Letters to the Editor 1321 pluripotent stem cell. However, the fact that not all CD34- Bobigny, France; 5 positive or all BFU-E or CFU-GM colonies are positive for Third Department of Internal Medicine, Semmelweis University, Budapest, Hungary; V617F suggests that the of this patient is not 6 monoclonal with respect to the JAK2 . Department of Haematology, University Hospital Linko¨ping, Linko¨ping, Sweden; In summary, the JAK2 V617F mutation was detected in a 7Genomics center, School of Biomedical and Health Sciences, cohort of patients with 5qÀ syndrome and a hypercellular Kings College London, London, UK; marrow. Despite no statistical difference, a higher median 8National Health Centre, Semmelweis University, platelet count was observed in the mutant cases with 50% (3/6) Budapest, Hungary; showing a platelet count 4700 Â 109/l compared with only 3% 9Institute of Pathology and Cancer Research, Semmelweis (3/91) in the wild-type cases. The lack of clinical response to University, Budapest, Hungary and 10 erythropoietin in the two cases described fails to support Department of and Clinical Immunology, previous in vitro studies documenting hypersensitivity to St-Johannes-Hospital, Duisburg, Germany erythropoietin in the presence of the mutation. E-mail: [email protected] 11These authors contributed equally to this work 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 References months in one case analysed, suggesting that the mutation occurred as an early event. Longer follow-up is however 1 James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C necessary to determine the prognostic significance of JAK2 et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 2005; 434: 1144– mutation and in particular, whether these cases will show 1148. favourable response to lenalidomide as previously demonstrated 2 Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ in 5qÀ chromosomal abnormalities.8 et al. Activating mutation in the tyrosine kinase JAK2 in , essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 2005; 7: 387–397. Acknowledgements 3 Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR et al. A gain-of-function mutation of JAK2 in myeloproliferative W Ingram is supported by the Leukaemia Research Fund, UK. 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 W Ingram1,11, NC Lea1,11, J Cervera2, U Germing3, P Fenaux4, myeloproliferative disorders. Lancet 2005; 365: 1054–1061. B Cassinat4, JJ Kiladjian4, J Varkonyi5, P Antunovic6, 5 Steensma DP, Dewald GW, Lasho TL, Powell HL, McClure RF, NB Westwood1, MJ Arno7, A Mohamedali1, J Gaken1, Levine RL et al. The JAK2 V617F activating tyrosine kinase T Kontou1, BH Czepulkowski1, NA Twine1, J Tamaska8, mutation is an infrequent event in both ‘atypical’ myeloprolife- J Csomer9, S Benedek5, N Gattermann3, E Zipperer3, rative disorders and myelodysplastic syndromes. Blood 2005; 106: 1207–1209. A Giagounidis10, Z Garcia-Casado2, G Sanz2 and GJ Mufti1 1 6 Jones AV, Kreil S, Zoi K, Waghorn K, Curtis C, Zhang L et al. Department of Haematological Medicine, Kings College Widespread occurrence of the JAK2 V617F mutation in chronic Hospital and Kings College London, London, UK; 2 myeloproliferative disorders. Blood 2005; 106: 2162–2168. The Servicio de Hematologia y Hemoterapia, Hospital 7 James C, Delhommeau F, Marzac C, Teyssandier I, Couedic JP, Universitario La Fe, Valencia, Spain; 3 Giraudier S et al. Detection of JAK2 V617F as a first intention Department of Haematology, Oncology and Clinical diagnostic test for erythrocytosis. 2006; 20: 350–353. Immunology, Heinrich-Heine-University, 8 List A, Kurtin S, Roe DJ, Buresh A, Mahadevan D, Fuchs D et al. Du¨sseldorf, Germany; Efficacy of lenalidomide in myelodysplastic syndromes. N Engl J 4Hematology Department at Hopital Avicenne, Med 2005; 352: 549–557.

