- Email: [email protected]
Prevalence and Clinical Effect of IDH1 and IDH2 Mutations Among Cytogenetically Normal Acute Myeloid Leukemia Patients
Original Study
Prevalence and Clinical Effect of IDH1 and IDH2 Mutations Among Cytogenetically Normal Acute Myeloid Leukemia Patients Salah Aref,1 El Sayed Kamel Areida,2 Mohamed Fathy Abdel Aaal,2 Ola Mohamed Adam,2 Mohamed Sabry El-Ghonemy,1 Mohamed Ali El-Baiomy,3 Tarek Abou Zeid4 Abstract This study aimed to determine the frequencies and clinical impact of isocitrate dehydrogenase (IDH) 1 and IDH2 mutations among Egyptian acute myeloid leukemia (AML) patients. The exon 4 of IDH1 and IDH2 were sequenced for detection of mutations in 211 AML bone marrow samples. The IDH1 and IDH2 mutations were detected in 8.5%, and 10.4% respectively. IDH1 and IDH2 mutations are negative prognostic markers in AML patients. A novel mutation (R132V) was detected in IDH1 in our cohort of AML patients. Background: The frequencies of isocitrate dehydrogenase (IDH) 1 and IDH2 mutations among patients with de novo acute myeloid leukemia (AML) are different among different ethnic groups. The aim of this study was to determine the frequencies of IDH1 and IDH2 mutations among Egyptian AML patients and its effect on patient outcomes. Patients and Methods: This study was conducted in 211 adult patients (104 men; 107 women; age range, 18-68 years) with cytogenetically normal AML. DNA was extracted from bone marrow samples at the time of diagnosis. The exon 4 of IDH1 and IDH2 were amplified using polymerase chain reaction and sequenced for detection of mutations. Results: IDH1 mutations were detected in 18 of 211 AML patients (8.5%) in the form of 8 cases, R132H; 6 cases, R132C; 2 cases, R132S; 1 case, R132G; and 1 case, R132V mutations). IDH2 mutations were detected in 22 of 211 AML patients (10.4%) in the form of 20 cases, R140Q; and 2 cases, R172K mutations. The overall survival after correction for nucleophosmin 1 and fims- related tyrosine kinase internal tandem duplication was significantly shorter in AML patients with the IDH mutation compared with those with wild type (P ¼ .02). Conclusion: IDH1 and IDH2 mutations are negative prognostic markers in AML patients. A novel mutation (R132V) was detected in IDH1 in our cohort of AML patients. We recommend molecular testing for IDH1 and IDH2 mutations for proper risk stratification of AML patients before the start of therapy. Clinical Lymphoma, Myeloma & Leukemia, Vol. -, No. -, --- ª 2015 Elsevier Inc. All rights reserved. Keywords: AML, Prognosis, Mutation, IDH
Introduction 1 Hematology Unit, Clinical Pathology Department, Mansoura University, Mansoura, Egypt 2 Faculty of Science, Mansoura University, Mansoura, Egypt 3 Medical Oncology Unit, Mansoura University Oncology Center, Mansoura Faculty of Medicine, Mansoura, Egypt 4 Clinical Hematology Unit, Mansoura University Oncology Center, Mansoura Faculty of Medicine, Mansoura, Egypt
Submitted: Mar 25, 2015; Revised: May 11, 2015; Accepted: May 29, 2015 Address for correspondence: Salah Aref, MD, Hematology Unit, Clinical Pathology Department, Mansoura Faculty of Medicine, Mansoura, Egypt E-mail contact: [email protected]
2152-2650/$ - see frontmatter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clml.2015.05.009
Acute myeloid leukemia (AML) is a clonal malignant disease of hematopoietic tissue caused by somatic mutations in genes that control normal cell proliferation and differentiation.1-4 The molecular genetic alterations are one of the most important prognostic factors that have been identified in AML and the role of these genetic alterations has been emphasized by the 2008 revised World Health Organization classification of AML like nucleophosmin (NPM) 1, and CCAAT enhancer-binding protein a (CEBPA).1,2,5,6 Among these are epigenetic mutations that include isocitrate dehydrogenase (IDH) 1 and IDH2 mutations. The IDH is an enzyme that catalyzes oxidative decarboxylation
Clinical Lymphoma, Myeloma & Leukemia Month 2015
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IDH1 and IDH2 Mutations in Cytogenetically Normal AML Table 1 Clinical and Demographic Characteristics of the AML Patients With Wild Type and Mutant IDH1 IDH1 Wild Type (n [ 193) Characteristic
n
%
IDH1 Mutant Type (n [ 18) n
%
P >.05
FAB Type M0 (n ¼ 3)
3
1.6
0
0
M1 (n ¼ 49)
48
24.9
1
5.6
M2 (n ¼ 63)
50
25.9
13
72.2
M3 (n ¼ 29)
25
12.9
4
22.2
M4 (n ¼ 32)
32
16.6
0
0
M5 (n ¼ 24)
24
12.4
0
0
M6 (n ¼ 9)
9
4.7
0
0
M7 (n ¼ 2)
2
1.0
0
0
<60 Years (n ¼ 178)
172
96.6
6
3.4
60 Years (n ¼ 33)
21
63.6
12
36.4
Female (n ¼ 107)
94
87.9
13
12.1
Male (n ¼ 104)
99
95.2
5
4.8
<.001
Age
Sex .047
Extramedullary Disease Absent (n ¼ 187)
171
91.4
16
8.6
Present (n ¼ 24)
22
91.7
2
8.3
144
93.5
10
6.4
49
85.9
8
14.1
Normal (n ¼ 154)
142
92.2
12
7.8
Abnormal (n ¼ 57)
51
89.5
6
10.5
159
90.9
16
9.1
34
94.4
2
5.6
125
91.2
12
8.8
68
91.9
6
8.1
153
90.5
16
9.5
40
95.2
2
4.8
>.05
Performance Status 0 and 1 (n ¼ 154) 2 (n ¼ 57)
>.05
Karyotyping >.05
Cytogenetics Favorable/intermediate (n ¼ 175) Adverse (n ¼ 36)
>.05
NPM1 Mutation Wild type (n ¼ 137) Mutant (n ¼ 74)
>.05
FLT3 ITD Mutation Wild type (n ¼ 169) Mutant (n ¼ 42)
>.05
7.6 1.5
8.04 2.78
>.05
Platelets (gm/dl), Mean ± SD
51.8 14.4
96 32.7
<.001
WBC Count (gm/dl), Mean ± SD
62.1 75.59
52.1 88.6
>.05
BM Blast (gm/dl), Mean ± SD
33.9 21.5
75.4 17.75
<.001
Hemoglobin (gm/dl), Mean ± SD
P < .05 is significant; P < .001 is highly significant, and P > .05 is not significant. Abbreviations: AML ¼ acute myeloid leukemia; BM ¼ bone marrow; FAB ¼ French-American-British; FLT3 ITD ¼ fims- related tyrosine kinase internal tandem duplication; WBC ¼ white blood cell.
