IDH1 and IDH2 mutation studies in 1473 patients with chronic-, fibrotic- or blast-phase essential thrombocythemia, polycythemia vera or myelofibrosis

ArticleinLeukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 24(7):1302-9 · July 2010with20 Reads
Impact Factor: 10.43 · DOI: 10.1038/leu.2010.113 · Source: PubMed
Abstract

In a multi-institutional collaborative project, 1473 patients with myeloproliferative neoplasms (MPN) were screened for isocitrate dehydrogenase 1 (IDH1)/IDH2 mutations: 594 essential thrombocythemia (ET), 421 polycythemia vera (PV), 312 primary myelofibrosis (PMF), 95 post-PV/ET MF and 51 blast-phase MPN. A total of 38 IDH mutations (18 IDH1-R132, 19 IDH2-R140 and 1 IDH2-R172) were detected: 5 (0.8%) ET, 8 (1.9%) PV, 13 (4.2%) PMF, 1 (1%) post-PV/ET MF and 11 (21.6%) blast-phase MPN (P<0.01). Mutant IDH was documented in the presence or absence of JAK2, MPL and TET2 mutations, with similar mutational frequencies. However, IDH-mutated patients were more likely to be nullizygous for JAK2 46/1 haplotype, especially in PMF (P=0.04), and less likely to display complex karyotype, in blast-phase disease (P<0.01). In chronic-phase PMF, JAK2 46/1 haplotype nullizygosity (P<0.01; hazard ratio (HR) 2.9, 95% confidence interval (CI) 1.7-5.2), but not IDH mutational status (P=0.55; HR 1.3, 95% CI 0.5-3.4), had an adverse effect on survival. This was confirmed by multivariable analysis. In contrast, in both blast-phase PMF (P=0.04) and blast-phase MPN (P=0.01), the presence of an IDH mutation predicted worse survival. The current study clarifies disease- and stage-specific IDH mutation incidence and prognostic relevance in MPN and provides additional evidence for the biological effect of distinct JAK2 haplotypes.

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ORIGINAL ARTICLE
IDH1 and IDH2 mutation studies in 1473 patients with chronic-, fibrotic- or blast-phase
essential thrombocythemia, polycythemia vera or myelofibrosis
A Tefferi
1
, TL Lasho
1
, O Abdel-Wahab
2
, P Guglielmelli
3
, J Patel
2
, D Caramazza
4
, L Pieri
3
, CM Finke
1
, O Kilpivaara
2
,
M Wadleigh
5
, M Mai
6
, RF McClure
6
, DG Gilliland
5
, RL Levine
2
, A Pardanani
1
and AM Vannucchi
3
1
Division of Hematology, Department of Medicine, Mayo Clinic, Rochester, MN, USA;
2
Human Oncology and Pathogenesis
Program and Leukemia Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, USA;
3
UF di
Ematologia, Dipartimento di Area Critica Medico-Chirurgica, Universita
`
degli Studi, Azienda Ospedaliera-Universitaria Careggi,
Istituto Toscano Tumori, Firenze, Italy;
4
Cattedra ed U.O. di Ematologia, Policlinico Universitario di Palermo, Palermo, Italy;
5
Division of Hematology, Department of Medicine, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Harvard
Medical School, Boston, MA, USA and
6
Division of Hematopathology, Department of Laboratory Medicine, Mayo Clinic,
Rochester, MN, USA
In a multi-institutional collaborative project, 1473 patients
with myeloproliferative neoplasms (MPN) were screened for
isocitrate dehydrogenase 1 (IDH1)/IDH2 mutations: 594 essen-
tial thrombocythemia (ET), 421 polycythemia vera (PV), 312
primary myelofibrosis (PMF), 95 post-PV/ET MF and 51 blast-
phase MPN. A total of 38 IDH mutations (18 IDH1-R132, 19 IDH2-
R140 and 1 IDH2-R172) were detected: 5 (0.8%) ET, 8 (1.9%)
PV, 13 (4.2%) PMF, 1 (1%) post-PV/ET MF and 11 (21.6%)
blast-phase MPN (Po0.01). Mutant IDH was documented in the
presence or absence of JAK2, MPL and TET2 mutations, with
similar mutational frequencies. However, IDH-mutated patients
were more likely to be nullizygous for JAK2 46/1 haplotype,
especially in PMF (P ¼ 0.04), and less likely to display complex
karyotype, in blast-phase disease (Po0.01). In chronic-phase
PMF, JAK2 46/1 haplotype nullizygosity (Po0.01; hazard ratio
(HR) 2.9, 95% confidence interval (CI) 1.7–5.2), but not IDH
mutational status (P ¼ 0.55; HR 1.3, 95% CI 0.5–3.4), had an
adverse effect on survival. This was confirmed by multivariable
analysis. In contrast, in both blast-phase PMF (P ¼ 0.04) and
blast-phase MPN (P ¼ 0.01), the presence of an IDH mutation
predicted worse survival. The current study clarifies disease-
and stage-specific IDH mutation incidence and prognostic
relevance in MPN and provides additional evidence for the
biological effect of distinct JAK2 haplotypes.
