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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2017  |  Volume : 60  |  Issue : 3  |  Page : 355-359
NPM1 and FLT3 mutations in acute myeloid leukemia with normal karyotype: Indian perspective


1 Department of Hematology, All India Institute of Medical Sciences, New Delhi, India
2 Department of Haematology, Gauhati Medical College and Hospital, Guwahati, Assam, India

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Date of Web Publication22-Sep-2017
 

   Abstract 

Background: FLT3-ITD and NPM1 mutations are considered to be the major determinants of the patient response to therapy and outcome. The primary aim of this study was to establish the correlation between these molecular mutations and the clinico-hematologic parameters as well as the prognostic outcome of the Indian acute myeloid leukemia (AML) patients. Materials and Methods: This prospective study involved newly diagnosed nonpromyelocytic AML patients who had undergone complete diagnostic workup, including immunophenotyping, conventional cytogenetics and molecular analysis for NPM1 and FLT3-ITD mutation by reverse transcriptase polymerase chain reaction at presentation. Results: Overall, the prevalence of NPM1 and FLT3-ITD mutations was found to be 14.4% and 10.8%, respectively. Among patients with normal karyotype, leukocytosis was significantly associated with NPM1+ group than the NPM1− group (P = 0.0019) and more severe degree of anemia was observed in the FLT3-ITD+ patients than the other groups (P = 0.025). No significant correlation was found in terms of age at presentation (P = 0.56), sex ratio (P = 0.467), median platelet count (P = 0.27), and blast percentage between NPM1+ and FLT3-ITD+ groups. Complete remission (CR) rates were better in the NPM1+/FLT3-ITD− group than the other three groups. Unlike most other studies, improved CR rates as well as disease-free survival were observed in the NPM−/FLT3-ITD− group than the FLT3-ITD+ groups although not reaching statistically significant levels. Conclusion: Some differences in the clinical behavior of the Indian AML patients in comparison to that of the West in the presence of NPM1 and FLT3-ITD suggests that comprehensive studies are required to confirm the definitive role of these mutations among AML patients, especially with normal karyotype.

Keywords: Acute myeloid leukemia, cytogenetics, disease-free survival, reverse transcriptase polymerase chain reaction

How to cite this article:
Sazawal S, Singh N, Jain S, Chhikara S, Chaubey R, Bhattacharyya J, Saikia KK, Mahapatra M, Saxena R. NPM1 and FLT3 mutations in acute myeloid leukemia with normal karyotype: Indian perspective. Indian J Pathol Microbiol 2017;60:355-9

How to cite this URL:
Sazawal S, Singh N, Jain S, Chhikara S, Chaubey R, Bhattacharyya J, Saikia KK, Mahapatra M, Saxena R. NPM1 and FLT3 mutations in acute myeloid leukemia with normal karyotype: Indian perspective. Indian J Pathol Microbiol [serial online] 2017 [cited 2019 Dec 15];60:355-9. Available from: http://www.ijpmonline.org/text.asp?2017/60/3/355/215388



   Introduction Top


Acute myeloid leukemia (AML) describes a group of hematopoietic stem cell disorders characterized by the expansion of undifferentiated myeloid progenitors.[1] As knowledge regarding leukemogenesis has increased, so has the acceptance that the genetic abnormalities leading to leukemia are not only heterogeneous but also complex, and multiple aberrations often cooperate in a multistep process to initiate the complete leukemia phenotype. On the molecular level, several specific changes have been identified. Some of the mutations such as NPM1 and CEBPA involve transcription factors while others such as FLT3, c-KIT, RAS, etc., affect signal transduction pathways. Not only have these mutations led to a better understanding of leukemogenesis but also have proved to be powerful prognostic factors.[2] Constitutive activation of the FLT3 receptor tyrosine kinase, either by internal tandem duplication (ITD) mutations of the juxtamembrane domain or point mutations clustering in the second tyrosine kinase domain (TKD mutations), has been found in 20%–30% of patients with AML and in 30%–45% of patients with normal karyotype. ITD mutations have been associated with an increased risk of treatment failure after conventional chemotherapy whereas the prognostic relevance of FLT3-TKD mutations is less evident. In contrast to FLT3-ITD mutations, alterations of the myeloid transcription factor NPM1 detectable in about 35% of patients with AML and normal karyotype have been associated with a better outcome after treatment. However, in approximately 50% of AML patients with normal karyotype, the molecular basis of leukemic development is still poorly understood.[3] The primary aim of this prospective study was to establish the correlation between molecular mutations such as FLT3-ITD, NPM1, and the clinico-hematologic parameters as well as the prognostic outcome of the Indian nonpromyelocytic AML (nonacute promyelocytic leukemia [APL]) patients with normal karyotype.


