International Journal of Hematology

, Volume 90, Issue 1, pp 1–5

Non-A type nucleophosmin 1 gene mutation predicts poor clinical outcome in de novo adult acute myeloid leukemia: differential clinical importance of NPM1 mutation according to subtype

Authors

  • Youngil Koh
    • Department of Internal MedicineSeoul National University Hospital
  • Juwon Park
    • Cancer Research InstituteSeoul National University College of Medicine
  • Eun-Kyung Bae
    • Department of Biomedical ScienceHanyang University College of Medicine
  • Kwang-Sung Ahn
    • Cancer Research InstituteSeoul National University College of Medicine
  • Inho Kim
    • Department of Internal MedicineSeoul National University Hospital
    • Cancer Research InstituteSeoul National University College of Medicine
    • Clinical Research InstituteSeoul National University Hospital
  • Soo-Mee Bang
    • Department of Internal MedicineSeoul National University Bundang Hospital
  • Jae-Hoon Lee
    • Department of Internal MedicineGachon University College of Medicine Gil Hospital
    • Department of Internal MedicineSeoul National University Hospital
    • Cancer Research InstituteSeoul National University College of Medicine
    • Clinical Research InstituteSeoul National University Hospital
  • Dong Soon Lee
    • Department of Clinical PathologySeoul National University Hospital
  • Young Yiul Lee
    • Department of Internal MedicineHanyang University Hospital
  • Seonyang Park
    • Department of Internal MedicineSeoul National University Hospital
    • Cancer Research InstituteSeoul National University College of Medicine
    • Clinical Research InstituteSeoul National University Hospital
  • Byoung Kook Kim
    • Department of Internal MedicineSeoul National University Hospital
    • Cancer Research InstituteSeoul National University College of Medicine
    • Clinical Research InstituteSeoul National University Hospital
Rapid Communication

DOI: 10.1007/s12185-009-0350-1

Cite this article as:
Koh, Y., Park, J., Bae, E. et al. Int J Hematol (2009) 90: 1. doi:10.1007/s12185-009-0350-1
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Abstract

Mutations of nucleophosmin gene (NPM1) are known to be related to good prognosis in AML patients lacking FLT3 internal tandem duplication (FLT3-ITD). Recently, NPM1 mutations other than type A were reported, but their clinical significance is not well known. Retrospective medical record review of 106 de novo AML patients lacking FLT3-ITD, who received induction chemotherapy from three centers in Korea between 1997 and 2007, was performed. Direct sequencing of NPM1 and RT-PCR for FLT3-ITD was performed on genomic DNA derived from blood samples of patients before induction chemotherapy for detection of mutations. NPM1 mutation was detected in 18 patients, where 13 were type A mutants and 5 were non-type A mutants. CR, CR1-D and OS was not different according to NPM1 mutational status overall. But, non-type A NPM1 mutation was related to shorter CR1-D when compared with NPM1 wild types and NPM1 type A mutation (p = 0.004). OS was shorter in non-type A mutants when compared with NPM1 wild-type patients and NPM1 type A mutants (p = 0.001). The type of mutation of NPM1 is important for prognosis in de novo AML lacking FLT3-ITD. Non-A type NPM1 mutation is a poor prognostic factor.

Keywords

AMLNPM1Prognosis

1 Introduction

Nucleophosmin gene (NPM1) in parallel with FMS-like tyrosine kinase 3 (FLT3) is a genetic change investigated recently, and it is shown to be a predictive marker in acute myeloid leukemia (AML). NPM1 is mutated in one-third to half of adult patients with AML and thus the most frequent genetic lesions identified both in de novo AML and normal karyotype AML [1, 2]. NPM1 mutation frequently translocates NPM to cytoplasm, which is called cytoplasmic NPM (NPMc+) (Fig. 1).
https://static-content.springer.com/image/art%3A10.1007%2Fs12185-009-0350-1/MediaObjects/12185_2009_350_Fig1_HTML.gif
Fig. 1

1-DFS and OS of patients according to subtypes of NPM1 mutation. a and b show 1-DFS and OS of specific NPM1 mutations, respectively. c and d show 1-DFS and OS according to type A NPM1 mutation, wild-type NPM1, and non-A type NPM1 mutation, respectively

Recent studies found that NPMc+ or NPM1 mutation in the absence of FLT3 internal tandem duplication (FLT3-ITD) is associated with improved outcome [3]. NPM1 mutations in NPMc+ are restricted to exon 12, and more than 95% of mutations occur around nucleotide position 960 [2]. The most common mutation in NPMc+ patients is a type A mutation, which duplicates a TCTG tetranucleotide at positions 956 to 959 of the reference sequence. This accounts for 75–80% of adult AML with NPM1 mutation. Therefore, previously reported favorable influence of NPM1 mutation or NPMc+ in FLT3-ITD-negative AML patients may be attributed to type A NPM1 mutation. Other than type A mutation, type B, C and D mutations in NPMc+ have been previously reported [4]. Recent studies reveal new NPM1 mutations other than type A–D, and some of them do not accompany NPMc+ [5]. However, significance of these various types of NPM1 mutation on AML is not defined. So, we analyzed the differential significance of various subtypes of NPM1 mutation in adult de novo AML patients lacking FLT3-ITD who received induction chemotherapy. Our aim is to identify clinical meaning of non-A type NPM1 mutants as prognostic and predictive markers to chemotherapy (Table 1).
Table 1

Clinical characteristics and treatment results of non-A type NPM1 mutants

Patient no.

