Letters to the Editor
1319 modulate Bcl2 phosphorylation and suggest that PKC a-mediated Bcl2 phosphorylation may be relevant to chemoresistance in AML.
S Kurinna1, M Konopleva2, SL Palla3, W Chen2, S Kornblau2, R Contractor2, X Deng4, WS May4, M Andreeff2 and PP Ruvolo1,2 1 Division of Cell Signaling, Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX, USA; 2 Department of Blood and Marrow Transplantation, MD Anderson Cancer Center, Houston, TX, USA; 3 Department of Biostatistics and Applied Mathematics, MD Anderson Cancer Center, Houston, TX, USA and 4 Shands Cancer Center, University of Florida, Gainesville, FL, USA E-mail:
[email protected]
References 1 Yang E, Korsmeyer SJ. Molecular thanatopsis: a discourse on the Bcl2 family and cell death. Blood 1996; 88: 386–401.
2 Deng X, Kornblau SM, Ruvolo PP, May WS. Regulation of Bcl2 phosphorylation and potential significance for leukemic cell chemoresistance. J Natl Cancer Inst 2000; Monograph 28: 30–37. 3 Haldar S, Jena N, Croce CM. Inactivation of Bcl2 by phosphorylation. Proc Natl Acad Sci USA 1995; 92: 4507–4511. 4 Ito T, Deng X, Carr BK, May WS. Bcl2 phosphorylation required for anti-apoptosis function. J Biol Chem 1997; 272: 11671–11673. 5 Ruvolo PP, Deng X, Carr BK, May WS. A functional role for mitochondrial PKC a in Bcl2 phosphorylation and suppression of apoptosis. J Biol Chem 1998; 273: 25436–25442. 6 Deng X, Ruvolo P, Carr B, May WS. Survival function of ERK1/2 as IL-3-activated staurosporine-resistant Bcl2 kinases. Proc Natl Acad Sci USA 2000; 97: 1578–1583. 7 Konopleva M, Tsao T, Ruvolo P, Stiouf I, Estrov Z, Leysath CE et al. Novel triterpenoid CDDO-Me is a potent inducer of apoptosis and differentiation in acute myelogenous leukemia. Blood 2002; 99: 326–335. 8 Kornblau SM, Vu HT, Ruvolo P, Estrov Z, O’Brien S, Cortes J et al. Bax and PKCa modulate the prognostic impact of Bcl2 expression in acute myelogenous leukemia. Clinical Cancer Res 2000; 6: 1401–1409. 9 Pagano M, Halvorsen K. An algorithm for finding the exact significance levels of r x c contingency tables. J Am Stat Assoc 1981; 76: 731–934.
The JAK2 V617F mutation identifies a subgroup of MDS patients with isolated deletion 5q and a proliferative bone marrow
Leukemia (2006) 20, 1319–1321. doi:10.1038/sj.leu.2404215; published online 13 April 2006
The detection of JAK2 V617F somatic mutation has greatly enhanced our understanding of the pathogenesis of the bcr/ablnegative chronic myeloproliferative disorders (MPD). An increased sensitivity to erythropoietin and growth factor independence is reported in the presence of the mutation.1–3 Four independent groups recently report JAK2 V617F mutation in the majority of patients with polycythaemia vera and up to 50% of essential thrombocythaemia and idiopathic myelofibrosis.1–4 An increased incidence of thrombosis, haemorrhage and fibrotic transformation in the presence of the mutant allele is reported.3 Larger studies are however required to elicit the true prognostic significance of the mutation. The finding of JAK2 V617F mutation outside of the classical MPDs is uncommon with reports of low incidence in chronic myelomonocytic leukaemia, atypical chronic myeloid leukaemia, hypereosinophilic syndrome and chronic neutrophilic leukaemia.5 The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal haematopoietic stem cell disorders characterised by peripheral blood cytopenias, ineffective erythropoiesis and increased apoptosis. The 5q syndrome is a subgroup of MDS characterised by an interstitial deletion of the long