   Materials and Methods Top


It was a prospective, observational study carried out in the Department of Hematology during 2012–2014. Only the newly diagnosed patients of non-APL AML were enrolled in which details of complete diagnostic workup, including the molecular mutations such as NPM1 and FLT3-ITD, were available. Patients with therapy-related or postmyelodysplastic syndrome AMLs or relapsed AMLs were excluded from the study. Patients were classified according to the French-American-British (FAB) and WHO classification and their baseline demographic characteristics and treatment details at least for up to 1 year of follow-up were compiled. Written informed consent was taken from all the patients, and ethical clearance was obtained before the conduct of this study.

All materials investigated were obtained at diagnosis. Bone marrow (BM) aspirate sample was used whenever available. In all other cases, peripheral blood samples were examined. Three to five milliliters samples were collected in ethylenediaminetetraacetic acid vials for performing molecular mutations and immunophenotyping and in heparinized syringes for conventional karyotyping and processed within 6–24 h of collection.

Immunophenotyping using 6-color flowcytometry was carried out on the freshly collected BM/blood samples as per standard protocols, using a comprehensive panel of antibodies. Flow cytometric analysis was performed on a FACSCalibur Flow Cytometer (Becton-Dickinson, USA).

Karyotyping by G-banding method was performed on BM cells after short culture. All cases with normal karyotype were confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) to exclude common translocations [t(8;21), t(9;11), t(15;17), t(16;16), and Inv(16)].

Mononuclear cells were isolated from BM and peripheral blood samples by Ficoll-Hypaque centrifugation (Histopaque 1077; Sigma diagnostics, USA). RNA was extracted (Qiagen, GmbH, Germany) and reverse-transcribed to cDNA with random hexamers (Roche diagnostics, GmbH, Germany). The quality of cDNA was assessed by the amplification of the β2 microglobulin gene. RT-PCR was performed to identify NPM1 and FLT3 ITD.[4] The presence of fragment sizes of 320 base pairs (bp) for the exon 12 of NPM was considered positive for this transcript, and the presence of sizes >366 bp (wild type) was considered positive for FLT3/ITD, as shown in [Figure 1] and [Figure 2]. PCR reactions were setup in GeneAmp PCR system 9700 (Applied Biosystems).
Figure 1: Reverse transcriptase polymerase chain reaction analysis of the FLT3/ITD fusion transcript. Lane M, 100-bp ladder; lanes 1–8 are patient samples. FLT3/ITD mutation shows 387-bp fragment (lanes 5 and 7), and wild-type allele shows a 366-bp fragment

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Figure 2: Reverse transcriptase polymerase chain reaction analysis of the NPM1 mutant transcript. Lane M, 100-bp ladder; lanes 1–6 are patient samples. NPM1 mutation shows 320-bp fragment (lanes 1, 4 and 5), and wild-type allele shows a 298-bp fragment

