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

IDH1 and IDH2 mutation studies in 1473 patients with chronic-, fibrotic- or blast-phase essential... Leukemia (2010) 24, 1302–1309 & 2010 Macmillan Publishers Limited All rights reserved 0887-6924/10 www.nature.com/leu ORIGINAL ARTICLE IDH1 and IDH2 mutation studies in 1473 patients with chronic-, fibrotic- or blast-phase essential thrombocythemia, polycythemia vera or myelofibrosis 1 1 2 3 2 4 3 1 2 A Tefferi , TL Lasho , O Abdel-Wahab , P Guglielmelli , J Patel , D Caramazza , L Pieri , CM Finke , O Kilpivaara , 5 6 6 5 2 1 3 M Wadleigh , M Mai , RF McClure , DG Gilliland , RL Levine , A Pardanani and AM Vannucchi 1 2 Division of Hematology, Department of Medicine, Mayo Clinic, Rochester, MN, USA; Human Oncology and Pathogenesis Program and Leukemia Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, USA; UF di Ematologia, Dipartimento di Area Critica Medico-Chirurgica, Universita ` degli Studi, Azienda Ospedaliera-Universitaria Careggi, Istituto Toscano Tumori, Firenze, Italy; Cattedra ed U.O. di Ematologia, Policlinico Universitario di Palermo, Palermo, Italy; Division of Hematology, Department of Medicine, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA and Division of Hematopathology, Department of Laboratory Medicine, Mayo Clinic, Rochester, MN, USA In a multi-institutional collaborative project, 1473 patients currently under investigation. The glioma-associated isocitrate with myeloproliferative neoplasms (MPN) were screened for dehydrogenase 1 (IDH1) and IDH2 mutations are the latest to isocitrate dehydrogenase 1 (IDH1)/IDH2 mutations: 594 essen- 9 be added to the ‘MPN mutations list’. tial thrombocythemia (ET), 421 polycythemia vera (PV), 312 IDH1, located on chromosome 2q33.3, and IDH2, located on primary myelofibrosis (PMF), 95 post-PV/ET MF and 51 blast- chromosome 15q26.1, encode enzymes that catalyze oxidative phase MPN. A total of 38 IDH mutations (18 IDH1-R132, 19 IDH2- decarboxylation of isocitrate to a-ketoglutarate. IDH1 (cyto- 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%) plasm and peroxisome) and IDH2 (mitochondria) use NADP blast-phase MPN (Po0.01). Mutant IDH was documented in the as a co-factor to generate NADPH, which is important in the presence or absence of JAK2, MPL and TET2 mutations, with production of intracellular glutathione. Intact IDH activity is similar mutational frequencies. However, IDH-mutated patients therefore necessary for cellular protection from oxidative stress. were more likely to be nullizygous for JAK2 46/1 haplotype, Mutant IDH has decreased affinity to isocitrate, but displays especially in PMF (P¼ 0.04), and less likely to display complex neomorphic catalytic activity toward a-ketoglutarate, the net karyotype, in blast-phase disease (Po0.01). In chronic-phase PMF, JAK2 46/1 haplotype nullizygosity (Po0.01; hazard ratio result being decreased supply of a-ketoglutarate and accumula- 10–13 (HR) 2.9, 95% confidence interval (CI) 1.7–5.2), but not IDH tion of 2-hydroxyglutarate. It is currently believed that these mutational status (P¼ 0.55; HR 1.3, 95% CI 0.5–3.4), had an intracellular changes facilitate oncogenic pathways including adverse effect on survival. This was confirmed by multivariable activation of HIF-1a. analysis. In contrast, in both blast-phase PMF (P¼ 0.04) and IDH1 and IDH2 mutations were first described in low-grade blast-phase MPN (P¼ 0.01), the presence of an IDH mutation gliomas/secondary glioblastomas and subsequently in acute predicted worse survival. The current study clarifies disease- and stage-specific IDH mutation incidence and prognostic myeloid leukemia (AML), with respective mutational frequen- relevance in MPN and provides additional evidence for the cies of B70 and 8%. We recently screened 200 patients biological effect of distinct JAK2 haplotypes. with either chronic- or blast-phase MPN for IDH mutations, and Leukemia (2010) 24, 1302–1309; doi:10.1038/leu.2010.113; identified 9 patients with either IDH1 (n¼ 5) or IDH2 (n¼ 4) published online 27 May 2010 mutations. Mutational frequencies were B21% for blast-phase Keywords: JAK2; MPL; TET2; myeloproliferative 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 Introduction 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 Despite the seminal discovery of JAK2 or MPL mutations in the majority of patients with BCR-ABL1-negative myeloproliferative and TET2 mutation and JAK2 haplotype status) phenotype, as 1–4 well as their prognostic relevance. neoplasms (MPN), it is becoming increasingly evident that these mutations do not signify either disease-initiating or 5,6 leukemia-promoting events. It is therefore important to keep looking for additional molecular alterations to clarify the genetic Materials and methods underpinnings of both chronic- and blast-phase MPN. In the last 2 years, mutations involving TET2, ASXL1 and CBL have This study was approved by the Mayo Clinic institutional review been described in some patients with BCR-ABL1-negative MPN, board. All patients provided authorization for use of their including polycythemia vera (PV), essential thrombocythemia medical records for research purposes, and the research was (ET) and primary myelofibrosis (PMF). The precise pathogenetic carried out according to the principles of the Declaration of contribution of these mutations and their clinical relevance are Helsinki. Patient samples were obtained from the Mayo Clinic, Harvard Medical Institute and University of Florence. Muta- Correspondence: Professor A Tefferi, Division of Hematology, tional analyses were performed on DNA derived from either Department of Medicine, Mayo Clinic, Rochester, MN 55905, USA. bone marrow or peripheral blood granulocytes. JAK2 46/1 E-mail: tefferi.ayalew@mayo.edu haplotype analysis on patient samples accrued from Harvard was Received 1 April 2010; accepted 23 April 2010; published online 27 May 2010 performed on germline DNA. Diagnoses of MPN, post-PV/ET MF Isocitrate dehydrogenase mutations in MPN A Tefferi et al and AML, in patient samples accrued from the Mayo Clinic and were analyzed using the following primers for IDH1, which the University of Florence, were according to the World Health cover amino acid residues 41–138: sense, 5 -TGTGTTGAGAT 7,15 0 0 Organization and International Working Group criteria. GGACGCCTA-3 and anti-sense, 5 -GGTGTACTCAGAGCCTTC Diagnoses in patients accrued from Harvard were self-reported GC-3 . Sequencing of IDH2 used primers that covered amino during an internet-based collection of samples, as previously acid residues 125–226: sense, 5 -CTGCCTCTTTGTGGCCTA 0 0 0 detailed. AG-3 and anti-sense, 5 -ATTCTGGTTGAAAGATGGCG-3 . DNA from either bone marrow (Mayo Clinic samples) or Sequence analysis was performed using Mutation Surveyor granulocytes (samples from Harvard and the University (SoftGenetics, State College, PA, USA) and all mutations were of Florence) was extracted using conventional methods. MPL, validated by repeat PCR and sequencing on unamplified DNA JAK2 and TET2 mutation and JAK2 haplotype analyses were from the archival sample. 