arm of chromosome 5(q31–q33) with macrocytic anaemia, normal or elevated platelet count, hypolobated megakaryocytes and associated with a favourable prognosis. However, cases presenting with 5q deletion and marked elevation of the platelet count in association with a hypercellular bone marrow display characteristics more suggestive of an overlap syndrome (MDS/ MPD). We analysed 97 patients from six European centres known to have a diagnosis of MDS and deletion, 5q abnormality for the presence of JAK2 V617F mutation. Isolated deletion of 5q was
detected in 81/97 cases, whereas additional cytogenetic abnormalities were noted in 16/97. The diagnosis of MDS was based on the World Health Organization Classification. A summary of patient characteristics is outlined in Table 1. Ethical approval was obtained before study commencement. Samples for analysis were obtained from archived bone marrow aspirate slides, archived cytogenetic samples or peripheral blood. The mutant JAK2 allele was detected using an allele-specificpolymerase chain reaction (AS-PCR).4,6,7 The presence of the mutation and ratio of mutant to wild-type JAK2 allele was confirmed in 2/6 mutant cases using pyrosequencing. Polymerase chain reaction products were generated using AS-PCR primer sequences. Sequences were read from a reverse sequencing primer 50 -TCTCGTCTCCACAGA-30 . Pyrosequencing reactions were run on a Biotage PSQ HS 96 pyrosequencer. In a patient with JAK2 mutation, cells from bone aspirate were subjected to progenitor cell culture (CFU-GM- and BFU-Ederived colonies) or subfractionated into CD34 þ ve cells, followed by molecular analysis. Peripheral blood from the same case was also subfractionated into CD14, CD15, CD3 and CD19 þ cells using antigen-specific microbeads followed by selection using the AutoMacs cell separator Milteny. In the case of progenitor cultures, PCR was performed directly on the cells without prior DNA purification. DNA was extracted from subfractionated cells using Charge Switch reagents (Invitrogen, Inchinnan Business Park, Fountain Drive, Paisley, UK). All DNA extraction and cell separation kits were used according to the manufacturer’s instructions. Samples were processed in accordance with standard cytogenetic methods. CD34 þ cells were probed for 5q using the Vysis LSI EGR1 (5q31), spectrum orange D5S23 and spectrum green D5S721 probe, according to the manufacturer’s instructions (Abbot Laboratories). 100K Affymetrix SNP analysis of DNA extracted from CD34-positive cells was performed using Leukemia
Letters to the Editor
1320 Table 1
Summary of clinical data in all patients analysed JAK2 wild type
JAK2 mutant
67 years (36–92 years)
67 years (46–80 years)
Sex Male Female
26 65
2 4
WHO classification RCMLD RARS 5q syndrome RAEB-I RAEB-II AML (RAEB-t by FAB criteria)
4 1 59 13 10 4
5 1
8.7 (4.3–14) 4.45 (1.2–11.6) 250 (28–919)
9.0 (6.7–11.5) 5.21 (1.8–13.6) 475 (125–969)
82/9
6/0
86/5
6/0
25 months (1–228)
53 months (2–192)
Median age
FBC at diagnosis Hb (g/dl) WCC ( 109/l) Platelets ( 109/l) Chemotherapy administered (no/yes) Bone marrow transplantation (no/yes) Median time to follow-up Transformation of disease No Yes to AML Yes progressive cytopenia
70 14 7
5 0 1
Abbreviations: AML, acute myeloid leukemia; FAB, French, American, British; FBC, full blood count; Hb, haemoglobin; RAEB-1, refractory anaemia with excess blast-1; RARS, refractory anaemia with ringed sideroblast; RCMCD, Refractory cytopenia with multilineage dysplasia, WCC, white cell count; WHO, World Health Organization.