2
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of isocitrate into ketoglutarate by using NAD or NADP as a cofactor to yield NADH or NADPH, respectively. There are 3 classes of IDH isoenzymes (mitochondrial NAD-dependent IDH, mitochondrial NADP-dependent IDH, and cytosolic NADP-dependent IDH). The NADP-dependent IDH1 enzyme is encoded by the IDH1 gene at chromosome 2 and is localized in cytoplasm and peroxisomes, and the mitochondrial NADPdependent IDH2 enzyme is encoded by the IDH2 gene at chromosome 15.5-7
Clinical Lymphoma, Myeloma & Leukemia Month 2015
The IDH1 and IDH2 mutations have been identified in glioma, cartilaginous tumors, thyroid carcinomas, cholangiocarcinoma, prostate cancers, paragangliomas, melanoma, chronic-, fibrotic-, and blastphase essential thrombocythemia, polycythemia vera or myelofibrosis, and AML. In AML the IDH1 and IDH2 mutations are frequently associated with blastic transformation or aggressive forms.4,8-10 The aim of this study was to evaluate the prevalence, clinical, and prognostic effect of IDH1 and IDH2 mutations in AML patients treated at Mansoura University Oncology Center.
Salah Aref et al Table 2 Clinical and Demographic Characteristics of the AML Patients With Wild Type and Mutant IDH2 IDH2 Wild Type (n [ 189) Characteristic
n
%
IDH2 Mutant Type (n [ 22) n
%
FAB type
P .26
M0 (n ¼ 3)
2
1.1
1
4.5
M1 (n ¼ 49)
48
25.4
1
4.5
M2 (n ¼ 63)
47
24.9
16
72.7
M3 (n ¼ 29)
26
13.7
3
13.6
M4 (n ¼ 32)
31
16.4
1
4.5
M5 (n ¼ 24)
24
12.7
0
0
M6 (n ¼ 9)
9
4.8
0
0
M7 (n ¼ 2)
2
1.0
0
0
<60 Years (n ¼ 178)
169
95.0
9
5.0
60 Years (n ¼ 33)
20
60.6
13
39.4
Female (n ¼ 107)
95
88.8
12
11.2
Male (n ¼ 104)
94
90.4
10
9.6
Absent (n ¼ 187)
167
89.3
20
10.7
Present (n ¼ 24)
22
91.7
2
8.3
139
90.2
15
9.8
50
87.7
7
12.3
Normal (n ¼ 154)
132
85.7
22
14.3
Abnormal (n ¼ 57)
57
100
0
0
154
88
21
12
Age
.005
>.05
Sex
>.05
Extramedullary Disease
>.05
Performance Status 0 and 1 (n ¼ 154) 2 (n ¼ 57) Karyotyping
.001
>.05
Cytogenetics Favorable/intermediate (n ¼ 175) Adverse (n ¼ 36)
35
97.2
1
2.8
120
87.6
17.0
12.4
69
93.2
5.0
6.8
149
88.2
20
11.8
40
95.2
2
4.8
NPM1 Mutation
>.05
Wild type (n ¼ 137) Mutant (n ¼ 74) FLT3 ITD Mutation
>.05
Wild type (n ¼ 169) Mutant (n ¼ 42) Hemoglobin, Mean ± SD (gm/dl)
7.7 1.75
7.6 1.38
>.05
Platelets, Mean ± SD (106/cmm)
23 12.0
114 30.0
<.001
6
WBC, Mean ± SD (10 /cmm) BM Blast, Mean ± SD (%)
66.9 19.4
22.9 8.42
37.06 23.85
36.8 20.9
.002 >.05
P < .05 is significant; P < .001 is highly significant, and P > .05 is not significant. Abbreviations: AML ¼ acute myeloid leukemia; BM ¼ bone marrow; FAB ¼ French-American-British; FLT3 ITD ¼ fims- related tyrosine kinase internal tandem duplication; WBC ¼ white blood cell.
Patients and Methods Patients and Treatment Protocols This study was carried out in 211 adult patients (aged 17-68 years), included 104 men and 107 women diagnosed with AML on the basis of bone marrow (BM) and peripheral blood morphology, cytochemistry, and immunophenotyping. Immunophenotyping using a Coulter Epics XL flow cytometer (PN 42372238; B. Coulter Corp, Miami, FL) for confirmation
of diagnosis using a cytoplasmic myeloperoxidase, CD13, CD33, and CD117 primary panel for myeloid lineage, CD14, CD36, and CD11b for M4 and M5, CD61, and glycophorin A for M6, and CD41, and CD42 for M7. The AML patients were divided according to the French-American-British subtypes into 3 M0, 49 M1, 36 M2, 32 M4, 24 M5, 9 M6, and 2 M7. All patients gave informed consent for treatment and genetic analysis. All patients received intensive induction therapy (cytarabine 100 mg/m2/d for
Clinical Lymphoma, Myeloma & Leukemia Month 2015
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IDH1 and IDH2 Mutations in Cytogenetically Normal AML 7 days intravenous [I.V.] continuous infusion and daunorubicin 90 mg/m2/d for 3 days I.V.), and consolidation therapy (cytarabine 1 g/m2 for 12 hours on the first, third, and fifth days with daunorubicin 45 mg/m2/d for 3 days I.V.). The patients who achieved complete remission with poor risk cytogenetics or in whom induction therapy failed or had disease relapse were prepared for BM transplantation. All patients were observed for 24 months or until death.