4,17–19 performed according to previously published methods. Mayo Clinic and University of Florence patient samples were With regard to IDH mutation analysis, Harvard patient samples screened for IDH1 and IDH2 mutations by direct sequencing IDH1 HRM IDH2 HRM 15.003 12.484 13.503 10.984 IDH1R132S IDH2R140Q 12.003 9.484 10.503 7.984 9.003 6.484 7.503 4.984 6.003 IDH1R132G 3.484 4.503 1.984 3.003 0.484 1.503 0.003 -1.016 -1.497 -2.516 -2.997 76.5 77.5 78.5 79.5 80.5 81.5 82.5 83.5 77.5 78 78.5 79 79.5 80 80.5 81 81.5 82 82.5 77 78 79 80 81 82 83 Temperature (°C) Temperature (°C) 5’ sequence 5’ sequence 3’ sequence IDH1R132S IDH1R132G 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 Males JAK2 mutation MPL mutation TET2 mutation JAK2 46/1 nullizygous in years (range) (%) frequency frequency frequency frequency Florence PV 150 62 (16–91) 66 83% (123/149) 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) 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) 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. Includes JAK2 exon 12 mutations: two cases from Mayo clinic and one case from Harvard. Leukemia Relative Signal Difference Relative Signal Difference Isocitrate dehydrogenase mutations in MPN A Tefferi et al and/or high-resolution melting assay. Direct sequencing for Correlation of IDH mutations with other IDH1 exon 4 mutations was carried out using the following MPN-associated mutations and JAK2 46/1 haplotype 0 0 primer sequences: sense, 5 -CGGTCTTCAGAGAAGCCATT-3 Considering the preponderance of informative cases with 0 0 and anti-sense, 5 -CACATTATTGCCAACATGAC-3 . IDH2 centrally confirmed diagnosis and availability of a more exon 4 was amplified using sense, 5 -CCACTATTATCTCTGTC complete laboratory data, the current analysis was limited to 0 0 0 CTC-3 and anti-sense, 5 -GCTAGGCGAGGAGCTCCAGT-3 . patients from the Mayo Clinic cohort (n ¼ 629). IDH mutational Both reactions were performed in 25 ml volume containing frequencies were similar among JAK2- (3.6%), MPL- (4.3%) and 100 ng of DNA, 0.25 U Taq polymerase, 0.3 mM each of dATP, TET2 (3.2%)-mutated patients and their respective mutation- dCTP, dGTP and dTTP, 5 mlof a 10 PCR buffer (Roche negative counterparts (4.2, 5.3 and 6.3%; Table 3). In other Diagnostics, Indianapolis, IN, USA) and 0.2 mM each of sense words, mutant IDH was shown to co-occur with a JAK2, MPL or and anti-sense primers. The reaction was denatured at 94 1C for TET2 mutation, and mutational frequency did not appear to 3 min followed by 35 cycles of denaturing at 94 1C for 30 s, be influenced by either the type of the coexisting mutation annealing at 57 1C for 30 s and extension at 72 1C for 40 s. (P ¼ 0.96) or the presence or absence of each specific mutation After a final extension at 72 1C for 2 min, the products (Table 3). However, IDH-mutated cases were more likely to be were confirmed by running on 1.3% agarose gel and purified nullizygous for JAK2 46/1 haplotype, especially when analysis using Qiagen’s PCR Quick Purification Kit. The product was was restricted to informative (that is, with JAK2 46/1 haplotype sequenced using the ABI PRISM 3730xl analyzer (Applied information) patients with chronic- (n ¼ 158) or blast (n ¼ 23)- Biosystems Inc, Foster City, CA, USA) to screen for the presence phase PMF, analyzed together (P ¼ 0.007) or separately of mutations. (P ¼ 0.04; Table 4). 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 Clinical correlates and prognostic relevance primers for IDH2 mutations (R140 and R172): R140 sense, To avoid disease- or stage-specific confounding factors, as well 0 0 0 5 -GCTGAAGAAGATGTGGAA-3 and anti-sense, 5 -TGATGG as assure adequate sample size of informative cases, clinical 0 0 GCTCCCGGAAGA-3 ; R172 sense, 5 -CCAAGCCCATCACCAT correlative and prognostic studies were limited to PMF. In this 0 0 0 TG-3 and anti-sense, 5 -CCCAGGTCAGTGGATCCC-3 (Figure 1). patient cohort, detailed clinical information was available in Conventional statistical procedures were used (SAS Institute, 111 patients with chronic-phase PMF (including 7 IDH-mutated Cary, NC, USA). All statistically analyzed data were obtained at cases) and 27 patients with blast-phase PMF (including 8 IDH- time of IDH mutation analysis. All P-values were two-tailed mutated cases), both patient populations were accrued from the and statistical significance was set at the level of Po0.05. Mayo Clinic cohort. In both chronic- and blast-phase PMF, the Categorical variables were described as count and relative presence of IDH mutations was not influenced by either age frequency and compared by w statistics. Comparison of (P ¼ 0.51 and 0.70, respectively) or gender (P ¼ 0.09 and 0.3, continuous variables between categories was performed by respectively). In chronic-phase disease, comparison of prog- the Mann–Whitney U-test. Survival analysis was performed nostically relevant disease variables at diagnosis revealed that by the Kaplan–Meier method taking the interval from the date cytogenetic findings in IDH-mutated cases often belonged to a of diagnosis, for chronic-phase disease, or from the date of low- or intermediate-risk category, although the difference leukemic transformation, for blast-phase disease, to death was not statistically significant (Table 4). Similarly, IDH-mutated or last contact. The log-rank test was used to compare blast-phase PMF was less likely to display complex karyotype survival data. Cox regression model was used for multivariable (0 vs 64% in IDH-unmutated cases; P¼ 0.001). analysis. In addition to biological implications, the aforementioned associations of IDH mutations with favorable cytogenetic profile and JAK2 46/1 haplotype nullizygosity, both which have 19,20 previously been shown to be prognostically relevant, Results mandated their inclusion as covariates during multivariable survival analysis. In chronic-phase PMF, univariate analysis Disease- and stage-specific IDH mutational frequencies showed statistically significant adverse survival effect from JAK2 A total of 1473 patients with BCR–ABL1-negative MPN were 46/1 haplotype nullizygosity (P¼ 0.0001; 34 nullizygous vs 74 recruited from the Mayo Clinic, Rochester, MN, USA (n¼ 629), not