standard methods (Affymetrix, Santa Clara, CA, USA). Data were analysed using DChipSNP software http://biosun1.harvard.edu/ complab/dchip/. The JAK2 V617F mutation was detected in 6/97 (6.2%) of the samples analysed. All six mutant cases had a diagnosis of 5q syndrome (isolated deletion 5q) with stable disease in 5/6 and RAEB-I in 1/6 at a median follow-up of 53 months (range 2–192 months). On analysis of the haemoglobin (Hb), platelet count and total white cell count (WCC) in the JAK2 mutant versus wild-type groups, no difference in median Hb (9.0 vs 8.7 g/dl, P ¼ 0.272), a trend towards a higher platelet count (475 vs 250 109/l, P ¼ 0.15) and a significant elevation in WCC (5.21 vs 4.45 109/l, P ¼ 0.012) was observed in the JAK2 mutant cases. In two JAK2 mutant cases, thrombocytosis warranted treatment with hydroxyurea or anagrelide. In addition to the higher indices, all six mutant cases were associated with a moderate/marked increase in bone marrow cellularity with evidence of granulocytic hyperplasia in 3/6, eosinophilia in 1/6 and dysplastic features in keeping with MDS in all six cases (Figure 1). Regular blood transfusion support was required in 4/6 JAK2 mutant cases with recombinant erythropoietin administered in two cases with no response.The V617F mutation was detected in CD34, CD14, CD15 and CD61 þ cells, but was absent in CD3 and CD19 þ cells. In addition, pyrosequencing, affymetrix 100K SNP analysis and fluorescence in situ hybridisation (FISH) analysis were performed on the same CD34 þ cell sample. Leukemia
Figure 1 (a) Bone marrow trephine image demonstrating increased cellularity in patient with JAK2 V617F mutation and 5q syndrome ( 10 magnification). (b) Bone marrow trephine demonstrating abnormal megakaryocyte morphology, hypercellularity and excess haemosiderin pigment in a transfusion-dependent case of JAK2 V617F mutation and 5q syndrome ( 40 magnification).
Fluorescence in situ hybridisation analysis showed the 5q abnormality in 91% (137/150) of CD34 þ cells. The frequency of the JAK2 allele within this same population is 20%. Microarray 100K SNP analysis did not reveal either uniparental disomy (UPD) or loss of heterozygosity (LOH) at the JAK2 locus. The relatively low percentage of the mutant JAK2 allele call in purified cell lineages and the lack of LOH or UPD support the notion that this patient is heterozygous for the mutant JAK2 V617F allele. We can also conclude that the 5q and JAK2 V617F mutant clones must overlap, that is, some cells that contain the 5q abnormality must also contain the JAK2 mutation. However, the data do not demonstrate that all 5q cells are also JAK2 V617F mutant. BFU-E- and CFU-GM-derived colonies were grown from the bone marrow of the above case. CFU-GM-derived colonies were more numerous than BFU-E-derived colonies. A total of 14 colonies were picked for AS-PCR, only 8/14 colonies gave analysable AS-PCR data with 2/3 BFU-E colonies and 4/8 CFUGM colonies containing the JAK2 mutation. The finding of JAK2 mutation in CD34 þ cells, BFU-E colonies and CFU-GM colonies supports the notion that JAK2 mutation arises from a
Letters to the Editor
pluripotent stem cell. However, the fact that not all CD34positive or all BFU-E or CFU-GM colonies are positive for V617F suggests that the bone marrow of this patient is not monoclonal with respect to the JAK2 mutation. In summary, the JAK2 V617F mutation was detected in a cohort of patients with 5q syndrome and a hypercellular marrow. Despite no statistical difference, a higher median platelet count was observed in the mutant cases with 50% (3/6) showing a platelet count 4700 109/l compared with only 3% (3/91) in the wild-type cases. The lack of clinical response to erythropoietin in the two cases described fails to support previous in vitro studies documenting hypersensitivity to erythropoietin in the presence of the mutation. Whether the JAK2 mutation occurs as an early or late event during the disease course is unclear. We detected the JAK2 mutation both at time of diagnosis and at a follow-up of 132 months in one case analysed, suggesting that the mutation occurred as an early event. Longer follow-up is however necessary to determine the prognostic significance of JAK2 mutation and in particular, whether these cases will show favourable response to lenalidomide as previously demonstrated in 5q chromosomal abnormalities.8
Acknowledgements W Ingram is supported by the Leukaemia Research Fund, UK.