Leukemia (2010) 24, 1302–1309; doi:10.1038/leu.2010.113;
published online 27 May 2010
Keywords: JAK2; MPL; TET2; myeloproliferative
Introduction
Despite the seminal discovery of JAK2 or MPL mutations in the
majority of patients with BCR-ABL1-negative myeloproliferative
neoplasms (MPN),
1–4
it is becoming increasingly evident that
these mutations do not signify either disease-initiating or
leukemia-promoting events.
5,6
It is therefore important to keep
looking for additional molecular alterations to clarify the genetic
underpinnings of both chronic- and blast-phase MPN. In the
last 2 years, mutations involving TET2, ASXL1 and CBL have
been described in some patients with BCR-ABL1-negative MPN,
including polycythemia vera (PV), essential thrombocythemia
(ET) and primary myelofibrosis (PMF).
7
The precise pathogenetic
contribution of these mutations and their clinical relevance are
currently under investigation. The glioma-associated
8
isocitrate
dehydrogenase 1 (IDH1) and IDH2 mutations are the latest to
be added to the ‘MPN mutations list’.
9
IDH1, located on chromosome 2q33.3, and IDH2, located on
chromosome 15q26.1, encode enzymes that catalyze oxidative
decarboxylation of isocitrate to a-ketoglutarate. IDH1 (cyto-
plasm and peroxisome) and IDH2 (mitochondria) use NADP
þ
as a co-factor to generate NADPH, which is important in the
production of intracellular glutathione. Intact IDH activity is
therefore necessary for cellular protection from oxidative stress.
Mutant IDH has decreased affinity to isocitrate, but displays
neomorphic catalytic activity toward a-ketoglutarate, the net
result being decreased supply of a -ketoglutarate and accumula-
tion of 2-hydroxyglutarate.
10–13
It is currently believed that these
intracellular changes facilitate oncogenic pathways including
activation of HIF-1a.
10
IDH1 and IDH2 mutations were first described in low-grade
gliomas/secondary glioblastomas
8
and subsequently in acute
myeloid leukemia (AML),
14
with respective mutational frequen-
cies of B70 and 8%. We recently screened 200 patients
with either chronic- or blast-phase MPN for IDH mutations, and
identified 9 patients with either IDH1 (n ¼ 5) or IDH2 (n ¼ 4)
mutations.
9
Mutational frequencies were B21% for blast-phase
MPN and B4% for PMF. In the current study, we expanded our
study cohort to include 1473 patients recruited from three MPN
centers of excellence, with the intent to accurately describe the
prevalence of IDH mutations in chronic-, fibrotic- and blast-
phase PV, ET and PMF. In addition, IDH-mutated patients were
analyzed for their cytogenetic and molecular (that is, JAK2, MPL
and TET2 mutation and JAK2 haplotype status) phenotype, as
well as their prognostic relevance.
Materials and methods
This study was approved by the Mayo Clinic institutional review
board. All patients provided authorization for use of their
medical records for research purposes, and the research was
carried out according to the principles of the Declaration of
Helsinki. Patient samples were obtained from the Mayo Clinic,
Harvard Medical Institute and University of Florence. Muta-
tional analyses were performed on DNA derived from either
bone marrow or peripheral blood granulocytes. JAK2 46/1
haplotype analysis on patient samples accrued from Harvard was
performed on germline DNA. Diagnoses of MPN, post-PV/ET MF
Received 1 April 2010; accepted 23 April 2010; published online 27
May 2010
Correspondence: Professor A Tefferi, Division of Hematology,
Department of Medicine, Mayo Clinic, Rochester, MN 55905, USA.
E-mail: tefferi.ayalew@mayo.edu
Leukemia (2010) 24, 1302–1309
& 2010 Macmillan Publishers Limited All rights reser ved 0887-6924/10
www.nature.com/leu
Page 1
and AML, in patient samples accrued from the Mayo Clinic and
the University of Florence, were according to the World Health
Organization and International Working Group criteria.
7,15
Diagnoses in patients accrued from Harvard were self-reported
during an internet-based collection of samples, as previously
detailed.
16
DNA from either bone marrow (Mayo Clinic samples) or
granulocytes (samples from Harvard and the University
of Florence) was extracted using conventional methods. MPL,
JAK2 and TET2 mutation and JAK2 haplotype analyses were
performed according to previously published methods.
4,17–19
With regard to IDH mutation analysis, Harvard patient samples
were analyzed using the following primers for IDH1, which
cover amino acid residues 41–138: sense, 5
0
-TGTGTTGAGAT
GGACGCCTA-3
0
and anti-sense, 5
0
-GGTGTACTCAGAGCCTTC
GC-3
0
. Sequencing of IDH2 used primers that covered amino
acid residues 125–226: sense, 5
0
-CTGCCTCTTTGTGGCCTA
AG-3
0
and anti-sense, 5
0
-ATTCTGGTTGAAAGATGGCG-3
0
.
Sequence analysis was performed using Mutation Surveyor
(SoftGenetics, State College, PA, USA) and all mutations were
validated by repeat PCR and sequencing on unamplified DNA
from the archival sample.