Cytogenetic and Molecular Genetic Analyses Blood samples from all patients collected before treatment were studied using chromosome banding analysis to improve the accuracy of cytogenetic diagnosis. The specimens were also analyzed using fluorescence in situ hybridization for the presence of t(8;21)(q22;q22) for M2, t(15;17)(q22;q12) for M3, inv(16)(p13q22) for M4e, and 11q23 for M5.
Determination of IDH1 and IDH2 Mutations Using Polymerase Chain Reaction The exon 4 of IDH1 and IDH2 were amplified using polymerase chain reaction (PCR) from extracted DNA using the primer pairs: IDH1f (50 -AGCTCTATATGCCATCACTGC-30 ), IDH1r (50 -AACATGCAAAATCACATTATTGCC-30 ), IDH2f (50 -AATTTTAGGACCCCCGTCTG-30 ), and IDH2r (50 -CTGCAGAGACAAGAGGATGG-30 ). Cycling conditions were initial denaturation at 95 C for 4 minutes, 35 cycles at 95 C for 10 seconds, 60 C for 30 seconds and 72 C for 30 seconds, and final extension at 72 C for 5 minutes. The PCR products were sequenced for mutation screens. Neucleophesmin 1 and fims- related tyrosine kinase internal tandem duplication (FLT3-ITD) mutations were assessed using the sequencing process as previously described.5
Statistical Analysis The statistical analysis of data was done using Excel (IBM Corp, Chicago, IL) version 2007 and SPSS (IBM Corp, Chicago, IL) version 16. Qualitative data were described in the form of numbers and percentages. Quantitative data were described in the form of mean SD. Statistical analyses on comparisons between groups were done using the c2 test regarding qualitative data and quantitative nonparametric data comparisons were performed using 1-way
analysis of variance and paired sample t test. The probability of being by chance (P value) was calculated for all parameters (P was considered significant if .05 at 95% confidence interval).
Results Prevalence of IDH1 and IDH2 Mutations Isocitrate dehydrogenase 1 mutations were detected in 18 cases of 211 patients with de novo AML (8.5%) in the form of in 8 cases R132H, 6 cases R132C, 2 cases R132S, 1 case R132G, and 1 case R132V mutations. Moreover, IDH2 mutations were detected in 22 cases of 211 patients with de novo AML (10.4%) in the form of in 20 cases R140Q and 2 cases R172K (Table 1). No single AML patient had combined mutations (both IDH1 and IDH2).
Clinical and Demographic Characteristics of Patients With IDH1 and IDH2 Mutations The clinical and demographic characteristics of the IDHmutated AML patients are shown in Tables 2 and 3. It was noticed that the IDH1 mutant patients were of older age, mostly female, had high platelet counts, and BM blast cell counts compared with the mutated NPM1 AML patients. However, there was no difference between the 2 groups with regard to French-AmericanBritish subtypes, performance status, extramedullary disease, cytogenetic status, hemoglobin concentration, and FLT3 ITD mutation. In the IDH2 mutant group, there was no difference between the 2 groups with regard to sex, French-American-British subtype, performance status, extramedullary disease, cytogenetic status, hemoglobin concentration, and BM blast cell counts and were not related to NPM1 or FLT3 mutations. However, the IDH2 mutation was more common in patients with older age, normal karyotyping, low white blood cell (WBC) count, and high platelet count.
Response to Therapy and Clinical Outcome in IDH-Mutant Patients There was no difference in induction or remission rate between the mutant and wild type groups of patients. The induction or remission rate in the wild type and mutant patients were 114 (66.4%) versus 26 (65%), respectively (Table 4).
Table 3 Type of IDH1 and IDH2 Mutations Identified in 40 Acute Myeloid Leukemia Patients Mutation
Nucleotide Change
Predicted Protein Change
Patient n
G395A
CGT-CAT
R132H
8
C394T
CGT-TGT
R132C
6
C394A
CGT-AGT
R132S
2
C394G
CGT-GGT
R132G
1
G395T
CGT-CTT
IDH1 (n [ 18)
CG394-395GT
R132L
0
R132V
1
IDH2 (n [ 22)
4
-
G419A
CGG-CAG
R140Q
20
G515A
AGG-AAG
R172K
2
Clinical Lymphoma, Myeloma & Leukemia Month 2015
Salah Aref et al Table 4 Response to Therapy in IDH-Mutant AML Patients Versus Wild Type Patients Remission After First Cycle IDH
Not in Remission
In Remission
Total
P >.05
Wild Type AML n
57
%
33.3
114 66.7
171
n
14
26
40
%
35
65
100.0
100.0
Mutant AML
These findings are in agreement with those reported by Chotirat et al,11 who stated that the overall frequency of IDH mutations appears to vary between 2% and 14% for IDH1 and 1% and 19% for IDH2 in studies from the Asia continent (Taiwan and China), the United States, Canada, France, Germany, The Netherlands, and the United Kingdom. Likewise, Paschka et al5 reported that the frequency of IDH1 mutations among AML patients was 7.6%, and IDH2 was 8.7%, and that 2 patients had IDH1 and IDH2 mutations. However, Nomdedéu et al4 reported that IDH1 or IDH2 mutations were identified in 23.3% of AML patients and in 22.5% of those with a normal karyotype. This difference could be attributed to the different ethnic groups. In the present study, IDH1 mutations were in the form of 8 cases R132H, 6 cases R132C, 2 cases R132S, 1 case R132G, and 1 case R132V mutations; moreover, the IDH2 mutations were in the form of 20 cases R140Q and 2 cases R172K. Paschka et al5 found that all but 1 IDH1 mutation caused substitutions of residue R132; IDH2
Survival Analysis With regard to overall survival (OS), the patients with the IDH mutation had shorter OS than patients with wild type (10 months vs. 18 months; P ¼ .02; Figure 1).