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All AML patients were given induction with 3 + 7 regimen comprising cytarabine 100 mg/m 2 daily as a 7-day continuous intravenous infusion and daunorubicin 60 mg/m 2 intravenously daily for 3 days. Patients who achieved complete remission (CR) received further 2–3 cycles of consolidation, each of which included intravenous cytarabine 3 g/m 2 over 3 h every 12 h for six doses on days 1, 3, and 5 in combination with intravenous daunorubicin 45 mg/m 2 daily for 2 days. Patients who did not achieve CR with induction therapy and who were deemed fit to receive a reinduction regimen received combinations of mitoxantrone, and high-dose cytarabine (HiDAC or HAM).[5] Most of the patients in the Indian settings did not opt for BM transplantation due to financial constraints or nonavailability of suitable donor which had a negative impact on the overall survival of these patients. Response to treatment was assessed using International Working Group criteria. CR was defined as a postinduction BM with <5% blasts, a peripheral blood platelet count >100 × 109/L, an absolute neutrophil count >1.0 × 109/L, and the absence of extramedullary disease.

SPSS software (Stata Statistical Software, Release 12.0; Illinois, USA) was used to analyze clinical characteristics and molecular features. Categorical data were compared among the comparison groups by Chi-square test and Fisher exact test. Continuous data following nonnormal distribution were compared in the two groups depending on the presence of cytogenetic abnormalities by Wilcoxon rank-sum test and among the four comparison groups depending on mutational status by Kruskal–Wallis equality-of-populations rank test, followed by Wilcoxon rank-sum test with Bonferroni correction. All tests were two-sided, and P < 0.05 was considered statistically significant. Kaplan–Meier survival analysis was used to see the effect of mutation on the long-term survival of the patients and log-rank test was used to compare their median survival time.


   Results Top


A total of 111 non-APL AMLs were enrolled in the study. The male-female ratio was 1.8:1. FAB classification of the 111 cases showed that majority of the patients belonged to M2 subtype (43.2%), followed by M4 (25.2%). Twenty-seven AML (24.3%) cases were associated with cytogenetic abnormalities while the remaining eighty-four cases (75.7%) were cytogenetically normal (CN). Overall, 16/111 (14.4%) patients were positive for NPM1 mutation and 12/111 (10.8%) showed positivity for FLT3-ITD mutation. Among the AML patients with cytogenetic abnormalities, six belonged to good-risk group, four were poor-risk type, and the rest 17 patients were of the intermediate-risk group [Figure 3]. Molecular mutations were less commonly observed in association with cytogenetic abnormalities (3.7% NPM1+, 11.1% FLT3-ITD+).
Figure 3: The frequency of various cytogenetic abnormalities found in our acute myeloid leukemia patients (n = 27)

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Among the AML patients with normal karyotype (CN-AML), 14.3% (12/84) patients were NPM1+/FLT3-ITD−, 7.1% (6/84) were NPM1−/FLT3-ITD+, 3.6% (3/84) were NPM1+/FLT3-ITD+, while rest 75% (63/84) belonged to NPM1−/FLT3-ITD−group. [Table 1] shows the clinico-hematologic characteristics of CN-AML patients on the basis of NPM1 and FLT3-ITD mutations. Leukocytosis was significantly associated with NPM1+ group as compared to NPM1− groups (P = 0.0019), and more severe degree of anemia was observed in the FLT3-ITD+ patients than the other groups (P = 0.025). No significant correlation was found in terms of age at presentation (P = 0.56), sex ratio (P = 0.467), median platelet count (P = 0.27), and blast percentage between NPM1+ and FLT3-ITD+ groups although there was a slight predilection for males in the NPM1−/FLT3-ITD+ group. FAB-M4 morphology was most commonly associated with NPM1−/FLT3-ITD+ (50%). Of the 63/84 patients, who opted for treatment, CR rates and survival at 1 year was better in the NPM1+/FLT3-ITD− group than the other groups (P = 0.45). [Figure 4] represents the Kaplan–Meier curves for survival estimates in CN-AML. It was also observed that the CR rates, as well as survival at 1 year or last follow-up was better in the NPM−/FLT3-ITD−group than the FLT3-ITD + groups although not reaching statistically significant levels. There was no correlation of NPM or FLT3 mutations with the need for intensive therapy with HiDAC or HAM regimens for re-induction of patients (P = 0.317).
Table 1: Characteristics of acute myeloid leukemia patients with normal karyotype (n=84)