Mayo Clinic and University of Florence patient samples were
screened for IDH1 and IDH2 mutations by direct sequencing
IDH1 HRM IDH2 HRM
12.484
10.984
9.484
7.984
6.484
4.984
3.484
1.984
0.484
-1.016
-2.516
76.5
77
77.5
78
78.5
79
79.5
80
80.5
81
81.5
82
82.5
83
83.5
5’ sequence 5’ sequence
3’ sequence
77.5 78 78.5 79 79.5 81 81.5 82 82.580 80.5
Temperature (°C)
Temperature (°C)
Relative Signal Difference
Relative Signal Difference
15.003
13.503
9.003
7.503
6.003
4.503
3.003
1.503
0.003
-1.497
-2.997
12.003
10.503
IDH1R132S
IDH1R132G
IDH1R132S IDH1R132G
IDH2R140Q
IDH2R140Q
Figure 1 High-resolution melting (HRM) normalized and temperature-shifted difference plot for IDH1 (a) and IDH2 (b) and corresponding
sequences (c and d).
Table 1 Specific diagnoses, age/sex distribution, JAK2, MPL and TET2 mutational status and JAK2 non-46/1 haplotype frequency in 1473
patients with polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), post-PV MF, post-ET MF, post-PV acute
myeloid leukemia (post-PV AML), post-ET AML or post-PMF AML
MPN center Diagnosis N Median age
in years (range)
Males
(%)
JAK2 mutation
frequency
MPL mutation
frequency
TET2 mutation
frequency
JAK2 46/1 nullizygous
frequency
Florence PV 150 62 (16–91) 66 83% (123/149)
a
NA NA 6% (1/18)
(n ¼ 522) ET 199 56 (13–93) 37 63% (124/198) 2.2% (4/184) NA 52% (25/48)
PMF 107 63 (16–90) 67 65% (69/106) 4% (4/98) NA 39% (37/96)
Post-PV MF 32 62 (48–78) 47 100% (32/32) 0% (0/28) NA 0% (0/16)
Post-ET MF 26 63 (33–82) 50 39% (10/26) 13% (3/24) NA 27% (3/11)
Post-PV AML 1 66 0 100% (1/1) 0% (0/1) NA NA
Post-ET AML 2 65–70 0 50% (1/2) 0% (0/1) NA NA
Post-PMF AML 5 73 (67–83) 80 20% (1/5) 0% (0/5) NA 20% (1/5)
Harvard PV 159 59 (32–85) 48 93% (139/150)
a
0% (0/159) 9.4% (15/159) 23% (29/125)
(n ¼ 322) ET 124 57 (31–84) 26 31% (35/114) 3.2% (4/124) 8% (10/124) 42% (41/98)
PMF 39 64 (50–70) 49 42% (16/38) 5.1% (2/39) 7.7% (3/39) 22% (5/23)
Mayo PV 112 66 (21–95) 48 95% (106/112)
a
1.8% (1/56) 15.7% (14/89) 25% (25/99)
(n ¼ 629) ET 271 63 (15–87) 38 49% (132/271) 4.9% (7/143) 5.7% (3/53) 34% (91/266)
PMF 166 62 (35–82) 67 57% (95/166) 10% (11/108) 18% (10/57) 35% (55/158)
Post-PV MF 22 65 (47–75) 64 100% (22/22) 7.7% (1/13) 7.7% (1/13) 5% (1/20)
Post-ET MF 15 63 (39–75) 80 47% (7/15) 10% (1/10) 12.5% (1/8) 31% (4/13)
Post-PV AML 11 64 (48–87) 64 100% (11/11) 0% (0/7) 20% (1/5) 36% (4/11)
Post-ET AML 5 64 (50–75) 60 60% (3/5) 0% (0/5) 25% (1/4) 20% (1/5)
Post-PMF AML 27 66 (49–83) 74 48% (13/27) 9% (2/22) 0% (0/7) 35% (8/23)
Abbreviation: NA, not done or not available.
a
Includes JAK2 exon 12 mutations: two cases from Mayo clinic and one case from Harvard.
Isocitrate dehydrogenase mutations in MPN
A Tefferi et al
1303
Leukemia
Page 2
and/or high-resolution melting assay. Direct sequencing for
IDH1 exon 4 mutations was carried out using the following
primer sequences: sense, 5
0
-CGGTCTTCAGAGAAGCCATT-3
0
and anti-sense, 5
0
-CACATTATTGCCAACATGAC-3
0
.
18
IDH2
exon 4 was amplified using sense, 5
0
-CCACTATTATCTCTGTC
CTC-3
0
and anti-sense, 5
0
-GCTAGGCGAGGAGCTCCAGT-3
0
.
19
Both reactions were performed in 25 ml volume containing
100 ng of DNA, 0.25 U Taq polymerase, 0.3 m
M each of dATP,
dCTP, dGTP and dTTP, 5 mlofa10 PCR buffer (Roche
Diagnostics, Indianapolis, IN, USA) and 0.2 m
M each of sense
and anti-sense primers. The reaction was denatured at 94 1C for
3 min followed by 35 cycles of denaturing at 94 1C for 30 s,
annealing at 57 1 C for 30 s and extension at 72 1 C for 40 s.