Immunophenotypic identification of acute with monocytic differentiation

Leukemia (2006) 20, 1321–1324. doi:10.1038/sj.leu.2404242; biologic diversity within FAB AML subtypes led to the World published online 27 April 2006 Health Organization (WHO) classification of AML, published in 2001, in which morphologic, immunophenotypic, genetic, and clinical features of AML were included in defining disease In 1976, the French-American-British (FAB) Cooperative entities.3 By WHO criteria, cases previously diagnosed as FAB Group published a morphologic classification of acute myeloid M4 or M5 may be classified as AML with recurrent cytogenetic leukemia (AML).1 A revision of this classification published in abnormalities (inv(16)(p13q22)/t(16;16)(p13;q22)(CBFb/MYH11), 1985 was widely used and recognized as the standard for AML 11q23(MLL), or t(8;21)(q22;q22)(AML1/ETO)), AML with multi- classification for over 15 years.2 Included in the FAB classifica- lineage dysplasia, therapy related AML, and AMML or AMoL tion were two groups of AML that exhibited monocytic subtypes of AML not otherwise categorized (NOC). Appropriate differentiation, acute myelomonocytic leukemia (AMML; M4), classification of AML is important for clinical management and and acute monoblastic/ (AMoL; M5). A allows for future studies to expand and refine our understanding subset of M4 with abnormal and increased eosinophils (M4EO) of these diseases. was found to be associated with 16 abnormalities, While immunophenotypic features are included in the WHO either inv(16)(p13q22) or t(16;16)(p13;q22). Recognition of the classification of AML, immunophenotypic criteria for monocytic

Leukemia Letters to the Editor 1322 54 Figure 1 Frequency of antigen expression in blasts of acute HLA-DR 100 monoblastic/monocytic leukemia (AMoL) (n ¼ 23) and nonmonocytic 60 leukemia (NM-AML) (n ¼ 72). Solid bars: AMoL; open bars: CD64 100 NM ¼ AML. 26 CD56 85 17 AML have not been clearly defined. A high level of expression of CD36 06 CD14 is a specific feature of mature , but this antigen 68 CD34 is frequently absent or underexpressed on immature monocytic 17 cells. Furthermore, other antigens that are generally considered 94 to be -associated may be expressed in other types of CD33 100 AML. The goals of the present study were to identify 0 immunophenotypic patterns that reliably characterize mono-

Antigens CD16 38 cytic AMLs and to explore their immunophenotypic 74 heterogeneity in relationship to morphologic and cytogenetic CD15 100 subgroups. 0 A total of 126 cases of de novo AML diagnosed and analyzed CD14 61 with 4-color flow cytometry at our institution from 1998 to 2004 100 were included in the study. All cases were classified according CD13 83 to FAB criteria2 based on morphology and cytochemical 33 reactivity for and alpha naphthyl butyrate CD11b 100 esterase (ANBE), and also according to WHO criteria3 based on 30 clinical, morphologic, immunophenotypic, and cytogenetic CD4 100 features. By FAB criteria, 54 exhibited morphologic and cytochemical features of monocytic AML, including 31 cases 020406080 100 120 of M4 and 23 cases of M5 (15 monoblastic and eight Antigen Expression in % monocytic). Those 54 cases were classified by WHO criteria

Figure 2 Expression patterns of CD36 in blasts of (AML) with monocytic differentiation. (a) Blasts (red) in acute monoblastic leukemia coexpressed CD36 and CD64. (b) Group 1 acute myelomonocytic leukemia (AMML) with one population (red) in the blast/ monocytic region, which showed a spectrum of CD36 and CD64. The least differentiated blasts were CD36(À) and CD45(moderate), followed by acquisition of CD36, and finally progression to bright CD45 expression. (c) Group 2 AMML with distinct population of blasts (red) and monocytes (blue) in the CD5/SSC dot plot. Both populations formed distinct clusters in the expression of CD36 and CD64. Green: .