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Figure 4: Kaplan–Meier curves for survival estimates in different molecular groups of cytogenetically normal acute myeloid leukemia patients

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   Discussion Top


Molecular mutations in AML are considered to be major determinants of the patient response to therapy and outcome besides their role in the pathogenesis of the disease.[6] Since both FLT3/ITDs and NPM1 mutations are known to be commoner in AML patients with normal karyotype, clinical outcome was evaluated according to the presence or absence of these two mutations in CN-AML patients, by dividing them into FLT3-ITD+/NPM1−, FLT3-ITD−/NPM1+, FLT3-ITD+/NPM1+ and FLT3-ITD−/NPM1−groups.

The prevalence of FLT3-ITD and NPM1 mutations was found to be relatively lower in the Indian AML patients, i.e., 14.4% and 10.8%, respectively, overall and 17.9% and 10.7% respectively, in CN-AML. This is in striking contrast to several other published studies which have showed higher frequency of these mutations, i.e., 47.9% and 36.6% by Nafea et al., 2011; 35.2% and 26.9% by Falini et al., 2005.[7],[8] The difference in the frequency of these mutations might be due to the geographical distribution of the population studied. Another study from India [9] has also found a lower prevalence rate of these molecular mutations, but they differ from this study in their observation of much higher numbers of FLT3-ITD+/NPM1+ (dual-positive) AML patients (3.6% vs. 45%). Studies have suggested that FLT3ITD mutation occurs more often in cases of NPM + AML than without due to cooperation of these genetic lesions in the promoting leukemic transformation.[3],[10]

In terms of clinico-hematologic characteristics, the results of this study correlated well with the observations of most other studies worldwide.[3],[9] CR rates were better in the FLT3-ITD−/NPM+ group (80%) than the other groups. This may be due to their tendency to form heterodimers in mutated state with the wild-type NPM1 leading to alterations in signal transduction pathways. However, long-term survival was not significantly different between the NPM1+ and NPM1− patients. It might be due to the high association of NPM1 mutation with other bad prognostic factors such as high WBC count, FAB M5 and the presence of FLT3 mutations.[7],[9] This may also account for the importance of investigating for some associated molecular markers such as c-kit or WT-1 which are known to worsen prognosis even in the presence of NPM1 mutations. Moreover, patients belonging to both FLT3-ITD+/NPM1− as well as the FLT3-ITD+/NPM1+ groups had poor CR rates which is similar to other studies,[3],[11] signifying the fact that NPM1 mutation confers favorable prognosis only in the absence of a co-existing FLT3/ITD mutation. Conversely, NPM1 does not have any impact on the adverse prognosis conferred by FLT3/ITD. Few studies have shown that the poorer outcomes of the FLT3 positive patients may also be related to the allele burden of the mutant gene.[12] Levels of mutant alleles were not performed in this study.

Unlike most other studies,[3],[12] improved CR rates as well as disease-free survival were observed in the NPM−/FLT3-ITD− group than the FLT3-ITD+ groups although not reaching statistically significant levels. Few other Indian studies [9],[13] also showed similar trend toward better outcome of dual-positive patients. However, the small sample size and short duration of follow-up of patients precludes any statistically significant conclusions on the impact of the double-negative status on the long-term prognosis of AML patients. Comprehensive studies on a wider range of mutations and their interplay with the more common molecular mutations as well as epigenetic alterations are required to gain insight into the differences in the pathogenesis of the Indian AML patients, which can have an impact on the treatment outcome and survival rates in the Indian settings.


   Conclusion Top


The more common molecular mutations in AML such as NPM1, FLT3-ITD have definite impact on the long-term treatment outcome of the patients, especially in patients with normal karyotype. However, there are some differences in the clinical behavior of the Indian AML patients with respect to these mutations in comparison to that of the West, which reinstate the fact that the biology of AMLs is very diverse and requires larger extensive studies with long follow-up durations to throw deeper insight into the pathogenesis of leukemias.