After a final extension at 72 1C for 2 min, the products
were confirmed by running on 1.3% agarose gel and purified
using Qiagen’s PCR Quick Purification Kit. The product was
sequenced using the ABI PRISM 3730xl analyzer (Applied
Biosystems Inc, Foster City, CA, USA) to screen for the presence
of mutations.
High-resolution melting was performed using the LightCycler
480 real-time PCR system (Roche Diagnostics), using the above-
mentioned primers for IDH1 mutations (R130) and the following
primers for IDH2 mutations (R140 and R172): R140 sense,
5
0
-GCTGAAGAAGATGTGGAA-3
0
and anti-sense, 5
0
-TGATGG
GCTCCCGGAAGA-3
0
; R172 sense, 5
0
-CCAAGCCCATCACCAT
TG-3
0
and anti-sense, 5
0
-CCCAGGTCAGTGGATCCC-3
0
(Figure 1).
Conventional statistical procedures were used (SAS Institute,
Cary, NC, USA). All statistically analyzed data were obtained at
time of IDH mutation analysis. All P-values were two-tailed
and statistical significance was set at the level of Po0.05.
Categorical variables were described as count and relative
frequency and compared by w
2
statistics. Comparison of
continuous variables between categories was performed by
the Mann–Whitney U-test. Survival analysis was performed
by the Kaplan–Meier method taking the interval from the date
of diagnosis, for chronic-phase disease, or from the date of
leukemic transformation, for blast-phase disease, to death
or last contact. The log-rank test was used to compare
survival data. Cox regression model was used for multivariable
analysis.
Results
Disease- and stage-specific IDH mutational frequencies
A total of 1473 patients with BCR–ABL1-negative MPN were
recruited from the Mayo Clinic, Rochester, MN, USA (n ¼ 629),
University of Florence, Florence, Italy (n ¼ 522) and Harvard
Medical Institute, Boston, Massachusetts, USA (n ¼ 322).
Specific diagnoses included ET (n ¼ 594), PV (n ¼ 421), PMF
(n ¼ 312), post-PV MF (n ¼ 54), post-ET MF (n ¼ 41), post-PV
AML (n ¼ 12), post-ET AML (n ¼ 7) and post-PMF AML (n ¼ 32).
Table 1 provides clinical and laboratory details of the study
population including age and sex distribution, specific diag-
noses and JAK2, MPL and TET2 mutational and JAK2 46/1
haplotype status, stratified by center of patient recruitment.
A total of 38 IDH mutations were documented (Table 2): 18
involved IDH1 (10 R132S, 7 R132C and 1 R132G) and 20 IDH2
(18 R140Q, 1 R140W and 1 R172G). IDH mutations were
infrequent in chronic- or fibrotic-phase disease and significantly
more prevalent in blast-phase disease (Po0.01; Table 3):
5 (0.8%) in ET, 8 (1.9%) in PV, 13 (4.1%) in PMF, 1 (1%) in
post-ET/PV MF, none in blast-phase ET, 3 (25%) in blast-phase
PV and 8 (25%) in blast-phase PMF.
Correlation of IDH mutations with other
MPN-associated mutations and JAK2 46/1 haplotype
Considering the preponderance of informative cases with
centrally confirmed diagnosis and availability of a more
complete laboratory data, the current analysis was limited to
patients from the Mayo Clinic cohort (n ¼ 629). IDH mutational
frequencies were similar among JAK2- (3.6%), MPL- (4.3%) and
TET2 (3.2%)-mutated patients and their respective mutation-
negative counterparts (4.2, 5.3 and 6.3%; Table 3). In other
words, mutant IDH was shown to co-occur with a JAK2, MPL or
TET2 mutation, and mutational frequency did not appear to
be influenced by either the type of the coexisting mutation
(P ¼ 0.96) or the presence or absence of each specific mutation
(Table 3). However, IDH-mutated cases were more likely to be
nullizygous for JAK2 46/1 haplotype, especially when analysis
was restricted to informative (that is, with JAK2 46/1 haplotype
information) patients with chronic- (n ¼ 158) or blast (n ¼ 23)-
phase PMF, analyzed together (P ¼ 0.007) or separately
(P ¼ 0.04; Table 4).
Clinical correlates and prognostic relevance
To avoid disease- or stage-specific confounding factors, as well
as assure adequate sample size of informative cases, clinical
correlative and prognostic studies were limited to PMF. In this
patient cohort, detailed clinical information was available in
111 patients with chronic-phase PMF (including 7 IDH-mutated
cases) and 27 patients with blast-phase PMF (including 8 IDH-
mutated cases), both patient populations were accrued from the
Mayo Clinic cohort. In both chronic- and blast-phase PMF, the
presence of IDH mutations was not influenced by either age
(P ¼ 0.51 and 0.70, respectively) or gender (P ¼ 0.09 and 0.3,
respectively). In chronic-phase disease, comparison of prog-
nostically relevant disease variables at diagnosis revealed that
cytogenetic findings in IDH-mutated cases often belonged to a
low- or intermediate-risk category,
20
although the difference
was not statistically significant (Table 4). Similarly, IDH-mutated
blast-phase PMF was less likely to display complex karyotype
(0 vs 64% in IDH-unmutated cases; P ¼ 0.001).