Leukemia Letters to the Editor 1323 as AML with t(8;21) (3), inv(16)/t(16;16) (7), or 11q23 (11); However, all AMoLs in our series were positive for CD11b AML with multilineage dysplasia (3); or AML NOC (30). Of and HLA-DR, while none of the APLs expressed both antigens the 30 AML NOC cases, 17 were classified as AMML and 13 (data not shown). Furthermore, intense myeloperoxidase ex- as AMoL. pression, either by cytochemistry or flow cytometry, should help By flow cytometric analysis, blasts and monocytes were to distinguish microgranular APL from AMoL. identified using CD45/side scatter (SSC)/forward scatter (FSC) Cases of FAB M4 AML could be divided into two groups characteristics in combination with various cell surface anti- based on the definitive separation of discrete subpopulation of gens. The frequency of antigen expression in 23 AMoLs was blasts and monocytic cells in the analysis of CD45 and light compared to 72 nonmonocytic AMLs (NM-AML) (Figure 1). scatter (Figure 2b and c). One group showed a single Overall, the blasts in AMoL more frequently expressed CD4, (indistinguishable) population composed of both blasts and CD11b, CD14, CD16, CD36, CD56, CD64, and HLA-DR, and monocytic cells (designated group 1; 11 cases) (Figure 2b). The less often expressed CD34 compared to NM-AMLs (each immature cells in all cases of group 1 coexpressed CD36 and Po0.01). As single markers, none of these were sufficiently CD64. Thus, immunophenotypically, these closely resembled sensitive and specific for identifying monocytic AML. CD14 the AMoLs in our series, but were distinguished by a substantial expression in the blast/immature monocytic population was population of maturing granulocytes. Although discrete popula- seen in all eight cases of , but in eight tion of blasts and monocytic cells could not be discriminated in of 15 cases of acute monoblastic leukemia (P ¼ 0.021). There these cases based on CD45 and light scatters, a continuous was no difference in expression of other monocyte-associated maturation spectrum could be appreciated in a CD36/CD45 dot antigens between cases of acute monocytic leukemia and acute plot (Figure 2b). In the other group (group 2; 20 cases), there monoblastic leukemia. When the expression patterns of multiple were distinct populations of blasts and monocytes (Figure 2c). antigens were explored, coexpression of CD36/FITC and CD64/ The immunophenotypically defined blasts in 15 of the 20 cases PE in blasts (Figure 2a) was found in 22 (96%) of 23 cases of lacked the coexpression pattern of CD36 and CD64. However, AMoL, but in only six (8%) of 72 cases of NM-AML (Po0.001). all of these cases demonstrated a large proportion of monocytes, CD36 was expressed in both mature and immature monocytic comprising 10–42% of all events (mean 24%). cells, making it a useful marker of monocytic differentiation in We have recently shown that monocytic cells from cases of immature monocytes that lack CD14 expression. All six cases of chronic myelomonocytic leukemia (CMML) bear multiple NM-AML with CD36/CD64 coexpression demonstrated a immunophenotypic aberrancies, including under-expression of recurrent cytogenetic abnormality: t(8;21) in two cases, CD13, CD15, CD36, or HLA-DR, and aberrant expression of t(16;16) in one case, and t(15;17) (acute promyelocytic CD2 or CD56.4 In a similar fashion, we evaluated the monocytic leukemia; APL) in three cases. Of note, each of the three cases cells from group 2 AMML cases. They also exhibited an aberrant containing the t(15;17) with coexpression of CD36 and CD64 immunophenotype in 16 (80%) of 20 cases. The aberrancies showed microgranular features, a variant of APL that may be included underexpression of CD13 (3), CD36 (2), or HLA-DR confused morphologically with acute monocytic leukemia. (13), and aberrant expression of CD2 (7), CD5 (1), or CD56(1)

Table 1 Comparison of monocyte aberrancies in cases of group 2 acute myelomonocytic leukemia with inv(16)/t(16;16) (M4EO) to those without the translocations (M4)

Cases Aberrancies Aberrant immunophenotypes

CD2(+) 0 X1 X2

M4EO 6/6(100%) 0/7(0%) 7/7(100%) 6/7(86%) Case-1a  HLA-DR(partial +) Case-2 ÂÂÂCD2(+), CD5(+), HLA-DR(partial +) Case-3 ÂÂÂCD2(+), HLA-DR(partial +) Case-4 ÂÂÂCD2(+), HLA-DR(partial +) Case-5 ÂÂÂCD2(+), HLA-DR(partial +) Case-6 ÂÂÂCD2(+), HLA-DR(partial +) Case-7 ÂÂÂCD2(+), HLA-DR(partial +)