W Ingram1,11, NC Lea1,11, J Cervera2, U Germing3, P Fenaux4, B Cassinat4, JJ Kiladjian4, J Varkonyi5, P Antunovic6, NB Westwood1, MJ Arno7, A Mohamedali1, J Gaken1, T Kontou1, BH Czepulkowski1, NA Twine1, J Tamaska8, J Csomer9, S Benedek5, N Gattermann3, E Zipperer3, A Giagounidis10, Z Garcia-Casado2, G Sanz2 and GJ Mufti1 1 Department of Haematological Medicine, Kings College Hospital and Kings College London, London, UK; 2 The Servicio de Hematologia y Hemoterapia, Hospital Universitario La Fe, Valencia, Spain; 3 Department of Haematology, Oncology and Clinical Immunology, Heinrich-Heine-University, Du¨sseldorf, Germany; 4 Hematology Department at Hopital Avicenne,
Bobigny, France; Third Department of Internal Medicine, Semmelweis University, Budapest, Hungary; 6 Department of Haematology, University Hospital Linko¨ping, Linko¨ping, Sweden; 7 Genomics center, School of Biomedical and Health Sciences, Kings College London, London, UK; 8 National Health Centre, Semmelweis University, Budapest, Hungary; 9 Institute of Pathology and Cancer Research, Semmelweis University, Budapest, Hungary and 10 Department of Oncology and Clinical Immunology, St-Johannes-Hospital, Duisburg, Germany E-mail:
[email protected] 11 These authors contributed equally to this work
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References 1 James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 2005; 434: 1144– 1148. 2 Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 2005; 7: 387–397. 3 Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005; 352: 1779–1790. 4 Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005; 365: 1054–1061. 5 Steensma DP, Dewald GW, Lasho TL, Powell HL, McClure RF, Levine RL et al. The JAK2 V617F activating tyrosine kinase mutation is an infrequent event in both ‘atypical’ myeloproliferative disorders and myelodysplastic syndromes. Blood 2005; 106: 1207–1209. 6 Jones AV, Kreil S, Zoi K, Waghorn K, Curtis C, Zhang L et al. Widespread occurrence of the JAK2 V617F mutation in chronic myeloproliferative disorders. Blood 2005; 106: 2162–2168. 7 James C, Delhommeau F, Marzac C, Teyssandier I, Couedic JP, Giraudier S et al. Detection of JAK2 V617F as a first intention diagnostic test for erythrocytosis. Leukemia 2006; 20: 350–353. 8 List A, Kurtin S, Roe DJ, Buresh A, Mahadevan D, Fuchs D et al. Efficacy of lenalidomide in myelodysplastic syndromes. N Engl J Med 2005; 352: 549–557.
Immunophenotypic identification of acute myeloid leukemia with monocytic differentiation
Leukemia (2006) 20, 1321–1324. doi:10.1038/sj.leu.2404242; published online 27 April 2006
In 1976, the French-American-British (FAB) Cooperative Group published a morphologic classification of acute myeloid leukemia (AML).1 A revision of this classification published in 1985 was widely used and recognized as the standard for AML classification for over 15 years.2 Included in the FAB classification were two groups of AML that exhibited monocytic differentiation, acute myelomonocytic leukemia (AMML; M4), and acute monoblastic/monocytic leukemia (AMoL; M5). A subset of M4 with abnormal and increased eosinophils (M4EO) was found to be associated with chromosome 16 abnormalities, either inv(16)(p13q22) or t(16;16)(p13;q22). Recognition of the
biologic diversity within FAB AML subtypes led to the World Health Organization (WHO) classification of AML, published in 2001, in which morphologic, immunophenotypic, genetic, and clinical features of AML were included in defining disease entities.3 By WHO criteria, cases previously diagnosed as FAB M4 or M5 may be classified as AML with recurrent cytogenetic abnormalities (inv(16)(p13q22)/t(16;16)(p13;q22)(CBFb/MYH11), 11q23(MLL), or t(8;21)(q22;q22)(AML1/ETO)), AML with multilineage dysplasia, therapy related AML, and AMML or AMoL subtypes of AML not otherwise categorized (NOC). Appropriate classification of AML is important for clinical management and allows for future studies to expand and refine our understanding of these diseases. While immunophenotypic features are included in the WHO classification of AML, immunophenotypic criteria for monocytic Leukemia