In addition to biological implications, the aforementioned
associations of IDH mutations with favorable cytogenetic profile
and JAK2 46/1 haplotype nullizygosity, both which have
previously been shown to be prognostically relevant,
19,20
mandated their inclusion as covariates during multivariable
survival analysis. In chronic-phase PMF, univariate analysis
showed statistically significant adverse survival effect from JAK2
46/1 haplotype nullizygosity (P ¼ 0.0001; 34 nullizygous vs 74
not nullizygous), high-risk karyotype (Po0.0001; 13 high-risk vs
98 not high-risk) and higher International Prognostic Scoring
System (IPSS; 27 high, 29 intermediate-2, 30 intermediate-1 and
25 low-risk patients)
21
risk score (Po0.0001), but not from IDH
mutational status (P ¼ 0.54; 7 mutated vs 104 unmutated;
Figure 2). Multivariable analysis confirmed the independent
prognostic value of JAK2 46/1 haplotype status (hazard ratio
(HR) 2.2, 95% confidence interval (CI) 1.2–4.2), karyotype
(HR 2.8, 95% CI 1.3–5.9) and IPSS risk score (HR 4.8, 95% CI
2.0–11.5).
In blast-phase PMF, despite its association with noncomplex
karyotype, the presence of mutant IDH predicted shortened
survival, calculated from the time of disease transformation
(P ¼ 0.04), and there was a similar trend for JAK2 non-46/1
haplotype (P ¼ 0.14; Figure 3). Significance was lost for both
during multivariable analysis, probably because of small sample
size. IDH mutation status also predicted worse survival when
the analysis included all blast-phase MPN cases from the Mayo
Isocitrate dehydrogenase mutations in MPN
A Tefferi et al
1304
Leukemia
Page 3
Table 2 Clinical, cytogenetic and molecular details, at time of mutation analysis, of 38 IDH-mutated patients with chronic- or advanced-phase polycythemia vera, essential thrombocythemia or
primary myelofibrosis
Specific diagnosis Age (years)
and sex
IDH mutation
variant
JAK2 V617F
burden
MPL mutation
status
TET2 mutation
status
JAK2 haplotype
status
Karyotype Antecedent MPN
diagnosis to IDH analysis
IDH analysis
to last f/u
Status at
last f/u
1 ET (Mayo) 26/F IDH2 R140Q 0% WT WT Heterozygous NN 0 month 6 years Alive with ET
2 ET (Mayo) 38/F IDH2 R140Q 5% WT WT Heterozygous NA 5 years NA Lost to follow-up
3 ET (Florence) 80/F IDH2 R140Q 73% WT NA NA NA 1.1 years 4 months Alive with ET
4 ET (Florence) 79/M IDH2 R140Q 64% WT NA Nullizygous NA 7 months 2 years Alive with ET
5 ET (Florence) 65/F IDH1 R132C 0% NA NA NA NA 7.2 years 1 month Alive with ET
6 PV (Harvard) 52/F IDH2 R140Q 58% WT WT Heterozygous NN NA NA NA
7 PV (Harvard) 47/M IDH2 R140Q 90% WT WT Homozygous NN NA NA NA
8 PV (Florence) 79/F IDH1 R132S 50% NA ND NA NA 0 month 0 month Alive with PV
9 PV (Florence) 49/M IDH2 R140Q 25% NA ND NA NA 1 month 0 month Alive with PV
10 PV (Mayo) 82/M IDH1 R132C 1% WT WT Heterozygous NN 4 years 5 months Dead with AML
11 PV (Mayo) 50/M IDH2 R140Q 25% WT WT NA NN 4 months 5 years Alive with PV
12 PV (Mayo) 75/M IDH2 R140Q 11% WT WT Heterozygous NN 0 month 1 month Alive with PV
13 PV (Mayo) 82/F IDH2 R140Q 51% WT WT Homozygous NN 29 months 3.4 years Dead with PV
14 PMF (Harvard) 76/M IDH2 R140Q 72% WT WT Homozygous NN NA NA NA
15 PMF (Florence) 57/M IDH1 R132S 65% WT ND Nullizygous NA 0 month 6 years Alive with PMF
16 PMF (Florence) 80/M IDH1 R132S 50% WT ND Nullizygous NA 0 month 1 year Alive with PMF
17 PMF (Florence) 62/M IDH1 R132G 10% WT ND NA NA 2 months 4 months Alive with PMF
18 PMF (Florence) 72/M IDH2 R140Q 70% WT NA Heterozygous NA 0 month 1.8 years Alive with PMF
19 PMF (Florence) 50/M IDH1 R132S 54% WT NA Heterozygous NA 0 month 5.2 years Alive with PMF
20 PMF (Mayo) 74/M IDH1 R132S 0% WT Mutated Nullizygous NN 0 month 2.8 years Dead with PMF
21 PMF (Mayo) 69/M IDH2 R140Q 22% WT NA Nullizygous NN 4 months 11 months Dead with AML
22 PMF (Mayo) 73/M IDH1 R132S 26% WT NA Nullizygous NN 1 month 6 months Dead from
unknown cause
23 PMF (Mayo) 69/M IDH2 R140Q 0% WT WT Nullizygous NN 2 months 3 months Dead from
unknown cause