nullizygous), high-risk karyotype (Po0.0001; 13 high-risk vs University of Florence, Florence, Italy (n¼ 522) and Harvard 98 not high-risk) and higher International Prognostic Scoring Medical Institute, Boston, Massachusetts, USA (n¼ 322). System (IPSS; 27 high, 29 intermediate-2, 30 intermediate-1 and Specific diagnoses included ET (n¼ 594), PV (n ¼ 421), PMF 25 low-risk patients) risk score (Po0.0001), but not from IDH (n ¼ 312), post-PV MF (n ¼ 54), post-ET MF (n ¼ 41), post-PV mutational status (P ¼ 0.54; 7 mutated vs 104 unmutated; AML (n¼ 12), post-ET AML (n ¼ 7) and post-PMF AML (n¼ 32). Figure 2). Multivariable analysis confirmed the independent Table 1 provides clinical and laboratory details of the study prognostic value of JAK2 46/1 haplotype status (hazard ratio population including age and sex distribution, specific diag- (HR) 2.2, 95% confidence interval (CI) 1.2–4.2), karyotype noses and JAK2, MPL and TET2 mutational and JAK2 46/1 (HR 2.8, 95% CI 1.3–5.9) and IPSS risk score (HR 4.8, 95% CI haplotype status, stratified by center of patient recruitment. 2.0–11.5). A total of 38 IDH mutations were documented (Table 2): 18 In blast-phase PMF, despite its association with noncomplex involved IDH1 (10 R132S, 7 R132C and 1 R132G) and 20 IDH2 karyotype, the presence of mutant IDH predicted shortened (18 R140Q, 1 R140W and 1 R172G). IDH mutations were survival, calculated from the time of disease transformation infrequent in chronic- or fibrotic-phase disease and significantly (P ¼ 0.04), and there was a similar trend for JAK2 non-46/1 more prevalent in blast-phase disease (Po0.01; Table 3): haplotype (P¼ 0.14; Figure 3). Significance was lost for both 5 (0.8%) in ET, 8 (1.9%) in PV, 13 (4.1%) in PMF, 1 (1%) in during multivariable analysis, probably because of small sample post-ET/PV MF, none in blast-phase ET, 3 (25%) in blast-phase size. IDH mutation status also predicted worse survival when PV and 8 (25%) in blast-phase PMF. the analysis included all blast-phase MPN cases from the Mayo Leukemia Isocitrate dehydrogenase mutations in MPN A Tefferi et al Leukemia 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) IDH mutation JAK2 V617F MPL mutation TET2 mutation JAK2 haplotype Karyotype Antecedent MPN IDH analysis Status at and sex variant burden status status status diagnosis to IDH analysis to last f/u 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 o1 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 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. Multiple trisomies: +2, +3, +6, +8, +10, +11, +12, +13, +14, +19, +20, +21. Isocitrate dehydrogenase mutations in MPN A Tefferi et al 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 IDH mutated IDH1 IDH2 P-value patients (IDH1 or IDH2), n (%) mutated, n mutated, n 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 (n¼ 629) 389 vs 240 14 (3.6%) vs 10 (4.2%) 0.72 MPL mutated vs wild type (n¼ 364) 23 vs 341 1 (4.3%) vs 18 (5.3%) 0.85 TET2 mutated vs wild type (n¼ 237) 31 vs 206 1 (3.2%) vs 13 (6.3%) 0.5 JAK2 46/1 nullizygous vs not nullizygous (n ¼ 596) 189 vs 407 11 (5.8%) vs 12 (2.9%) 0.09 Abbreviations: AML, acute myeloid leukemia; IDH, isocitrate dehydrogenase; PMF, primary myelofibrosis. 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 (n¼ 166) or blast-phase (n¼ 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 111 (13 vs 98) 7 (6.3%) (0 (0%) vs 7 (7.1%)) 0.32 vs not high-risk) Blast-phase PMF karyotype at transformation (complex karyotype 22 (11 vs 11) 7 (32%) (0 vs 7 (64%)) 0.001 vs not complex) Abbreviations: IDH, isocitrate dehydrogenase; N, number of patients evaluable; PMF, primary myelofibrosis. P=0.54 P<0.0001 0.8 0.8 0.6 0.6 0.4 0.4 IDH Low-or intermediate-risk mutated IDH karyotype unmutated High-risk karyotype 0 5 10 15 20 25 0 5 10 15 20 25 P=0.0001 P<0.0001 0.8 0.8 0.6 Low 0.6 risk Intermediate-1 0.4 0.4 risk Not nullizygous Intermediate-2 High risk risk Nullizygous 0 5 10 15 20 25 0 5 10 15 20 25 Years 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). Leukemia 0.2 0.2 0.2 0.2 Survival Isocitrate dehydrogenase mutations in MPN A Tefferi et al ab P=0.14 P=0.04 0.8 0.8 0.6 0.6 0.4 0.4 JAK2 46/1 haplotype 0.2 0.2 IDH not nullizygous unmutated Mutated Nullizygous 0 10203040506070 80 0 1020304050607080 Months Months Figure 3 Survival curves of patients with blast-phase primary myelofibrosis stratified by their isocitrate dehydrogenase (IDH) mutational (a; n¼ 27 including 8 mutated cases) or JAK2 46/1 haplotype (b; n¼ 23 including 8 nullizygous cases) status. 14,28 1 R132S, 15% R132G and 4% R132L). In both studies, IDH1 IDH un-mutated, n=32 mutations clustered with normal karyotype, NPM1 mutations IDH mutated, n=11 and trisomy 8. IDH1 mutations are rare in pediatric AML. P=0.01 0.8 12,13 More recently, IDH2 mutations, affecting R172 (R172K) or R140 (R140Q), were also shown to occur in primary 12,13 0.6 AML. 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. In 0.4 general, survival in primary AML did not seem to be affected by 13,14,28–30 the presence of IDH mutations. However, more recent 0.2 studies suggest that specific IDH mutation variants might be prognostically relevant in certain molecular subsets of AML. 0 The first reports of IDH mutations in MPN came from 9,32,33 0 1020304050607080 three independent groups. In one of these studies, IDH1 Months mutations were seen in B8% (5 of 63) of blast-phase MPN patients, mostly occurring in the absence of TET2 and ASXL1 Figure 4 Survival curves of 43 patients with blast-phase myelo- mutations. The second study was focused on blast-phase MPN proliferative neoplasm stratified by their isocitrate dehydrogenase that arose from JAK2-mutated chronic-phase MPN. In this (IDH) mutational status. 