Age/gender

Subtype of NPM1 mutationa

Mutation sequence (exon 12)

MRC risk group

FAB classification

Initial WBC

Initial BM blast %

Induction regimen

CR

TRMb

BMT

CR1-D (months)

Relapse

OS (months)

Alive

1

64.4/M

B

CATG

Standard

M2

27,260

80

AId

Yes

Yes

No

6.57

No

6.8

Dead

2

32.4/M

B

CATG

Standard

M5

118,460

88.7

AId

Yes

No

Yes

7.03

Yes

9.4

Dead

3

47.0/M

Q6

CCGA

Standard

M1

5,040

93.2

AId

No

Yes

No

NA

NA

0.8

Dead

4

20.1/M

B

CATG

Poor

M1

5,180

90.2

AId

Yes

Yes

Yes

1.73

Yes

11.7

Dead

5

37.0/F

B

CATG

UA

M1

189,900

UA

AIEc

No

Yes

No

NA

NA

1.5

Dead

UA unavailable, NA not applicable

a4-bp insertion at positions 956 through 959 of the reference sequence (GenBank accession number NM_002520)

bIncludes treatment-related mortality (TRM) during reinduction and consolidation chemotherapy

cAIE: cytarabine, idarubicin and etoposide

2 Materials and methods

2.1 Patients and treatment plan

We included adult (age ≥ 15) consecutive de novo AML patients referred to three centers in Korea between 1997 and 2007, who received induction chemotherapy. One hundred and sixty-six patients agreed to donate blood samples for molecular testing on research base with informed consent and were analyzed. Existence of FLT3-ITD was tested and 106 FLT3-ITD-negative patients were finally included in the study. Patients with acute promyelocytic leukemia were excluded. Data regarding characteristics of patients were obtained by medical record review.

Induction regimens consisted of cytarabine and idarubicin. An average of 1.7 cycles ranging from 0 to 3 cycles of consolidation chemotherapy comprised of combinations of idarubicin, daunorubicin, and cytarabine was given to the patients after complete remission (CR). Allogeneic stem cell transplantation was performed in 17 patients after CR according to donor availability and risk stratification [6].

Risk grouping based on karyotype was done by medical research council (MRC) criteria [7, 8]. Overall survival (OS) was calculated from diagnosis to death from any cause. Duration of first CR (CR1-D) was defined as a period from first successful induction chemotherapy to documentation of disease relapse. The study protocol was approved by the institutional review board of each hospital.

2.2 Molecular study for NPM1 mutation

For NPM1 mutational analysis, PCR was performed in a mixture of 1.25 pmol of each primer NPM1-ex12F (5′-TCTGAGTATAAATTTTCTTGGAGTCA-3′) and NPM1-ex12R (5′-TGGGAACACAGCACTAAATCC-3′), 50 ng genomic DNA, 250 μM dNTPs, and 0.15 U Taq DNA Polymerase (Applied Biosystems, Foster City, CA, USA) in the buffer provided by the manufacturer. Amplification was carried out in a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA) under touchdown conditions [9]. The PCR fragments were purified using Millipore PCR Cleanup Filter Plates (Millipore, Billerica, MA, USA). Sequencing reactions were run on an ABI 3730 sequencer with BigDye, Dye Terminator chemistry (Applied Biosystems, Foster City, CA, USA) and an internal sequencing primer NPM_seqR (5′-TTGGACAACACATTCTTGGCAATA-3′). Sequence variants were verified by chromatograms.

2.3 Statistical analysis

The included variables for analysis in this study were age, gender, cytogenetic analysis result, initial white blood cell (WBC) count, platelet (PLT) count, absolute neutrophil count (ANC), bone marrow cellularity, bone marrow blast percentage, chemotherapy regimen, NPM1 mutation status, outcome of induction chemotherapy, CR1-D and OS.

Statistical analysis of 2 × 2 contingency tables of categorical variables were performed using Fisher’s exact test. Median durations of CR1-D and OS were calculated using the Kaplan–Meier method and comparisons between groups were made using log-rank tests. Multivariate analysis was performed using a logistic regression model for response and Cox regression models for CR1-D and OS. All analyses were performed using SPSS for Windows Version 12.0 (SPSS Inc.).

3 Results

3.1 Patient characteristics and NPM1 mutation status

This study included 43 females and 63 males with a median age of 47.0 (range 16.0–76.5) years. 99 patients had information about karyotype, where 51 with standard 23 with poor risk disease.