24 PMF (Mayo) 58/F IDH1 R132C 0% WT NA Nullizygous NN 3 years 3.2 years Dead with AML
25 PMF (Mayo) 53/M IDH2 R140Q 0% WT WT Heterozygous NN 0 month 6 years Alive with PMF
26 PMF (Mayo) 50/F IDH2 R172G 0% WT WT Heterozygous +9 1 year 5 years Alive with PMF
27 Post-PV MF (Florence) 56/F IDH2 R140Q 84% WT NA Homozygous NA 1 year 1.4 years Dead with AML
28 Post-PMF AML (Mayo) 62/M IDH2 R140W 65% WT NA Nullizygous +21 8 months 2 months Dead with AML
29 Post-PMF AML (Mayo) 64/M IDH1 R132C 0% WT WT Heterozygous 15q 5.8 years 4 months Dead with AML
30 Post-PMF AML (Mayo) 73/M IDH1 R132C 0% WT WT Nullizygous NN 2 years 2.5 months Dead with AML
31 Post-PMF AML (Mayo) 61/M IDH2 R140Q 0% WT NA Nullizygous 7,20q 5 months 5 months Dead with AML
32 Post-PMF AML (Mayo) 66/M IDH1 R132S 96% WT WT Homozygous +2 4.5 years 1 month Dead with AML
33 Post-PMF AML (Mayo) 82/F IDH1 R132S 1% WT NA Nullizygous +9 2 months o 1 month Dead with AML
34 Post-PMF AML (Mayo) 80/M IDH1 R132S 1% WT NA Homozygous NA 2.2 years 1 month Dead with AML
35 Post-PMF AML (Mayo) 81/M IDH1 R132C 0% MPLW515L WT Nullizygous T(8;21) 2 years 2 months Dead with AML
36 Post-PV AML (Mayo) 82/M IDH1 R132C 7% WT NA Heterozygous 5q 2.8 years 3 months Dead with AML
37 Post-PV AML (Mayo) 67/M IDH2 R140Q 3% WT NA Heterozygous 5 and
a
NA 1.5 months Dead with AML
38 Post-PV AML (Mayo) 53/M IDH1 R132S 80% WT NA Nullizygous der(1;7),+8 16 years 8 months Dead with AML
Abbreviations: AML, acute myeloid leukemia; ET, essential thrombocythemia; IDH, isocitrate dehydrogenase; MPN, myeloproliferative neoplasm; NA, information not available; ND, not done; NN,
normal cytogenetics; PMF, primary myelofibrosis; PV, polycythemia vera; WT, wild type.
a
Multiple trisomies: +2, +3, +6, +8, +10, +11, +12, +13, +14, +19, +20, +21.
Isocitrate dehydrogenase mutations in MPN
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Table 3 IDH mutational frequencies in 1473 patients with polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis
(PMF), post-PV MF, post-ET MF, post-PV acute myeloid leukemia (post-PV AML), post-ET AML or post-PMF AML
Variables Number of
patients
IDH mutated
(IDH1 or IDH2), n (%)
IDH1
mutated, n
IDH2
mutated, n
P-value
All patients 1473 38 18 20 o0.01
PV 421 8 (1.9%) 2 6
ET 594 5 (0.8%) 1 4
PMF 312 13 (4.2%) 7 6
Post-PV MF 54 1 (1.9%) 0 1
Post-ET MF 41 0 0 0
Post-PV AML 12 3 (25%) 2 1
Post-ET AML 7 0 0 0
Post-PMF AML 32 8 (25%) 6 2
JAK2 mutated vs wild type (n ¼ 629)
a
389 vs 240 14 (3.6%) vs 10 (4.2%) 0.72
MPL mutated vs wild type (n ¼ 364)
a
23 vs 341 1 (4.3%) vs 18 (5.3%) 0.85
TET2 mutated vs wild type (n ¼ 237)
a
31 vs 206 1 (3.2%) vs 13 (6.3%) 0.5
JAK2 46/1 nullizygous vs not nullizygous (n ¼ 596)
a
189 vs 407 11 (5.8%) vs 12 (2.9%) 0.09
Abbreviations: AML, acute myeloid leukemia; IDH, isocitrate dehydrogenase; PMF, primary myelofibrosis.
a
Analysis limited to Mayo clinic patients only and n’ signifies number of patients evaluated.
Table 4 IDH mutational frequencies in 193 Mayo clinic patients with chronic-phase (n ¼ 166) or blast-phase (n ¼ 27) primary myelofibrosis
(PMF) stratified by JAK2 mutational, JAK2 46/1 haplotype or cytogenetic status
Variables N IDH mutated (IDH1 or IDH2), n (%) P-value
Chronic-phase PMF (JAK2V617F mutated vs wild type) 166 (95 vs 71) 7 (4.2%) (2 (2.1%) vs 5 (7%)) 0.12
Blast-phase PMF (JAK2V617F mutated vs wild type) 27 (13 vs 14) 8 (30%) (4 (31%) vs 4 (29%)) 0.9
Chronic-phase PMF (JAK2 46/1 nullizygous vs not nullizygous) 158 (55 vs 103) 7 (4.4%) (5 (9%) vs 2 (1.9%)) 0.04
Blast-phase PMF (JAK2 46/1 nullizygous vs not nullizygous) 23 (8 vs 15) 8 (35%) (5 (63%) vs 3 (20%)) 0.04
Chronic-phase PMF karyotype at diagnosis (high-risk karyotype
vs not high-risk)
111 (13 vs 98) 7 (6.3%) (0 (0%) vs 7 (7.1%)) 0.32
Blast-phase PMF karyotype at transformation (complex karyotype
vs not complex)
22 (11 vs 11) 7 (32%) (0 vs 7 (64%)) 0.001
Abbreviations: IDH, isocitrate dehydrogenase; N, number of patients evaluable; PMF, primary myelofibrosis.