Discussion Aberrant epigenetic programming is a hallmark of cancer. In this context IDH1 and IDH2 genes were described to be frequently mutated in multiple types of human cancer. Mutations result in simultaneous loss of their normal catalytic activity and gain of a new function.5,6 Several studies have been conducted to assess the incidence of IDH1 and IDH2 genes in multiple human tumors especially in AML, however, their prognostic effect and their prevalence in our locality remain to be elucidated. In the current study, IDH1 mutations were identified in 18 of 211 patients with de novo AML (8.5%) and IDH2 mutations were identified in 22 of 211 patients with de novo AML (10.4%).
Figure 1 Overall Survival (Months) of IDH-Mutant Acute Myeloid Leukemia (AML) Patients (n [ 35) Versus IDH Wild Type AML Patients. Patients (n [ 75) With Exclusion of NPM1- and FLT3-Mutant Patients
Survival Functions 1.0
Cumulative Survival
Wild IDH without NPM1 or FLT3 mutations Mutated IDH without NPM1 or FLT3 mutations
0.8
0.6
P = .02 0.4
0.2
0.0 10
20
30
40
OS
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IDH1 and IDH2 Mutations in Cytogenetically Normal AML mutations caused changes of R140 (n ¼ 48) or R172 (n ¼ 22). Moreover, Yamaguchi et al9 detected IDH1 R132 mutations in 20 (8.6%) patients with AML and IDH2 mutations in 19 (8.2%; 17 R140 and 2 R172) patients. Patel et al12 reported that IDH1 R132 mutation was detected in 12 of 199 patients with AML (6.0%), and the IDH2 R172 mutation was detected in 4 of 196 patients with AML (2.0%), and stated that no mutated cases had both IDH1 and IDH2 mutations, which suggests that these mutations are mutually exclusive. In our study we reported a novel mutation, which is the R132V mutation, detected in our patients. This mutation was not previously reported in AML but it was described in other tumors like glioma.13-15 The correlation of the demographic and laboratory data in the current studied cohort of AML patients revealed that there was significant difference between the wild and mutant IDH1 patients with regard to age, sex, platelet counts, BM blast cells, and the NPM1 mutation status. Similar findings were reported by Paschka et al,5 who found that IDH1-mutated patients were older, had higher platelet counts, had higher percentages of BM blasts, and were associated with mutated NPM1 than patients with wild type IDH1. The IDH2 mutation was detected more commonly in patients with older age, normal karyotyping, and low WBC count and high platelet counts and with no difference between the 2 groups with regard to sex, French-American-British subtype, performance status, extramedullary disease, cytogenetic status, hemoglobin concentration, BM blast cell counts, and NPM1 mutation. This in agreement with Chotirat et al11 and Paschka et al,5 who found that IDH2 mutation was associated with older age, a higher platelet count, and normal karyotype AML but they found that the IDH2 mutation was not associated with low WBC counts and was associated with the NPM1 mutation. Our study revealed that there was no significant difference between the IDH mutation in either IDH1 or IDH2 on the induction or remission rate (P ¼ .4). Similar findings were reported by Paschka et al,5 who found that the IDH mutations had no effect on the response to induction or treatment of AML. In the current study, the patients with IDH mutations after correction for NPM1 and FLT3 ITD had a significantly shorter OS compared with those in patients with wild type (P ¼ .02). This finding is in agreement with that reported by Yamaguchi et al,9 who concluded that IDH mutations have an unfavorable effect in AML, especially AML with the NPM1 wild type, and mentioned that younger AML patients with IDH mutations might benefit from allogeneic stem cell transplantation. Moreover, Nomdedéu et al4 also reported that AML patients with the IDH mutation were associated with short OS and stated that this adverse effect was even more evident in patients with the NPM- or CEBPA-mutated/FLT3 wild type genotype. In contrast, Paschka et al5 stated that IDH1 and IDH2 mutations had no effect on relapse-free survival or OS. This might be because of the simultaneous presence of other mutations like NPM1, which modify their effects.
6
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Clinical Lymphoma, Myeloma & Leukemia Month 2015
Conclusion Isocitrate dehydrogenase 1 and IDH2 mutations are negative prognostic markers in AML patients. A novel mutation (R132V) was detected in IDH1 in our cohort of AML patients. We recommend molecular testing for IDH1 and IDH2 mutations to make proper risk stratification of AML patients before treatment.
Clinical Practice Points The prevalence of IDH1 and IDH2 mutations among different
cohorts of AML patients in previous studies showed wide variations. The negative clinical effect of IDH mutations on cytogenetically normal AML patients was previously reported. In the current study the prevalence of IDH1 and IDH2 mutations was not previously reported in our cohort of Egyptian patients. A novel mutation (R132V) was detected in IDH1 in our cohort of AML patients. Our findings will be added to the data base concerning this subject.
Disclosure The authors have stated that they have no conflicts of interest.
