis and biological characteristics (1, 29).

The facts that NPM mutation is not present in leukemia with recurrent cytogenetic abnormalities and that the mutation disappears at complete remission imply a critical role of NPM mutation in leukemogenesis. Furthermore, we showed that none of the patients without the NPM mutation at diagnosis acquired the mutation during the follow-up period, suggesting that the NPM mutation might play little role in the progression of AML. In the sequential studies on a smaller population of patients, Boissel et al. (4) and Suzuki et al. (8) also found that none of their patients without NPM mutation at diagnosis acquired NPM mutation at relapse. Although the same NPM mutation as that detected at diagnosis could be identified again at first relapse in the NPM-mutated patients in our study, one patient lost the mutation at second relapse (patient 63; Table 1). The bone marrow blasts in this patient were 5.5% when the second relapse was diagnosed, but the relapse status was confirmed by the clonal chromosomal abnormalities of –7 and t(12;18), which were not seen at initial diagnosis. The disappearance of NPM mutation in this patient could be due to suppression of PCR amplification of mutant gene when there were only a small number of mutated cells within a major population of normal cells. Analysis by more sensitive quantitative real-time PCR may solve this problem. Another possible explanation was the emergence of a different clone of leukemia cells at that time. The presence of new chromosomal abnormalities –7, which was commonly seen in secondary leukemia, and the long interval (58 months) between initial diagnosis and this relapse (Table 1) supported the latter possibility. In the report of Boissel et al. (4), all 10 patients with NPM mutations at diagnosis still displayed NPM mutations at relapse. Of the 17 patients with NPM mutations at diagnosis reported by Suzuki et al. (8), 15 carried the same mutation at relapse. Two patients in that study lost the mutation at relapse; one of them acquired a chromosomal abnormality that was not present at diagnosis and the other showed normal karyotype at both diagnosis and relapse. Together, these findings suggest that NPM gene status is stable in contrast to FLT3/ITD (8, 18) and that NPM mutation can be a good marker for monitoring minimal residual disease in AML.

We found that NPM mutations were mutually exclusive with CEBPA mutations but were highly associated with FLT3/ITD. Recent studies argue for a multistep pathogenesis of AML (30). The genetic events underlying AML pathogenesis appear to fall into two broadly defined complementation groups: One comprises mutations that confer proliferation and/or survival advantage to hematopoietic cells, such as FLT3 and RAS mutations, and the other comprises mutations that affect transcription factors resulting in impaired hematopoietic differentiation, such as t(8;21)/AML1-ETO, inv(16)/CBFß-SMMHC, and t(15;17)/PML-RAR{alpha} fusions as well as CEBPA and AML1 mutations (30, 31). Like CEBPA, NPM mutation is not present in AML with t(8;21) t(15;17) or inv(16) (25, 31, 32). The mutation of NPM results in cytoplasmic translocation of NPM protein, which is normally located in the nucleoli (33) where it may serve as a chaperone to prevent protein aggregation (34). Although the pathogenetic role of NPM mutation in AML is not clear, the findings that NPM mutation is inversely associated with CEBPA mutation and chromosomal abnormalities, t(8;21), t(15;17) and inv(16), suggest that mutant NPM protein may serve to impair differentiation of hematopoietic cells in the multistep pathogenesis model of AML, like CEBPA and AML1 (6, 30, 31). Consistent with this hypothesis, a recent study on animal models revealed an indispensable role of NPM in embryonic hematopoiesis (35). In addition, haploinsufficiency of NPM in mice resulted in a syndrome mimicking human myelodysplastic syndrome, a hematologic disease of impaired differentiation (35). It was surmised that deletion of chromosome 5q commonly seen in myelodysplastic syndrome might be related to haploinsufficiency of NPM. Although there were no mutations at the exon 12 of NPM in >100 myelodysplastic syndrome patients tested (2), we cannot exclude the possibilities of haploinsufficiency by microdeletion of chromosome 5q involving this gene or a functional compromise by mutations of other genes in the same pathogenetic pathway.

In summary, NPM mutations were detected in 19.1% of a total of 173 de novo Chinese AML patients and 40.3% of those with a normal karyotype. Children had a significantly lower incidence of NPM mutations than adults. The mutation occurred infrequently in AML patients younger than 40 years. The same was also true if only the patients with normal karyotype were analyzed. The genetic changes underlying the childhood AML seem quite different from those in adult AML. Serial studies showed that the NPM mutation detected at diagnosis disappeared at complete remission and the same mutation reappeared at relapse. One patient lost the mutation at the second relapse. Whether this is due to an emergence of a new clone of leukemia cells or low sensitivity of the method to detect the mutation remains to be determined. No one acquired a new mutation during a median follow-up time of 16 months (range, 1-60 months), suggesting that the NPM mutation is probably an early event in the development of AML but may play little role in the progression of the disease. The findings that the NPM mutation was inversely associated with the CEBPA mutation and chromosomal abnormalities t(8;21), t(15;17), and inv(16), and was closely correlated with FLT3/ITD infer that the NPM mutants might impair the differentiation of hematopoietic cells. Further comprehensive studies on the biological effects of NPM mutants are needed to disclose the role of NPM mutations in the pathogenesis of AML and their interactions with other genetic alterations.


   Acknowledgments
 
Grant support: NSC 93-2320-B002-129, NSC 94-2314-B-002-307, and NSC 93-2314-B002-039 from National Science Council (Taiwan), Department of Medical Research in National Taiwan University Hospital (Taiwan), and NTUHYL 93S006 from the Yin-Lin Branch of The National Taiwan University Hospital.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 12/ 5/05. Revised 1/ 1/06. Accepted 1/16/06.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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[Abstract] [Full Text] [PDF]

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T. S. Laughlin, M. W. Becker, J. L. Liesveld, D. A. Mulford, C. N. Abboud, P. Brown, and P. G. Rothberg
Rapid Method for Detection of Mutations in the Nucleophosmin Gene in Acute Myeloid Leukemia
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B. Falini, M. P. Martelli, C. Mecucci, A. Liso, N. Bolli, B. Bigerna, A. Pucciarini, S. Pileri, G. Meloni, M. F. Martelli, et al.
Cytoplasmic mutated nucleophosmin is stable in primary leukemic cells and in a xenotransplant model of NPMc+ acute myeloid leukemia in SCID mice
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M. Palmisano, T. Grafone, E. Ottaviani, N. Testoni, M. Baccarani, and G. Martinelli
NPM1 mutations are more stable than FLT3 mutations during the course of disease in patients with acute myeloid leukemia
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P. Brown, E. McIntyre, R. Rau, S. Meshinchi, N. Lacayo, G. Dahl, T. A. Alonzo, M. Chang, R. J. Arceci, and D. Small
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B. Falini, I. Nicoletti, N. Bolli, M. P. Martelli, A. Liso, P. Gorello, F. Mandelli, C. Mecucci, and M. F. Martelli
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