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
0 5 10 15 20 25
0 5 10 15 20
25
0 5 10 15 20 25
0 5 10 15 20 25
Nullizygous
Not nullizygous
High-risk karyotype
Low-or intermediate-ris
k
karyotype
IDH
mutated
IDH
unmutated
High
risk
Intermediate-2
risk
Intermediate-1
risk
Low
risk
P=0.0001
P=0.54
P<0.0001
P<0.0001
Years
Survival
Figure 2 Survival curves of 111 patients with chronic-phase primary myelofibrosis stratified by their isocitrate dehydrogenase (IDH) mutational
status (a), cytogenetic risk (b), JAK2 46/1 haplotype status (c) or International Prognostic Scoring System risk category (d).
Isocitrate dehydrogenase mutations in MPN
A Tefferi et al
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cohort (Figure 4; n ¼ 43; P ¼ 0.01). In this instance, significance
was sustained during multivariable analysis that included
JAK2 46/1 haplotype as a covariate.
Discussion
IDH1 point mutations involving exon 4 occur in the majority
(60–90%) of patients with low-grade gliomas and secondary
glioblastomas, and always affect the amino acid arginine at
position 132 (B93% R132H, 4% R132C, 2% R132S and o1%
R132G, R132L or R132V).
8,22,23
These mutations are relatively
infrequent in primary glioblastoma (B7%)
22
and are usually not
seen in other solid tumors.
23,24
A small fraction (B4%) of
glioma-associated IDH mutations involves IDH2, specifically
the R132 analogous R172 residue on exon 4 (R172K, R172M,
R172G, R172W).
23,25
IDH mutations in glioma are hetero-
zygous, believed to constitute early genetic events and might be
mutually exclusive of EGFR and PTEN, but not TP53 mutations.
Clinical correlates of IDH mutations in glioma include younger
age, longer survival and reduced risk of disease progression after
conventional therapy.
8,22,23,26,27
The first study on IDH mutations in AML included 188
patients with primary AML and reported IDH1, but not IDH2,
mutations in 8.5% (n ¼ 16) of the cases and 16% of those with
normal karyotype: R132C in 8 patients, R132H in 7 and R132S
in 1.
14
In a subsequent AML study of 493 patients,
28
27 (5.5%)
expressed IDH1 mutations (37% R132C, 26% R132H, 19%
R132S, 15% R132G and 4% R132L). In both studies,
14,28
IDH1
mutations clustered with normal karyotype, NPM1 mutations
and trisomy 8. IDH1 mutations are rare in pediatric AML.
29
More recently, IDH2 mutations, affecting R172 (R172K)
12,13
or
R140 (R140Q),
13
were also shown to occur in primary
AML.
12,13
In one of these studies, IDH1 or IDH2 mutations
were seen in 18 (23%) of 78 AML cases and the majority of the
mutations (12 of 18) involved IDH2, primarily R140Q.
13
In
general, survival in primary AML did not seem to be affected by
the presence of IDH mutations.
13,14,28–30
However, more recent
studies suggest that specific IDH mutation variants might be
prognostically relevant in certain molecular subsets of AML.
31
The first reports of IDH mutations in MPN came from
three independent groups.
9,32,33
In one of these studies, IDH1
mutations were seen in B8% (5 of 63) of blast-phase MPN
patients, mostly occurring in the absence of TET2 and ASXL1
mutations.
32
The second study was focused on blast-phase MPN
that arose from JAK2-mutated chronic-phase MPN.
33
In this
study, mutant IDH was seen in 5 (31%) of 16 blast-phase MPN
(three cases with R132C and two with R140Q) and in none of
the 180 PV or ET patients.
33
The third study from the Mayo
Clinic included 200 MPN patients and showed IDH mutational
frequencies of B21% for blast-phase MPN, regardless of JAK2
mutational status, and B4% for PMF.
9
The specific IDH1
mutations found in the particular study included R132C and
R132S and the IDH2 mutations R140Q and R140W.
The current study is an extension of the above-mentioned
Mayo Clinic study and involves a large number of patients
(n ¼ 1473) recruited from three major MPN centers of excel-
lence. The results of the study clarify a number of issues
regarding IDH mutations in MPN. First, the study provides
robust incidence figures for IDH1 and IDH2 mutations across
different disease stages of specific MPN variants. Accordingly,
we now show that both IDH1 and IDH2 mutations can occur in
chronic-phase ET, PV or PMF, although infrequently. Mutational
frequency was equally low in post-PV/ET MF and this fact
combined with the significantly higher mutation incidence
observed in blast-phase disease suggests a pathogenetic
contribution to leukemic but not fibrotic disease transformation.