References 1. Byrd J, Mrozek K, Dodge R, et al. Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood 2002; 100:4325-36. 2. Appelbaum F, Gundacker H, Head D, et al. Age and acute myeloid leukemia. Blood 2006; 107:3481-5. 3. Foran J. New prognostic markers in acute myeloid leukemia: perspective from the clinic. Hematology 2010; 1:47-55. 4. Nomdedéu J, Hoyos M, Carricondo M, et al. Adverse impact of IDH1 and IDH2 mutations in primary AML: experience of the Spanish CETLAM group. Leuk Res 2012; 36:990-7. 5. Paschka P, Schlenk R, Gaidzik V, et al. IDH1 and IDH2 mutations are frequent genetic alterations in acute myeloid leukemia and confer adverse prognosis in cytogenetically normal acute myeloid leukemia with NPM1 mutation without FLT3 internal tandem duplication. J Clin Oncol 2010; 28:3636-43. 6. Sjöblom T, Jones S, Wood LD, et al. The consensus coding sequences of human breast and colorectal cancers. Science 2006; 314:268-74. 7. Cairns R, Mak T. Oncogenic isocitrate dehydrogenase mutations: mechanisms, models, and clinical opportunities. Cancer Discov 2013; 3:730-41. 8. Green A, Beer P. Somatic mutations of IDH1 and IDH2 in the leukemic transformation of myeloproliferative neoplasms. N Engl J Med 2010; 362:369-70. 9. Yamaguchi S, Iwanaga E, Tokunaga K, et al. IDH1 and IDH2 mutations confer an adverse effect in patients with acute myeloid leukemia lacking the NPM1 mutation. Eur J Haematol 2015; 92:471-7. 10. Tefferi A, Lasho T, Abdel-Wahab O, et al. IDH1 and IDH2 mutation studies in 1473 patients with chronic-, fibrotic- or blast-phase essential thrombocythemia, polycythemia vera or myelofibrosis. Leukemia 2010; 24:1302-9. 11. Chotirat S, Thongnoppakhun W, Promsuwicha O, Boonthimat C, Auewarakul C. Molecular alterations of isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) metabolic genes and additional genetic mutations in newly diagnosed acute myeloid leukemia patients. J Hematol Oncol 2012; 5:5. 12. Patel KP, Ravandi F, Ma D, et al. Acute myeloid leukemia with IDH1 or IDH2 mutation: frequency and clinicopathologic features. Am J Clin Pathol 2011; 135: 35-45. 13. Yang H, Ye D, Guan K, Xiong Y. IDH1 and IDH2 mutations in tumorigenesis: mechanistic insights and clinical perspectives. Clin Cancer Res 2012; 18:5562-71. 14. Yan H, Parsons D, Jin G, et al. IDH1 and IDH2 mutations in gliomas. N Engl J Med 2009; 360:765-73. 15. Parsons D, Jones S, Zhang X, et al. An integrated genomic analysis of human glioblastoma multiforme. Science 2008; 321:1807-12.
Prevalence and Clinical Effect of IDH1 and IDH2 Mutations Among Cytogenetically Normal Acute Myeloid Leukemia Patients Salah Aref,1 El Sayed Kamel Areida,2 Mohamed Fathy Abdel Aaal,2 Ola Mohamed Adam,2 Mohamed Sabry El-Ghonemy,1 Mohamed Ali El-Baiomy,3 Tarek Abou Zeid4 Abstract This study aimed to determine the frequencies and clinical impact of isocitrate dehydrogenase (IDH) 1 and IDH2 mutations among Egyptian acute myeloid leukemia (AML) patients. The exon 4 of IDH1 and IDH2 were sequenced for detection of mutations in 211 AML bone marrow samples. The IDH1 and IDH2 mutations were detected in 8.5%, and 10.4% respectively. IDH1 and IDH2 mutations are negative prognostic markers in AML patients. A novel mutation (R132V) was detected in IDH1 in our cohort of AML patients. Background: The frequencies of isocitrate dehydrogenase (IDH) 1 and IDH2 mutations among patients with de novo acute myeloid leukemia (AML) are different among different ethnic groups. The aim of this study was to determine the frequencies of IDH1 and IDH2 mutations among Egyptian AML patients and its effect on patient outcomes. Patients and Methods: This study was conducted in 211 adult patients (104 men; 107 women; age range, 18-68 years) with cytogenetically normal AML. DNA was extracted from bone marrow samples at the time of diagnosis. The exon 4 of IDH1 and IDH2 were amplified using polymerase chain reaction and sequenced for detection of mutations. Results: IDH1 mutations were detected in 18 of 211 AML patients (8.5%) in the form of 8 cases, R132H; 6 cases, R132C; 2 cases, R132S; 1 case, R132G; and 1 case, R132V mutations). IDH2 mutations were detected in 22 of 211 AML patients (10.4%) in the form of 20 cases, R140Q; and 2 cases, R172K mutations. The overall survival after correction for nucleophosmin 1 and fims- related tyrosine kinase internal tandem duplication was significantly shorter in AML patients with the IDH mutation compared with those with wild type (P ¼ .02). Conclusion: IDH1 and IDH2 mutations are negative prognostic markers in AML patients. A novel mutation (R132V) was detected in IDH1 in our cohort of AML patients. We recommend molecular testing for IDH1 and IDH2 mutations for proper risk stratification of AML patients before the start of therapy. Clinical Lymphoma, Myeloma & Leukemia, Vol. -, No. -, --- ª 2015 Elsevier Inc. All rights reserved. Keywords: AML, Prognosis, Mutation, IDH
Introduction 1 Hematology Unit, Clinical Pathology Department, Mansoura University, Mansoura, Egypt 2 Faculty of Science, Mansoura University, Mansoura, Egypt 3 Medical Oncology Unit, Mansoura University Oncology Center, Mansoura Faculty of Medicine, Mansoura, Egypt 4 Clinical Hematology Unit, Mansoura University Oncology Center, Mansoura Faculty of Medicine, Mansoura, Egypt