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. The third study from the Mayo cohort (Figure 4; n¼ 43; P¼ 0.01). In this instance, significance Clinic included 200 MPN patients and showed IDH mutational was sustained during multivariable analysis that included frequencies of B21% for blast-phase MPN, regardless of JAK2 JAK2 46/1 haplotype as a covariate. mutational status, and B4% for PMF. The specific IDH1 mutations found in the particular study included R132C and R132S and the IDH2 mutations R140Q and R140W. Discussion The current study is an extension of the above-mentioned Mayo Clinic study and involves a large number of patients IDH1 point mutations involving exon 4 occur in the majority (n¼ 1473) recruited from three major MPN centers of excel- (60–90%) of patients with low-grade gliomas and secondary lence. The results of the study clarify a number of issues glioblastomas, and always affect the amino acid arginine at regarding IDH mutations in MPN. First, the study provides position 132 (B93% R132H, 4% R132C, 2% R132S and o1% robust incidence figures for IDH1 and IDH2 mutations across 8,22,23 R132G, R132L or R132V). These mutations are relatively different disease stages of specific MPN variants. Accordingly, infrequent in primary glioblastoma (B7%) and are usually not we now show that both IDH1 and IDH2 mutations can occur in 23,24 seen in other solid tumors. A small fraction (B4%) of chronic-phase ET, PV or PMF, although infrequently. Mutational glioma-associated IDH mutations involves IDH2, specifically frequency was equally low in post-PV/ET MF and this fact the R132 analogous R172 residue on exon 4 (R172K, R172M, combined with the significantly higher mutation incidence 23,25 R172G, R172W). IDH mutations in glioma are hetero- observed in blast-phase disease suggests a pathogenetic zygous, believed to constitute early genetic events and might be contribution to leukemic but not fibrotic disease transformation. mutually exclusive of EGFR and PTEN, but not TP53 mutations. Two additional observations support this contention (i) complex Clinical correlates of IDH mutations in glioma include younger karyotype was infrequently encountered in IDH-mutated blast- age, longer survival and reduced risk of disease progression after phase MPN, which suggests an independent pathogenetic 8,22,23,26,27 conventional therapy. contribution that might be tied to distinct molecular alterations, The first study on IDH mutations in AML included 188 such as, for example, overexpression of the APP (amyloid a ˆ (A4) patients with primary AML and reported IDH1, but not IDH2, precursor protein) gene, which has previously been shown in mutations in 8.5% (n ¼ 16) of the cases and 16% of those with AML to be associated with either complex karyotype or normal karyotype: R132C in 8 patients, R132H in 7 and R132S IDHR172 mutation and (ii) the absence of mutual exclusivity 14 28 in 1. In a subsequent AML study of 493 patients, 27 (5.5%) between IDH and other MPN-associated mutations (for exam- expressed IDH1 mutations (37% R132C, 26% R132H, 19% ple, TET2, MPL), which is consistent with the suggestion that Leukemia Survival Survival Isocitrate dehydrogenase mutations in MPN A Tefferi et al the former are later-arising cooperating mutations that are Acknowledgements more involved in disease progression rather than disease This study is supported in part by grants from the ‘Myelo- initiation. proliferative Disorders Foundation, Chicago, IL, USA’, ‘The The types of IDH mutations seen in our patients with MPN Henry J. Predolin Foundation for Research in Leukemia, Mayo (mostly IDH2R140Q and IDH1R132S/C) are distinctly different Clinic, Rochester, MN, USA’ and ‘Associazione Italiana per la than those seen in gliomas (mostly IDH1R132H) and more Ricerca sul Cancro-AIRC Milan, Italy, to AMV’. 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 References and blast-phase MPN. More studies are needed to confirm this apparent trend. Regardless, there is currently no good explana- 1 James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C tion for the observed diversity in IDH mutation variants among et al. A unique clonal JAK2 mutation leading to constitutive gliomas and myeloid malignancies and current information signalling causes polycythaemia vera. Nature 2005; 434: suggests similar biological consequences. Whether or not 1144–1148. different IDH mutations carry different prognostic relevance in 2 Scott LM, Tong W, Levine RL, Scott MA, Beer PA, Stratton MR et al. MPN is currently not known and we did not attempt to address JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis. N Engl J Med 2007; 356: 459–468. the particular issue because of our relatively small number of 3 Pikman Y, Lee BH, Mercher T, McDowell E, Ebert BL, Gozo M informative cases. Of note, in a recent study of primary AML et al. MPLW515L is a novel somatic activating mutation in with normal karyotype, different types of IDH mutations myelofibrosis with myeloid metaplasia. PLoS Med 2006; 3: e270. appeared to variably influence disease-free survival and 4 Pardanani AD, Levine RL, Lasho T, Pikman Y, Mesa RA, Wadleigh complete remission rates. M et al. MPL515 mutations in myeloproliferative and other One particularly interesting observation from the current myeloid disorders: a study of 1182 patients. Blood 2006; 108: 3472–3476. study was the significant association between mutant IDH and 5 Kilpivaara O, Levine RL. JAK2 and MPL mutations in myelopro- JAK2 non-46/1 haplotype. The latter phenomenon is further liferative neoplasms: discovery and science. Leukemia 2008; 22: evidence for the JAK2 mutation specificity of the previously 1813–1817. described association between the JAK2 46/1 haplotype and 6 Vannucchi AM, Antonioli E, Guglielmelli P, Pardanani A, Tefferi A. 19,34,35 19,35 MPN. In other words, whereas JAK2 exon 14 or exon Clinical correlates of JAK2V617F presence or allele burden in 12 mutations have been shown to be associated with JAK2 myeloproliferative neoplasms: a critical reappraisal. Leukemia 2008; 22: 1299–1307. 46/1 haplotype, we did not see the same effect involving MPL 34 37 7 Tefferi A, Vardiman JW. 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Impact of IDH1 R132 mutations and an IDH1 single nucleotide polymorphism in cytogenetically normal acute myeloid This work is licensed under the Creative Commons leukemia: SNP rs11554137 is an adverse prognostic factor. J Clin Attribution-NonCommercial-No Derivative Works Oncol 2010; 28: 2356–2364. JCO.2009.2027.6899. 3.0 Unported License. To view a copy of this license, visit http:// 31 Marcucci G, Maharry K, Wu Y-Z, Radmacher MD, Mrozek K, creativecommons.org/licenses/by-nc-nd/3.0/ Margeson D et al. IDH1 and IDH2 gene mutations identify novel Leukemia http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Leukemia Springer Journals

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

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Springer Journals
Copyright
Copyright © 2010 by The Author(s)
Subject
Medicine & Public Health; Medicine/Public Health, general; Internal Medicine; Intensive / Critical Care Medicine; Cancer Research; Oncology; Hematology
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0887-6924
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1476-5551
DOI
10.1038/leu.2010.113
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Abstract