Acknowledgment

The authors wish to acknowledge Mr Rajender and Mr. R. Jaiswal for technical assistance.

Financial support and sponsorship

This study was supported by grants from the Department of Biotechnology, New Delhi, India.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

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Gilliland DG, Jordan CT, Felix CA. The molecular basis of leukemia. Hematology Am Soc Hematol Educ Program 2004; 80-97.  Back to cited text no. 1
    
2.
Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ, Porwit A, et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: Rationale and important changes. Blood 2009;114:937-51.  Back to cited text no. 2
    
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Thiede C, Koch S, Creutzig E, Steudel C, Illmer T, Schaich M, et al. Prevalence and prognostic impact of NPM1 mutations in 1485 adult patients with acute myeloid leukemia (AML). Blood 2006;107:4011-20.  Back to cited text no. 3
    
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Ottone T, Ammatuna E, Lavorgna S, Noguera NI, Buccisano F, Venditti A, et al. An allele-specific rt-PCR assay to detect type A mutation of the nucleophosmin-1 gene in acute myeloid leukemia. J Mol Diagn 2008;10:212-6.  Back to cited text no. 4
    
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Cheson BD, Bennett JM, Kopecky KJ, Büchner T, Willman CL, Estey EH, et al. Revised recommendations of the International Working Group for diagnosis, standardization of response criteria, treatment outcomes, and reporting standards for therapeutic trials in acute myeloid leukemia. J Clin Oncol 2003; 21:4642-9.  Back to cited text no. 5
    
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Döhner H. Implication of the molecular characterization of acute myeloid leukemia. Hematology Am Soc Hematol Educ Program 2007; 412-9.  Back to cited text no. 6
    
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Nafea D, Rahman MA, Boris D, Perot C, Laporte JP, Isnard F, et al. Incidence and prognostic value of NPM1 and FLT3 gene mutations in AML with normal karyotype. Open Hematol J 2011;5:14-20.  Back to cited text no. 7
    
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Falini B, Mecucci C, Tiacci E, Alcalay M, Rosati R, Pasqualucci L, et al. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med 2005;352:254-66.  Back to cited text no. 8
    
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Dunna NR, Rajappa S, Digumarti R, Vure S, Kagita S, Damineni S, et al. Fms like tyrosine kinase (FLT3) and nucleophosmin 1 (NPM1) mutations in de novo normal karyotype acute myeloid leukemia (AML). Asian Pac J Cancer Prev 2010;11:1811-6.  Back to cited text no. 9
    
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Döhner K, Schlenk RF, Habdank M, Scholl C, Rücker FG, Corbacioglu A, et al. Mutant nucleophosmin (NPM1) predicts favorable prognosis in younger adults with acute myeloid leukemia and normal cytogenetics: Interaction with other gene mutations. Blood 2005;106:3740-6.  Back to cited text no. 10
    
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Schnittger S, Schoch C, Kern W, Mecucci C, Tschulik C, Martelli MF, et al. Nucleophosmin gene mutations are predictors of favorable prognosis in acute myelogenous leukemia with a normal karyotype. Blood 2005;106:3733-9.  Back to cited text no. 11
    
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Gale RE, Green C, Allen C, Mead AJ, Burnett AK, Hills RK, et al. The impact of FLT3 internal tandem duplication mutant level, number, size, and interaction with NPM1 mutations in a large cohort of young adult patients with acute myeloid leukemia. Blood 2008;111:2776-84.  Back to cited text no. 12
    
13.
Ghosh K, Swaminathan S, Madkaikar M, Gupta M, Kerketta L, Vundinti B. FLT3 and NPM1 mutations in a cohort of AML patients and detection of a novel mutation in tyrosine kinase domain of FLT3 gene from Western India. Ann Hematol 2012;91:1703-12.  Back to cited text no. 13
    

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Correspondence Address:
Renu Saxena
Department of Hematology, All India Institute of Medical Sciences, New Delhi - 110 029
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_501_15

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