NPM1 mutation results were not available in one patient due to low sample quality. NPM1 mutation was detected in 18 patients. 17 patients had previously reported NPM1 mutation, whereas 13 had type A mutation and 4 had B mutation [10]. One patient showed novel CCGA insertion in exon 12 of NPM1, which has not been reported yet and we designated it as Q6 mutation. Overall, five patients were non-A type mutant. NPM1 mutation was more frequently detected in standard risk patients, whereas only one patient had poor risk disease. Ratio of A and non-A type mutation was not different according to risk group.

3.2 Mutation status of NPM1 on treatment outcome

Risk group, NPM1 mutation status was not important for CR. No other single parameter predicted CR. The patient with Q6 mutation did not achieve CR. TRM during first induction chemotherapy was observed in seven patients and among them, two patients had non-A type NPM1 mutants. TRM rate was significantly high in non-A type NPM1 mutants (p = 0.021).

For 81 patients who achieved CR, median CR1-D was 16.8 months. Median CR1-D was not different between NPM1 mutants in general and NPM1 wild type (p = 0.644). But, patients with non-A type NPM1 mutation had significantly shorter median CR1-D when compared with NPM1 wild-type patients (7.0 vs. 16.8 months, p = 0.004), and type A NPM1 mutants (7.0 vs. 23.7 months, p = 0.001). There was no significant difference in CR1-D between patients with wild-type NPM1 and type A NPM1 mutation. In multivariate analysis regarding NPM1 mutation status and risk group, non-A type NPM1 mutation and type A mutation had divergent impact on CR1-D and predicted shorter and longer CR1-D, respectively (p = 0.023 and 0.034, respectively).

Median OS was 19.4 months, and NPM1 mutation in overall was not a prognostic factor (p = 0.969). Non-A type predicted shorter survival such that median OS was shorter in non-A type NPM1 mutants when compared with NPM1 wild-type patients (6.8 vs. 14.8 months, p = 0.001) and type A NPM1 mutants (6.8 vs. 33.8 months, p < 0.001). Lower WBC and higher ANC predicted longer OS (Cox, p = 0.003 and 0.047, respectively). In multivariate analysis, no single factor was associated with OS and non-A type NPM1 mutation predicted shorter OS without reaching statistical significance (p = 0.079).

During follow-up of median 51.7 months, 43 patients experienced relapse and 61 patients died.

4 Discussion

In this study, we introduced new insight into the nature of NPM1 mutation for the first time. Before large-scale analyses for confirmation of meaning of numerous molecules in AML, there were anecdotal reports with relatively small number of patients suggesting the existence of such molecules. From this point, overall 106 patients and 18 patients with NPM1 mutations are not small numbers when compared to those anecdotal studies.

Nucleophosmin control of cell growth and proliferation is assumed to be a result of several activities which include interactions with p53 and ARF proteins [1113]. How NPM1 mutation disrupts this cell control and induces leukemia is not well known. Generally, type A to D mutations of exon 12 in NPM1 are believed to share common mechanism in leukemogenesis, and thus to have a similar clinical impact. For other mutations, the significance is rarely known. In this report, we present those minor NPM1 mutations, B and Q6—other than type A—confer poor prognosis and predict shorter CR1-D in unselected AML patients lacking FLT3-ITD. Patient with type Q6 mutation had especially unfavorable prognosis with OS of 0.67 months, without achievement of CR. Besides, type B mutation also conferred poor prognosis in contrast to conventional conjecture. Interestingly, poor prognosis of non-A type NPM1 mutants was mainly due to treatment-related mortality.

The frequency of NPM1 mutation in our study was 17.0% (18/106), and this is relatively low when compared with previous western studies [1, 2]. However, our study included only FLT3-ITD-negative AML patients and the result concur with the findings of Japanese study which showed low-positive rate (13.2%) of NPM1 mutation in FLT3-ITD negative patients [14]. Similarly, low-positive rate of NPM1 mutation in these two studies may be a reflection of different pathophysiology of AML between Asian and western countries which may be attributed to ethnic diversity.

In contrast to previous studies, our results do not support favorable outcome of NPM1 mutations on FLT3-ITD-negative patients. Only tendency for favorable outcome with type A NPM1 mutation was observed without reaching statistical significance. This may be attributed to the small number of patients, but this may be also due to ethnic diversity. It is well known that characteristics of AML in Asian patients are different from that in western patients. Especially, frequency [15, 16] and prognostic impact of molecular markers can be different [17]. Previous Japanese study failed to show favorable impact of NPM1 mutation in AML patients [14].

In conclusion, subtypes of NPM1 mutation should be considered while analyzing clinical significance of NPM1 mutations in parallel with karyotype and FLT3-ITD status. Further study with larger number of patients is needed to ascertain the exact clinical meaning of this finding.

Acknowledgment

This work was supported by a grant from Seoul National University Hospital Research Fund (No. 03-2006-003-0). This work was also supported by a grant from Cancer Research Institute, Seoul National University College of Medicine (No.800-20070121).

Copyright information

© The Japanese Society of Hematology 2009