Submitted: Mar 25, 2015; Revised: May 11, 2015; Accepted: May 29, 2015 Address for correspondence: Salah Aref, MD, Hematology Unit, Clinical Pathology Department, Mansoura Faculty of Medicine, Mansoura, Egypt E-mail contact: [email protected]
2152-2650/$ - see frontmatter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clml.2015.05.009
Acute myeloid leukemia (AML) is a clonal malignant disease of hematopoietic tissue caused by somatic mutations in genes that control normal cell proliferation and differentiation.1-4 The molecular genetic alterations are one of the most important prognostic factors that have been identified in AML and the role of these genetic alterations has been emphasized by the 2008 revised World Health Organization classification of AML like nucleophosmin (NPM) 1, and CCAAT enhancer-binding protein a (CEBPA).1,2,5,6 Among these are epigenetic mutations that include isocitrate dehydrogenase (IDH) 1 and IDH2 mutations. The IDH is an enzyme that catalyzes oxidative decarboxylation
Clinical Lymphoma, Myeloma & Leukemia Month 2015
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IDH1 and IDH2 Mutations in Cytogenetically Normal AML Table 1 Clinical and Demographic Characteristics of the AML Patients With Wild Type and Mutant IDH1 IDH1 Wild Type (n [ 193) Characteristic
n
%
IDH1 Mutant Type (n [ 18) n
%
P >.05
FAB Type M0 (n ¼ 3)
3
1.6
0
0
M1 (n ¼ 49)
48
24.9
1
5.6
M2 (n ¼ 63)
50
25.9
13
72.2
M3 (n ¼ 29)
25
12.9
4
22.2
M4 (n ¼ 32)
32
16.6
0
0
M5 (n ¼ 24)
24
12.4
0
0
M6 (n ¼ 9)
9
4.7
0
0
M7 (n ¼ 2)
2
1.0
0
0
<60 Years (n ¼ 178)
172
96.6
6
3.4
60 Years (n ¼ 33)
21
63.6
12
36.4
Female (n ¼ 107)
94
87.9
13
12.1
Male (n ¼ 104)
99
95.2
5
4.8
<.001
Age
Sex .047
Extramedullary Disease Absent (n ¼ 187)
171
91.4
16
8.6
Present (n ¼ 24)
22
91.7
2
8.3
144
93.5
10
6.4
49
85.9
8
14.1
Normal (n ¼ 154)
142
92.2
12
7.8
Abnormal (n ¼ 57)
51
89.5
6
10.5
159
90.9
16
9.1
34
94.4
2
5.6
125
91.2
12
8.8
68
91.9
6
8.1
153
90.5
16
9.5
40
95.2
2
4.8
>.05
Performance Status 0 and 1 (n ¼ 154) 2 (n ¼ 57)
>.05
Karyotyping >.05
Cytogenetics Favorable/intermediate (n ¼ 175) Adverse (n ¼ 36)
>.05
NPM1 Mutation Wild type (n ¼ 137) Mutant (n ¼ 74)
>.05
FLT3 ITD Mutation Wild type (n ¼ 169) Mutant (n ¼ 42)
>.05
7.6 1.5
8.04 2.78
>.05
Platelets (gm/dl), Mean ± SD
51.8 14.4
96 32.7
<.001
WBC Count (gm/dl), Mean ± SD
62.1 75.59
52.1 88.6
>.05
BM Blast (gm/dl), Mean ± SD
33.9 21.5
75.4 17.75
<.001
Hemoglobin (gm/dl), Mean ± SD
P < .05 is significant; P < .001 is highly significant, and P > .05 is not significant. Abbreviations: AML ¼ acute myeloid leukemia; BM ¼ bone marrow; FAB ¼ French-American-British; FLT3 ITD ¼ fims- related tyrosine kinase internal tandem duplication; WBC ¼ white blood cell.
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of isocitrate into ketoglutarate by using NAD or NADP as a cofactor to yield NADH or NADPH, respectively. There are 3 classes of IDH isoenzymes (mitochondrial NAD-dependent IDH, mitochondrial NADP-dependent IDH, and cytosolic NADP-dependent IDH). The NADP-dependent IDH1 enzyme is encoded by the IDH1 gene at chromosome 2 and is localized in cytoplasm and peroxisomes, and the mitochondrial NADPdependent IDH2 enzyme is encoded by the IDH2 gene at chromosome 15.5-7
Clinical Lymphoma, Myeloma & Leukemia Month 2015
The IDH1 and IDH2 mutations have been identified in glioma, cartilaginous tumors, thyroid carcinomas, cholangiocarcinoma, prostate cancers, paragangliomas, melanoma, chronic-, fibrotic-, and blastphase essential thrombocythemia, polycythemia vera or myelofibrosis, and AML. In AML the IDH1 and IDH2 mutations are frequently associated with blastic transformation or aggressive forms.4,8-10 The aim of this study was to evaluate the prevalence, clinical, and prognostic effect of IDH1 and IDH2 mutations in AML patients treated at Mansoura University Oncology Center.
Salah Aref et al Table 2 Clinical and Demographic Characteristics of the AML Patients With Wild Type and Mutant IDH2 IDH2 Wild Type (n [ 189) Characteristic
n
%
IDH2 Mutant Type (n [ 22) n
%
FAB type
P .26
M0 (n ¼ 3)
2
1.1
1
4.5
M1 (n ¼ 49)
48
25.4
1
4.5
M2 (n ¼ 63)
47
24.9
16
72.7
M3 (n ¼ 29)
26
13.7
3
13.6
M4 (n ¼ 32)
31
16.4
1
4.5
M5 (n ¼ 24)
24
12.7
0
0
M6 (n ¼ 9)
9
4.8
0
0
M7 (n ¼ 2)
2
1.0
0
0
<60 Years (n ¼ 178)
169
95.0
9
5.0
60 Years (n ¼ 33)
20
60.6
13
39.4
Female (n ¼ 107)
95
88.8
12
11.2
Male (n ¼ 104)
94
90.4
10
9.6
Absent (n ¼ 187)
167
89.3
20
10.7
Present (n ¼ 24)
22
91.7
2
8.3
139
90.2
15
9.8
50
87.7
7
12.3
Normal (n ¼ 154)
132
85.7
22
14.3
Abnormal (n ¼ 57)
57
100
0
0
154
88
21
12
Age
.005
>.05
Sex
>.05
Extramedullary Disease
>.05
Performance Status 0 and 1 (n ¼ 154) 2 (n ¼ 57) Karyotyping
.001
>.05
Cytogenetics Favorable/intermediate (n ¼ 175) Adverse (n ¼ 36)
35
97.2
1
2.8
120
87.6
17.0
12.4
69
93.2
5.0
6.8
149
88.2
20
11.8
40
95.2
2
4.8
NPM1 Mutation
>.05
Wild type (n ¼ 137) Mutant (n ¼ 74) FLT3 ITD Mutation
>.05
Wild type (n ¼ 169) Mutant (n ¼ 42) Hemoglobin, Mean ± SD (gm/dl)
7.7 1.75
7.6 1.38
>.05
Platelets, Mean ± SD (106/cmm)
23 12.0
114 30.0
<.001
6
WBC, Mean ± SD (10 /cmm) BM Blast, Mean ± SD (%)
66.9 19.4
22.9 8.42
37.06 23.85
36.8 20.9
.002 >.05
P < .05 is significant; P < .001 is highly significant, and P > .05 is not significant. Abbreviations: AML ¼ acute myeloid leukemia; BM ¼ bone marrow; FAB ¼ French-American-British; FLT3 ITD ¼ fims- related tyrosine kinase internal tandem duplication; WBC ¼ white blood cell.