Leukemia (2010) 24, 1302–1309 & 2010 Macmillan Publishers Limited All rights reserved 0887-6924/10 www.nature.com/leu ORIGINAL ARTICLE IDH1 and IDH2 mutation studies in 1473 patients with chronic-, fibrotic- or blast-phase essential thrombocythemia, polycythemia vera or myelofibrosis 1 1 2 3 2 4 3 1 2 A Tefferi , TL Lasho , O Abdel-Wahab , P Guglielmelli , J Patel , D Caramazza , L Pieri , CM Finke , O Kilpivaara , 5 6 6 5 2 1 3 M Wadleigh , M Mai , RF McClure , DG Gilliland , RL Levine , A Pardanani and AM Vannucchi 1 2 Division of Hematology, Department of Medicine, Mayo Clinic, Rochester, MN, USA; Human Oncology and Pathogenesis Program and Leukemia Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, USA; UF di Ematologia, Dipartimento di Area Critica Medico-Chirurgica, Universita ` degli Studi, Azienda Ospedaliera-Universitaria Careggi, Istituto Toscano Tumori, Firenze, Italy; Cattedra ed U.O. di Ematologia, Policlinico Universitario di Palermo, Palermo, Italy; Division of Hematology, Department of Medicine, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA and Division of Hematopathology, Department of Laboratory Medicine, Mayo Clinic, Rochester, MN, USA In a multi-institutional collaborative project, 1473 patients currently under investigation. The glioma-associated isocitrate with myeloproliferative neoplasms (MPN) were screened for dehydrogenase 1 (IDH1) and IDH2 mutations are the latest to isocitrate dehydrogenase 1 (IDH1)/IDH2 mutations: 594 essen- 9 be added to the ‘MPN mutations list’. tial thrombocythemia (ET), 421 polycythemia vera (PV), 312 IDH1, located on chromosome 2q33.3, and IDH2, located on primary myelofibrosis (PMF), 95 post-PV/ET MF and 51 blast- chromosome 15q26.1, encode enzymes that catalyze oxidative phase MPN. A total of 38 IDH mutations (18 IDH1-R132, 19 IDH2- decarboxylation of isocitrate to a-ketoglutarate. IDH1 (cyto- 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%) plasm and peroxisome) and IDH2 (mitochondria) use NADP blast-phase MPN (Po0.01). Mutant IDH was documented in the as a co-factor to generate NADPH, which is important in the presence or absence of JAK2, MPL and TET2 mutations, with production of intracellular glutathione. Intact IDH activity is similar mutational frequencies. However, IDH-mutated patients therefore necessary for cellular protection from oxidative stress. were more likely to be nullizygous for JAK2 46/1 haplotype, Mutant IDH has decreased affinity to isocitrate, but displays especially in PMF (P¼ 0.04), and less likely to display complex neomorphic catalytic activity toward a-ketoglutarate, the net karyotype, in blast-phase disease (Po0.01). In chronic-phase PMF, JAK2 46/1 haplotype nullizygosity (Po0.01; hazard ratio result being decreased supply of a-ketoglutarate and accumula- 10–13 (HR) 2.9, 95% confidence interval (CI) 1.7–5.2), but not IDH tion of 2-hydroxyglutarate. It is currently believed that these mutational status (P¼ 0.55; HR 1.3, 95% CI 0.5–3.4), had an intracellular changes facilitate oncogenic pathways including adverse effect on survival. This was confirmed by multivariable activation of HIF-1a. analysis. In contrast, in both blast-phase PMF (P¼ 0.04) and IDH1 and IDH2 mutations were first described in low-grade blast-phase MPN (P¼ 0.01), the presence of an IDH mutation gliomas/secondary glioblastomas and subsequently in acute predicted worse survival. The current study clarifies disease- and stage-specific IDH mutation incidence and prognostic myeloid leukemia (AML), with respective mutational frequen- relevance in MPN and provides additional evidence for the cies of B70 and 8%. We recently screened 200 patients biological effect of distinct JAK2 haplotypes. with either chronic- or blast-phase MPN for IDH mutations, and Leukemia (2010) 24, 1302–1309; doi:10.1038/leu.2010.113; identified 9 patients with either IDH1 (n¼ 5) or IDH2 (n¼ 4) published online 27 May 2010 mutations. Mutational frequencies were B21% for blast-phase Keywords: JAK2; MPL; TET2; myeloproliferative 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 Introduction 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 Despite the seminal discovery of JAK2 or MPL mutations in the majority of patients with BCR-ABL1-negative myeloproliferative and TET2 mutation and JAK2 haplotype status) phenotype, as 1–4 well as their prognostic relevance. neoplasms (MPN), it is becoming increasingly evident that these mutations do not signify either disease-initiating or 5,6 leukemia-promoting events. It is therefore important to keep looking for additional molecular alterations to clarify the genetic Materials and methods underpinnings of both chronic- and blast-phase MPN. In the last 2 years, mutations involving TET2, ASXL1 and CBL have This study was approved by the Mayo Clinic institutional review been described in some patients with BCR-ABL1-negative MPN, board. All patients provided authorization for use of their including polycythemia vera (PV), essential thrombocythemia medical records for research purposes, and the research was (ET) and primary myelofibrosis (PMF). The precise pathogenetic carried out according to the principles of the Declaration of contribution of these mutations and their clinical relevance are Helsinki. Patient samples were obtained from the Mayo Clinic, Harvard Medical Institute and University of Florence. Muta- Correspondence: Professor A Tefferi, Division of Hematology, tional analyses were performed on DNA derived from either Department of Medicine, Mayo Clinic, Rochester, MN 55905, USA. bone marrow or peripheral blood granulocytes. JAK2 46/1 E-mail: tefferi.ayalew@mayo.edu haplotype analysis on patient samples accrued from Harvard was Received 1 April 2010; accepted 23 April 2010; published online 27 May 2010 performed on germline DNA. Diagnoses of MPN, post-PV/ET MF Isocitrate dehydrogenase mutations in MPN A Tefferi et al and AML, in patient samples accrued from the Mayo Clinic and were analyzed using the following primers for IDH1, which the University of Florence, were according to the World Health cover amino acid residues 41–138: sense, 5 -TGTGTTGAGAT 7,15 0 0 Organization and International Working Group criteria. GGACGCCTA-3 and anti-sense, 5 -GGTGTACTCAGAGCCTTC Diagnoses in patients accrued from Harvard were self-reported GC-3 . Sequencing of IDH2 used primers that covered amino during an internet-based collection of samples, as previously acid residues 125–226: sense, 5 -CTGCCTCTTTGTGGCCTA 0 0 0 detailed. AG-3 and anti-sense, 5 -ATTCTGGTTGAAAGATGGCG-3 . DNA from either bone marrow (Mayo Clinic samples) or Sequence analysis was performed using Mutation Surveyor granulocytes (samples from Harvard and the University (SoftGenetics, State College, PA, USA) and all mutations were of Florence) was extracted using conventional methods. MPL, validated by repeat PCR and sequencing on unamplified DNA JAK2 and TET2 mutation and JAK2 haplotype analyses were from the archival sample. 