Two additional observations support this contention (i) complex
karyotype was infrequently encountered in IDH-mutated blast-
phase MPN, which suggests an independent pathogenetic
contribution that might be tied to distinct molecular alterations,
such as, for example, overexpression of the APP (amyloid a
ˆ
(A4)
precursor protein) gene, which has previously been shown in
AML to be associated with either complex karyotype or
IDHR172 mutation
31
and (ii) the absence of mutual exclusivity
between IDH and other MPN-associated mutations (for exam-
ple, TET2, MPL), which is consistent with the suggestion that
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
0 1020304050607080
IDH
unmutated
Nullizygous
Mutated
JAK2 46/1 haplotype
not nullizygous
P=0.04
ab
P=0.14
Survival
Months
0 1020304050607080
Months
Figure 3 Survival curves of patients with blast-phase primary myelofibrosis stratified by their isocitrate dehydrogenase (IDH) mutational (a; n ¼ 27
including 8 mutated cases) or JAK2 46/1 haplotype (b; n ¼ 23 including 8 nullizygous cases) status.
0
0.2
0.4
0.6
0.8
1
0 1020304050607080
IDH un-mutated, n=32
IDH mutated, n=11
P=0.01
Months
Survival
Figure 4 Survival curves of 43 patients with blast-phase myelo-
proliferative neoplasm stratified by their isocitrate dehydrogenase
(IDH) mutational status.
Isocitrate dehydrogenase mutations in MPN
A Tefferi et al
1307
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Page 6
the former are later-arising cooperating mutations that are
more involved in disease progression rather than disease
initiation.
The types of IDH mutations seen in our patients with MPN
(mostly IDH2R140Q and IDH1R132S/C) are distinctly different
than those seen in gliomas (mostly IDH1R132H) and more
similar to those seen in AML, although IDH1R132H was
significantly more prevalent in AML. Within the context of
MPN, IDH2R140Q was over represented in chronic-phase ET
and PV, whereas IDH1 mutations were more prevalent in PMF
and blast-phase MPN. More studies are needed to confirm this
apparent trend. Regardless, there is currently no good explana-
tion for the observed diversity in IDH mutation variants among
gliomas and myeloid malignancies and current information
suggests similar biological consequences.
13
Whether or not
different IDH mutations carry different prognostic relevance in
MPN is currently not known and we did not attempt to address
the particular issue because of our relatively small number of
informative cases. Of note, in a recent study of primary AML
with normal karyotype, different types of IDH mutations
appeared to variably influence disease-free survival and
complete remission rates.
31
One particularly interesting observation from the current
study was the significant association between mutant IDH and
JAK2 non-46/1 haplotype. The latter phenomenon is further
evidence for the JAK2 mutation specificity of the previously
described association between the JAK2 46/1 haplotype and
MPN.
19,34,35
In other words, whereas JAK2 exon 14
19,35
or exon
12
36
mutations have been shown to be associated with JAK2
46/1 haplotype, we did not see the same effect involving MPL
mutations
34
(although others have shown otherwise),
37
and now
show an association with JAK2 non-46/1 haplotype for IDH
mutations. This latter observation is also consistent with our
previous report on the prognostically detrimental effect of
JAK2 non-46/1 haplotype in PMF;
19
it is possible that patients
with PMF who are nullizygous for JAK2 46/1 haplotype are
susceptible to additional adverse molecular events, such as IDH
mutations, which might lead to biologically more aggressive
disease. Consistent with this possible scenario, in the current
study, the negative prognostic impact of mutant IDH was
accounted for by the JAK2 46/1 genotype in PMF but not in
blast-phase MPN, in which risk factors other than JAK2 non-46/1
haplotype might have promoted the development of IDH
mutations.
It is becoming increasingly evident that there are many more
mutations than JAK2 and MPL mutations in BCR–ABL1-negative
MPN including those that involve TET2,
38,39
ASXL1,
40
IDH1,
32,33
IDH2,
9,33
CBL,
41
IKZF1
42
and LNK.
43
Some of these
mutations might be later-arising and more prevalent in blast-
phase disease. What is currently lacking is a composite
evaluation (that is, concurrent analysis of all relevant mutations),
which includes paired chronic- and blast-phase samples of a
large number of patients with blast-phase MPN. Such an
approach is essential for clarifying the individual pathogenetic
or prognostic contribution of the aforementioned mutations and
their chronological order of appearance. It is very likely that
additional mutations in MPN will be described soon, but
practical relevance in terms of either disease prognostication or
value as drug targets has so far been limited.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
This study is supported in part by grants from the ‘Myelo-
proliferative Disorders Foundation, Chicago, IL, USA’, ‘The
Henry J. Predolin Foundation for Research in Leukemia, Mayo
Clinic, Rochester, MN, USA’ and ‘Associazione Italiana per la
Ricerca sul Cancro-AIRC Milan, Italy, to AMV’.
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Isocitrate dehydrogenase mutations in MPN
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