Patients and Methods Patients and Treatment Protocols This study was carried out in 211 adult patients (aged 17-68 years), included 104 men and 107 women diagnosed with AML on the basis of bone marrow (BM) and peripheral blood morphology, cytochemistry, and immunophenotyping. Immunophenotyping using a Coulter Epics XL flow cytometer (PN 42372238; B. Coulter Corp, Miami, FL) for confirmation
of diagnosis using a cytoplasmic myeloperoxidase, CD13, CD33, and CD117 primary panel for myeloid lineage, CD14, CD36, and CD11b for M4 and M5, CD61, and glycophorin A for M6, and CD41, and CD42 for M7. The AML patients were divided according to the French-American-British subtypes into 3 M0, 49 M1, 36 M2, 32 M4, 24 M5, 9 M6, and 2 M7. All patients gave informed consent for treatment and genetic analysis. All patients received intensive induction therapy (cytarabine 100 mg/m2/d for
Clinical Lymphoma, Myeloma & Leukemia Month 2015
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IDH1 and IDH2 Mutations in Cytogenetically Normal AML 7 days intravenous [I.V.] continuous infusion and daunorubicin 90 mg/m2/d for 3 days I.V.), and consolidation therapy (cytarabine 1 g/m2 for 12 hours on the first, third, and fifth days with daunorubicin 45 mg/m2/d for 3 days I.V.). The patients who achieved complete remission with poor risk cytogenetics or in whom induction therapy failed or had disease relapse were prepared for BM transplantation. All patients were observed for 24 months or until death.
Cytogenetic and Molecular Genetic Analyses Blood samples from all patients collected before treatment were studied using chromosome banding analysis to improve the accuracy of cytogenetic diagnosis. The specimens were also analyzed using fluorescence in situ hybridization for the presence of t(8;21)(q22;q22) for M2, t(15;17)(q22;q12) for M3, inv(16)(p13q22) for M4e, and 11q23 for M5.
Determination of IDH1 and IDH2 Mutations Using Polymerase Chain Reaction The exon 4 of IDH1 and IDH2 were amplified using polymerase chain reaction (PCR) from extracted DNA using the primer pairs: IDH1f (50 -AGCTCTATATGCCATCACTGC-30 ), IDH1r (50 -AACATGCAAAATCACATTATTGCC-30 ), IDH2f (50 -AATTTTAGGACCCCCGTCTG-30 ), and IDH2r (50 -CTGCAGAGACAAGAGGATGG-30 ). Cycling conditions were initial denaturation at 95 C for 4 minutes, 35 cycles at 95 C for 10 seconds, 60 C for 30 seconds and 72 C for 30 seconds, and final extension at 72 C for 5 minutes. The PCR products were sequenced for mutation screens. Neucleophesmin 1 and fims- related tyrosine kinase internal tandem duplication (FLT3-ITD) mutations were assessed using the sequencing process as previously described.5
Statistical Analysis The statistical analysis of data was done using Excel (IBM Corp, Chicago, IL) version 2007 and SPSS (IBM Corp, Chicago, IL) version 16. Qualitative data were described in the form of numbers and percentages. Quantitative data were described in the form of mean SD. Statistical analyses on comparisons between groups were done using the c2 test regarding qualitative data and quantitative nonparametric data comparisons were performed using 1-way
analysis of variance and paired sample t test. The probability of being by chance (P value) was calculated for all parameters (P was considered significant if .05 at 95% confidence interval).
Results Prevalence of IDH1 and IDH2 Mutations Isocitrate dehydrogenase 1 mutations were detected in 18 cases of 211 patients with de novo AML (8.5%) in the form of in 8 cases R132H, 6 cases R132C, 2 cases R132S, 1 case R132G, and 1 case R132V mutations. Moreover, IDH2 mutations were detected in 22 cases of 211 patients with de novo AML (10.4%) in the form of in 20 cases R140Q and 2 cases R172K (Table 1). No single AML patient had combined mutations (both IDH1 and IDH2).
Clinical and Demographic Characteristics of Patients With IDH1 and IDH2 Mutations The clinical and demographic characteristics of the IDHmutated AML patients are shown in Tables 2 and 3. It was noticed that the IDH1 mutant patients were of older age, mostly female, had high platelet counts, and BM blast cell counts compared with the mutated NPM1 AML patients. However, there was no difference between the 2 groups with regard to French-AmericanBritish subtypes, performance status, extramedullary disease, cytogenetic status, hemoglobin concentration, and FLT3 ITD mutation. In the IDH2 mutant group, there was no difference between the 2 groups with regard to sex, French-American-British subtype, performance status, extramedullary disease, cytogenetic status, hemoglobin concentration, and BM blast cell counts and were not related to NPM1 or FLT3 mutations. However, the IDH2 mutation was more common in patients with older age, normal karyotyping, low white blood cell (WBC) count, and high platelet count.
Response to Therapy and Clinical Outcome in IDH-Mutant Patients There was no difference in induction or remission rate between the mutant and wild type groups of patients. The induction or remission rate in the wild type and mutant patients were 114 (66.4%) versus 26 (65%), respectively (Table 4).