4,17–19 performed according to previously published methods. Mayo Clinic and University of Florence patient samples were With regard to IDH mutation analysis, Harvard patient samples screened for IDH1 and IDH2 mutations by direct sequencing IDH1 HRM IDH2 HRM 15.003 12.484 13.503 10.984 IDH1R132S IDH2R140Q 12.003 9.484 10.503 7.984 9.003 6.484 7.503 4.984 6.003 IDH1R132G 3.484 4.503 1.984 3.003 0.484 1.503 0.003 -1.016 -1.497 -2.516 -2.997 76.5 77.5 78.5 79.5 80.5 81.5 82.5 83.5 77.5 78 78.5 79 79.5 80 80.5 81 81.5 82 82.5 77 78 79 80 81 82 83 Temperature (°C) Temperature (°C) 5’ sequence 5’ sequence 3’ sequence IDH1R132S IDH1R132G 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 Males JAK2 mutation MPL mutation TET2 mutation JAK2 46/1 nullizygous in years (range) (%) frequency frequency frequency frequency Florence PV 150 62 (16–91) 66 83% (123/149) 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) 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) 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. Includes JAK2 exon 12 mutations: two cases from Mayo clinic and one case from Harvard. Leukemia Relative Signal Difference Relative Signal Difference Isocitrate dehydrogenase mutations in MPN A Tefferi et al and/or high-resolution melting assay. Direct sequencing for Correlation of IDH mutations with other IDH1 exon 4 mutations was carried out using the following MPN-associated mutations and JAK2 46/1 haplotype 0 0 primer sequences: sense, 5 -CGGTCTTCAGAGAAGCCATT-3 Considering the preponderance of informative cases with 0 0 and anti-sense, 5 -CACATTATTGCCAACATGAC-3 . IDH2 centrally confirmed diagnosis and availability of a more exon 4 was amplified using sense, 5 -CCACTATTATCTCTGTC complete laboratory data, the current analysis was limited to 0 0 0 CTC-3 and anti-sense, 5 -GCTAGGCGAGGAGCTCCAGT-3 . patients from the Mayo Clinic cohort (n ¼ 629). IDH mutational Both reactions were performed in 25 ml volume containing frequencies were similar among JAK2- (3.6%), MPL- (4.3%) and 100 ng of DNA, 0.25 U Taq polymerase, 0.3 mM each of dATP, TET2 (3.2%)-mutated patients and their respective mutation- dCTP, dGTP and dTTP, 5 mlof a 10 PCR buffer (Roche negative counterparts (4.2, 5.3 and 6.3%; Table 3). In other Diagnostics, Indianapolis, IN, USA) and 0.2 mM each of sense words, mutant IDH was shown to co-occur with a JAK2, MPL or and anti-sense primers. The reaction was denatured at 94 1C for TET2 mutation, and mutational frequency did not appear to 3 min followed by 35 cycles of denaturing at 94 1C for 30 s, be influenced by either the type of the coexisting mutation annealing at 57 1C for 30 s and extension at 72 1C for 40 s. (P ¼ 0.96) or the presence or absence of each specific mutation After a final extension at 72 1C for 2 min, the products (Table 3). However, IDH-mutated cases were more likely to be were confirmed by running on 1.3% agarose gel and purified nullizygous for JAK2 46/1 haplotype, especially when analysis using Qiagen’s PCR Quick Purification Kit. The product was was restricted to informative (that is, with JAK2 46/1 haplotype sequenced using the ABI PRISM 3730xl analyzer (Applied information) patients with chronic- (n ¼ 158) or blast (n ¼ 23)- Biosystems Inc, Foster City, CA, USA) to screen for the presence phase PMF, analyzed together (P ¼ 0.007) or separately of mutations. (P ¼ 0.04; Table 4). 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 Clinical correlates and prognostic relevance primers for IDH2 mutations (R140 and R172): R140 sense, To avoid disease- or stage-specific confounding factors, as well 0 0 0 5 -GCTGAAGAAGATGTGGAA-3 and anti-sense, 5 -TGATGG as assure adequate sample size of informative cases, clinical 0 0 GCTCCCGGAAGA-3 ; R172 sense, 5 -CCAAGCCCATCACCAT correlative and prognostic studies were limited to PMF. In this 0 0 0 TG-3 and anti-sense, 5 -CCCAGGTCAGTGGATCCC-3 (Figure 1). patient cohort, detailed clinical information was available in Conventional statistical procedures were used (SAS Institute, 111 patients with chronic-phase PMF (including 7 IDH-mutated Cary, NC, USA). All statistically analyzed data were obtained at cases) and 27 patients with blast-phase PMF (including 8 IDH- time of IDH mutation analysis. All P-values were two-tailed mutated cases), both patient populations were accrued from the and statistical significance was set at the level of Po0.05. Mayo Clinic cohort. In both chronic- and blast-phase PMF, the Categorical variables were described as count and relative presence of IDH mutations was not influenced by either age frequency and compared by w statistics. Comparison of (P ¼ 0.51 and 0.70, respectively) or gender (P ¼ 0.09 and 0.3, continuous variables between categories was performed by respectively). In chronic-phase disease, comparison of prog- the Mann–Whitney U-test. Survival analysis was performed nostically relevant disease variables at diagnosis revealed that by the Kaplan–Meier method taking the interval from the date cytogenetic findings in IDH-mutated cases often belonged to a of diagnosis, for chronic-phase disease, or from the date of low- or intermediate-risk category, although the difference leukemic transformation, for blast-phase disease, to death was not statistically significant (Table 4). Similarly, IDH-mutated or last contact. The log-rank test was used to compare blast-phase PMF was less likely to display complex karyotype survival data. Cox regression model was used for multivariable (0 vs 64% in IDH-unmutated cases; P¼ 0.001). analysis. In addition to biological implications, the aforementioned associations of IDH mutations with favorable cytogenetic profile and JAK2 46/1 haplotype nullizygosity, both which have 19,20 previously been shown to be prognostically relevant, Results mandated their inclusion as covariates during multivariable survival analysis. In chronic-phase PMF, univariate analysis Disease- and stage-specific IDH mutational frequencies showed statistically significant adverse survival effect from JAK2 A total of 1473 patients with BCR–ABL1-negative MPN were 46/1 haplotype nullizygosity (P¼ 0.0001; 34 nullizygous vs 74 recruited from the Mayo Clinic, Rochester, MN, USA (n¼ 629), not nullizygous), high-risk karyotype (Po0.0001; 13 high-risk vs University of Florence, Florence, Italy (n¼ 522) and Harvard 98 not high-risk) and higher International Prognostic Scoring Medical Institute, Boston, Massachusetts, USA (n¼ 322). System (IPSS; 27 high, 29 intermediate-2, 30 intermediate-1 and Specific diagnoses included ET (n¼ 594), PV (n ¼ 421), PMF 25 low-risk patients) risk score (Po0.0001), but not from IDH (n ¼ 312), post-PV MF (n ¼ 54), post-ET MF (n ¼ 41), post-PV mutational status (P ¼ 0.54; 7 mutated vs 104 unmutated; AML (n¼ 12), post-ET AML (n ¼ 7) and post-PMF AML (n¼ 32). Figure 2). Multivariable analysis confirmed the independent Table 1 provides clinical and laboratory details of the study prognostic value of JAK2 46/1 haplotype status (hazard ratio population including age and sex distribution, specific diag- (HR) 2.2, 95% confidence interval (CI) 1.2–4.2), karyotype noses and JAK2, MPL and TET2 mutational and JAK2 46/1 (HR 2.8, 95% CI 1.3–5.9) and IPSS risk score (HR 4.8, 95% CI haplotype status, stratified by center of patient recruitment. 