Table 3 Type of IDH1 and IDH2 Mutations Identified in 40 Acute Myeloid Leukemia Patients Mutation
Nucleotide Change
Predicted Protein Change
Patient n
G395A
CGT-CAT
R132H
8
C394T
CGT-TGT
R132C
6
C394A
CGT-AGT
R132S
2
C394G
CGT-GGT
R132G
1
G395T
CGT-CTT
IDH1 (n [ 18)
CG394-395GT
R132L
0
R132V
1
IDH2 (n [ 22)
4
-
G419A
CGG-CAG
R140Q
20
G515A
AGG-AAG
R172K
2
Clinical Lymphoma, Myeloma & Leukemia Month 2015
Salah Aref et al Table 4 Response to Therapy in IDH-Mutant AML Patients Versus Wild Type Patients Remission After First Cycle IDH
Not in Remission
In Remission
Total
P >.05
Wild Type AML n
57
%
33.3
114 66.7
171
n
14
26
40
%
35
65
100.0
100.0
Mutant AML
These findings are in agreement with those reported by Chotirat et al,11 who stated that the overall frequency of IDH mutations appears to vary between 2% and 14% for IDH1 and 1% and 19% for IDH2 in studies from the Asia continent (Taiwan and China), the United States, Canada, France, Germany, The Netherlands, and the United Kingdom. Likewise, Paschka et al5 reported that the frequency of IDH1 mutations among AML patients was 7.6%, and IDH2 was 8.7%, and that 2 patients had IDH1 and IDH2 mutations. However, Nomdedéu et al4 reported that IDH1 or IDH2 mutations were identified in 23.3% of AML patients and in 22.5% of those with a normal karyotype. This difference could be attributed to the different ethnic groups. In the present study, IDH1 mutations were in the form of 8 cases R132H, 6 cases R132C, 2 cases R132S, 1 case R132G, and 1 case R132V mutations; moreover, the IDH2 mutations were in the form of 20 cases R140Q and 2 cases R172K. Paschka et al5 found that all but 1 IDH1 mutation caused substitutions of residue R132; IDH2
Survival Analysis With regard to overall survival (OS), the patients with the IDH mutation had shorter OS than patients with wild type (10 months vs. 18 months; P ¼ .02; Figure 1).
Discussion Aberrant epigenetic programming is a hallmark of cancer. In this context IDH1 and IDH2 genes were described to be frequently mutated in multiple types of human cancer. Mutations result in simultaneous loss of their normal catalytic activity and gain of a new function.5,6 Several studies have been conducted to assess the incidence of IDH1 and IDH2 genes in multiple human tumors especially in AML, however, their prognostic effect and their prevalence in our locality remain to be elucidated. In the current study, IDH1 mutations were identified in 18 of 211 patients with de novo AML (8.5%) and IDH2 mutations were identified in 22 of 211 patients with de novo AML (10.4%).
Figure 1 Overall Survival (Months) of IDH-Mutant Acute Myeloid Leukemia (AML) Patients (n [ 35) Versus IDH Wild Type AML Patients. Patients (n [ 75) With Exclusion of NPM1- and FLT3-Mutant Patients
Survival Functions 1.0
Cumulative Survival
Wild IDH without NPM1 or FLT3 mutations Mutated IDH without NPM1 or FLT3 mutations
0.8
0.6
P = .02 0.4
0.2
0.0 10
20
30
40
OS
Clinical Lymphoma, Myeloma & Leukemia Month 2015
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IDH1 and IDH2 Mutations in Cytogenetically Normal AML mutations caused changes of R140 (n ¼ 48) or R172 (n ¼ 22). Moreover, Yamaguchi et al9 detected IDH1 R132 mutations in 20 (8.6%) patients with AML and IDH2 mutations in 19 (8.2%; 17 R140 and 2 R172) patients. Patel et al12 reported that IDH1 R132 mutation was detected in 12 of 199 patients with AML (6.0%), and the IDH2 R172 mutation was detected in 4 of 196 patients with AML (2.0%), and stated that no mutated cases had both IDH1 and IDH2 mutations, which suggests that these mutations are mutually exclusive. In our study we reported a novel mutation, which is the R132V mutation, detected in our patients. This mutation was not previously reported in AML but it was described in other tumors like glioma.13-15 The correlation of the demographic and laboratory data in the current studied cohort of AML patients revealed that there was significant difference between the wild and mutant IDH1 patients with regard to age, sex, platelet counts, BM blast cells, and the NPM1 mutation status. Similar findings were reported by Paschka et al,5 who found that IDH1-mutated patients were older, had higher platelet counts, had higher percentages of BM blasts, and were associated with mutated NPM1 than patients with wild type IDH1. The IDH2 mutation was detected more commonly in patients with older age, normal karyotyping, and low WBC count and high platelet counts and with no difference between the 2 groups with regard to sex, French-American-British subtype, performance status, extramedullary disease, cytogenetic status, hemoglobin concentration, BM blast cell counts, and NPM1 mutation. This in agreement with Chotirat et al11 and Paschka et al,5 who found that IDH2 mutation was associated with older age, a higher platelet count, and normal karyotype AML but they found that the IDH2 mutation was not associated with low WBC counts and was associated with the NPM1 mutation. Our study revealed that there was no significant difference between the IDH mutation in either IDH1 or IDH2 on the induction or remission rate (P ¼ .4). Similar findings were reported by Paschka et al,5 who found that the IDH mutations had no effect on the response to induction or treatment of AML. In the current study, the patients with IDH mutations after correction for NPM1 and FLT3 ITD had a significantly shorter OS compared with those in patients with wild type (P ¼ .02). This finding is in agreement with that reported by Yamaguchi et al,9 who concluded that IDH mutations have an unfavorable effect in AML, especially AML with the NPM1 wild type, and mentioned that younger AML patients with IDH mutations might benefit from allogeneic stem cell transplantation. Moreover, Nomdedéu et al4 also reported that AML patients with the IDH mutation were associated with short OS and stated that this adverse effect was even more evident in patients with the NPM- or CEBPA-mutated/FLT3 wild type genotype. In contrast, Paschka et al5 stated that IDH1 and IDH2 mutations had no effect on relapse-free survival or OS. This might be because of the simultaneous presence of other mutations like NPM1, which modify their effects.
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Clinical Lymphoma, Myeloma & Leukemia Month 2015
Conclusion Isocitrate dehydrogenase 1 and IDH2 mutations are negative prognostic markers in AML patients. A novel mutation (R132V) was detected in IDH1 in our cohort of AML patients. We recommend molecular testing for IDH1 and IDH2 mutations to make proper risk stratification of AML patients before treatment.
Clinical Practice Points The prevalence of IDH1 and IDH2 mutations among different
cohorts of AML patients in previous studies showed wide variations. The negative clinical effect of IDH mutations on cytogenetically normal AML patients was previously reported. In the current study the prevalence of IDH1 and IDH2 mutations was not previously reported in our cohort of Egyptian patients. A novel mutation (R132V) was detected in IDH1 in our cohort of AML patients. Our findings will be added to the data base concerning this subject.
Disclosure The authors have stated that they have no conflicts of interest.
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