2.0–11.5). A total of 38 IDH mutations were documented (Table 2): 18 In blast-phase PMF, despite its association with noncomplex involved IDH1 (10 R132S, 7 R132C and 1 R132G) and 20 IDH2 karyotype, the presence of mutant IDH predicted shortened (18 R140Q, 1 R140W and 1 R172G). IDH mutations were survival, calculated from the time of disease transformation infrequent in chronic- or fibrotic-phase disease and significantly (P ¼ 0.04), and there was a similar trend for JAK2 non-46/1 more prevalent in blast-phase disease (Po0.01; Table 3): haplotype (P¼ 0.14; Figure 3). Significance was lost for both 5 (0.8%) in ET, 8 (1.9%) in PV, 13 (4.1%) in PMF, 1 (1%) in during multivariable analysis, probably because of small sample post-ET/PV MF, none in blast-phase ET, 3 (25%) in blast-phase size. IDH mutation status also predicted worse survival when PV and 8 (25%) in blast-phase PMF. the analysis included all blast-phase MPN cases from the Mayo Leukemia Isocitrate dehydrogenase mutations in MPN A Tefferi et al Leukemia 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) IDH mutation JAK2 V617F MPL mutation TET2 mutation JAK2 haplotype Karyotype Antecedent MPN IDH analysis Status at and sex variant burden status status status diagnosis to IDH analysis to last f/u 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 o1 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 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. Multiple trisomies: +2, +3, +6, +8, +10, +11, +12, +13, +14, +19, +20, +21. Isocitrate dehydrogenase mutations in MPN A Tefferi et al 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 IDH mutated IDH1 IDH2 P-value patients (IDH1 or IDH2), n (%) mutated, n mutated, n 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 (n¼ 629) 389 vs 240 14 (3.6%) vs 10 (4.2%) 0.72 MPL mutated vs wild type (n¼ 364) 23 vs 341 1 (4.3%) vs 18 (5.3%) 0.85 TET2 mutated vs wild type (n¼ 237) 31 vs 206 1 (3.2%) vs 13 (6.3%) 0.5 JAK2 46/1 nullizygous vs not nullizygous (n ¼ 596) 189 vs 407 11 (5.8%) vs 12 (2.9%) 0.09 Abbreviations: AML, acute myeloid leukemia; IDH, isocitrate dehydrogenase; PMF, primary myelofibrosis. 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 (n¼ 166) or blast-phase (n¼ 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 111 (13 vs 98) 7 (6.3%) (0 (0%) vs 7 (7.1%)) 0.32 vs not high-risk) Blast-phase PMF karyotype at transformation (complex karyotype 22 (11 vs 11) 7 (32%) (0 vs 7 (64%)) 0.001 vs not complex) Abbreviations: IDH, isocitrate dehydrogenase; N, number of patients evaluable; PMF, primary myelofibrosis. P=0.54 P<0.0001 0.8 0.8 0.6 0.6 0.4 0.4 IDH Low-or intermediate-risk mutated IDH karyotype unmutated High-risk karyotype 0 5 10 15 20 25 0 5 10 15 20 25 P=0.0001 P<0.0001 0.8 0.8 0.6 Low 0.6 risk Intermediate-1 0.4 0.4 risk Not nullizygous Intermediate-2 High risk risk Nullizygous 0 5 10 15 20 25 0 5 10 15 20 25 Years 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). Leukemia 0.2 0.2 0.2 0.2 Survival Isocitrate dehydrogenase mutations in MPN A Tefferi et al ab P=0.14 P=0.04 0.8 0.8 0.6 0.6 0.4 0.4 JAK2 46/1 haplotype 0.2 0.2 IDH not nullizygous unmutated Mutated Nullizygous 0 10203040506070 80 0 1020304050607080 Months Months Figure 3 Survival curves of patients with blast-phase primary myelofibrosis stratified by their isocitrate dehydrogenase (IDH) mutational (a; n¼ 27 including 8 mutated cases) or JAK2 46/1 haplotype (b; n¼ 23 including 8 nullizygous cases) status. 14,28 1 R132S, 15% R132G and 4% R132L). In both studies, IDH1 IDH un-mutated, n=32 mutations clustered with normal karyotype, NPM1 mutations IDH mutated, n=11 and trisomy 8. IDH1 mutations are rare in pediatric AML. P=0.01 0.8 12,13 More recently, IDH2 mutations, affecting R172 (R172K) or R140 (R140Q), were also shown to occur in primary 12,13 0.6 AML. 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. In 0.4 general, survival in primary AML did not seem to be affected by 13,14,28–30 the presence of IDH mutations. However, more recent 0.2 studies suggest that specific IDH mutation variants might be prognostically relevant in certain molecular subsets of AML. 0 The first reports of IDH mutations in MPN came from 9,32,33 0 1020304050607080 three independent groups. In one of these studies, IDH1 Months mutations were seen in B8% (5 of 63) of blast-phase MPN patients, mostly occurring in the absence of TET2 and ASXL1 Figure 4 Survival curves of 43 patients with blast-phase myelo- mutations. The second study was focused on blast-phase MPN proliferative neoplasm stratified by their isocitrate dehydrogenase that arose from JAK2-mutated chronic-phase MPN. In this (IDH) mutational status. 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. The third study from the Mayo cohort (Figure 4; n¼ 43; P¼ 0.01). In this instance, significance Clinic included 200 MPN patients and showed IDH mutational was sustained during multivariable analysis that included frequencies of B21% for blast-phase MPN, regardless of JAK2 JAK2 46/1 haplotype as a covariate. mutational status, and B4% for PMF. The specific IDH1 mutations found in the particular study included R132C and R132S and the IDH2 mutations R140Q and R140W. Discussion The current study is an extension of the above-mentioned Mayo Clinic study and involves a large number of patients IDH1 point mutations involving exon 4 occur in the majority (n¼ 1473) recruited from three major MPN centers of excel- (60–90%) of patients with low-grade gliomas and secondary lence. The results of the study clarify a number of issues glioblastomas, and always affect the amino acid arginine at regarding IDH mutations in MPN. First, the study provides position 132 (B93% R132H, 4% R132C, 2% R132S and o1% robust incidence figures for IDH1 and IDH2 mutations across 8,22,23 R132G, R132L or R132V). These mutations are relatively different disease stages of specific MPN variants. Accordingly, infrequent in primary glioblastoma (B7%) and are usually not we now show that both IDH1 and IDH2 mutations can occur in 23,24 seen in other solid tumors. A small fraction (B4%) of chronic-phase ET, PV or PMF, although infrequently. Mutational glioma-associated IDH mutations involves IDH2, specifically frequency was equally low in post-PV/ET MF and this fact the R132 analogous R172 residue on exon 4 (R172K, R172M, combined with the significantly higher mutation incidence 23,25 R172G, R172W). IDH mutations in glioma are hetero- observed in blast-phase disease suggests a pathogenetic zygous, believed to constitute early genetic events and might be contribution to leukemic but not fibrotic disease transformation. mutually exclusive of EGFR and PTEN, but not TP53 mutations. Two additional observations support this contention (i) complex Clinical correlates of IDH mutations in glioma include younger karyotype was infrequently encountered in IDH-mutated blast- age, longer survival and reduced risk of disease progression after phase MPN, which suggests an independent pathogenetic 8,22,23,26,27 conventional therapy. contribution that might be tied to distinct molecular alterations, The first study on IDH mutations in AML included 188 such as, for example, overexpression of the APP (amyloid a ˆ (A4) patients with primary AML and reported IDH1, but not IDH2, precursor protein) gene, which has previously been shown in mutations in 8.5% (n ¼ 16) of the cases and 16% of those with AML to be associated with either complex karyotype or normal karyotype: R132C in 8 patients, R132H in 7 and R132S IDHR172 mutation and (ii) the absence of mutual exclusivity 14 28 in 1. In a subsequent AML study of 493 patients, 27 (5.5%) between IDH and other MPN-associated mutations (for exam- expressed IDH1 mutations (37% R132C, 26% R132H, 19% ple, TET2, MPL), which is consistent with the suggestion that Leukemia Survival Survival Isocitrate dehydrogenase mutations in MPN A Tefferi et al the former are later-arising cooperating mutations that are Acknowledgements more involved in disease progression rather than disease This study is supported in part by grants from the ‘Myelo- initiation. proliferative Disorders Foundation, Chicago, IL, USA’, ‘The The types of IDH mutations seen in our patients with MPN Henry J. Predolin Foundation for Research in Leukemia, Mayo (mostly IDH2R140Q and IDH1R132S/C) are distinctly different Clinic, Rochester, MN, USA’ and ‘Associazione Italiana per la than those seen in gliomas (mostly IDH1R132H) and more Ricerca sul Cancro-AIRC Milan, Italy, to AMV’. 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 References and blast-phase MPN. More studies are needed to confirm this apparent trend. Regardless, there is currently no good explana- 1 James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C tion for the observed diversity in IDH mutation variants among et al. A unique clonal JAK2 mutation leading to constitutive gliomas and myeloid malignancies and current information signalling causes polycythaemia vera. Nature 2005; 434: suggests similar biological consequences. Whether or not 1144–1148. different IDH mutations carry different prognostic relevance in 2 Scott LM, Tong W, Levine RL, Scott MA, Beer PA, Stratton MR et al. MPN is currently not known and we did not attempt to address JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis. N Engl J Med 2007; 356: 459–468. the particular issue because of our relatively small number of 3 Pikman Y, Lee BH, Mercher T, McDowell E, Ebert BL, Gozo M informative cases. Of note, in a recent study of primary AML et al. MPLW515L is a novel somatic activating mutation in with normal karyotype, different types of IDH mutations myelofibrosis with myeloid metaplasia. PLoS Med 2006; 3: e270. appeared to variably influence disease-free survival and 4 Pardanani AD, Levine RL, Lasho T, Pikman Y, Mesa RA, Wadleigh complete remission rates. M et al. MPL515 mutations in myeloproliferative and other One particularly interesting observation from the current myeloid disorders: a study of 1182 patients. Blood 2006; 108: 3472–3476. study was the significant association between mutant IDH and 5 Kilpivaara O, Levine RL. JAK2 and MPL mutations in myelopro- JAK2 non-46/1 haplotype. The latter phenomenon is further liferative neoplasms: discovery and science. Leukemia 2008; 22: evidence for the JAK2 mutation specificity of the previously 1813–1817. described association between the JAK2 46/1 haplotype and 6 Vannucchi AM, Antonioli E, Guglielmelli P, Pardanani A, Tefferi A. 19,34,35 19,35 MPN. In other words, whereas JAK2 exon 14 or exon Clinical correlates of JAK2V617F presence or allele burden in 12 mutations have been shown to be associated with JAK2 myeloproliferative neoplasms: a critical reappraisal. Leukemia 2008; 22: 1299–1307. 46/1 haplotype, we did not see the same effect involving MPL 34 37 7 Tefferi A, Vardiman JW. Classification and diagnosis of myelo- mutations (although others have shown otherwise), and now proliferative neoplasms: the 2008 World Health Organization show an association with JAK2 non-46/1 haplotype for IDH criteria and point-of-care diagnostic algorithms. Leukemia 2008; mutations. This latter observation is also consistent with our 22: 14–22. previous report on the prognostically detrimental effect of 8 Parsons DW, Jones S, Zhang X, Lin JC, Leary RJ, Angenendt P et al. JAK2 non-46/1 haplotype in PMF; it is possible that patients An integrated genomic analysis of human glioblastoma multi- forme. Science 2008; 321: 1807–1812. with PMF who are nullizygous for JAK2 46/1 haplotype are 9 Pardanani A, Lasho T, Finke C, Mai M, McClure R, Tefferi A. IDH1 susceptible to additional adverse molecular events, such as IDH and IDH2 mutation analysis in chronic and blast phase myelo- mutations, which might lead to biologically more aggressive proliferative neoplasms. 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Impact of IDH1 R132 mutations and an IDH1 single nucleotide polymorphism in cytogenetically normal acute myeloid This work is licensed under the Creative Commons leukemia: SNP rs11554137 is an adverse prognostic factor. J Clin Attribution-NonCommercial-No Derivative Works Oncol 2010; 28: 2356–2364. JCO.2009.2027.6899. 3.0 Unported License. To view a copy of this license, visit http:// 31 Marcucci G, Maharry K, Wu Y-Z, Radmacher MD, Mrozek K, creativecommons.org/licenses/by-nc-nd/3.0/ Margeson D et al. IDH1 and IDH2 gene mutations identify novel Leukemia

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LeukemiaSpringer Journals

Published: May 27, 2010

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