M4 1/11(9%) 4/13(31%) 9/13(69%) 3/13(23%) Case-1a  HLA-DR(À) Case-2a  HLA-DR(partial +) Case-3  HLA-DR(partial +) Case-4 ÂÂCD36(partial +), HLA-DR(partial +) Case-5  CD13(partial +) Case-6  CD13(À) Case-7  N/A Case-8  CD36(partial +) Case-9  N/A Case-10 ÂÂCD56(+), HLA-DR(À) Case-11  N/A Case-12  N/A Case-13 ÂÂÂCD2(+), CD13(À), HLA-DR(partial +) P-valueb 0.001 0.249 0.249 0.003 aIn these cases, CD2 was not available in the antibody panel for assessment. M4EO Case-1: AML with t(16;16); M4EO Cases-2–7: AML with inv(16). bTwo-tailed w2-test was used for the statistical analyses.

Leukemia Letters to the Editor 1324 (Table 1). Thus, significant immunophenotypic aberrancy in the results. The coexpression pattern is seen in a small proportion of context of appears to be consistent with a non-monocytic AMLs, including microgranular APL. This could monocytic , and is a helpful feature in the diagnosis be a potential diagnostic pitfall and requires correlation with of borderline cases. The findings in the present study further other immunophenotypic features, cytomorphology, and cyto- indicate that even when a blast population lacks definitive genetics. Furthermore, a well-defined population of monocytes immunophenotypic evidence of monocytic differentiation, the (distinct from blasts) showing two or more aberrancies and CD2 presence of a large number of aberrant monocytes suggests a expression was seen in all cases of AML with inv(16)(p13q22) monocytic AML. and was 91% specific for this cytogenetic subgroup. Thus, in the All seven cases of AML with inv(16)/t(16;16) belonged to context of AML, the presence of a distinct monocytic population group 2 AMML, in that they had distinct populations of blasts with this unique immunophenotype should prompt evaluation and monocytes. Interestingly, the monocytic cells in this for CBFb rearrangement. subgroup exhibited immunophenotypic abnormalities that distinguished them from other group 2 AMMLs lacking Y Xu, RW McKenna, KS Wilson, NJ Karandikar, RA Schultz chromosome 16 abnormalities (Table 1). They more frequently and SH Kroft Department of Pathology, University of Texas Southwestern showed two or more monocyte aberrancies (6/7 vs 3/13; Medical Center, Dallas, TX, USA P ¼ 0.003). Specifically, CD2 expression in monocytes was E-mail: [email protected] identified in 6/6 cases with inv(16)(p13;q22), and was sig- nificantly correlated with chromosome 16 abnormalities among AMMLs (6/6 vs 1/11; P ¼ 0.001). The single case of AMML with References t(16;16) that exhibited one monocyte aberrancy was not assessed for CD2 expression due to the absence of anti-CD2 1 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, in the panel (Table 1). An association between CD2 expression Gralnick HR et al. Proposals for the classification of the acute in the blast population of AML and inv(16) was documented in a leukaemias. French-American-British (FAB) co-operative group. Br J Haematol 1976; 33: 451–458. prior study.5 However, that 2-color flow cytometry study used 2 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, gating techniques based purely on light scatter properties and Gralnick HR et al. Proposed revised criteria for the classification of probably could not distinguish a pure population of blasts versus acute myeloid leukemia. A report of the French-American-British some contaminating monocytes and lymphocytes. In the present Cooperative Group. Ann Intern Med 1985; 103: 620–625. study, CD2 was also detected on the blast population (as 3 Brunning RD, Matutes E, Flandrin G, Vardiman J. Acute myeloid distinguished from the monocyte population) in four of six leukemia. In: Jaffe ES et al. (eds). Pathology and Genetics of Tumors AMMLs with inv(16)/t(16;16). The blasts of NM-AML, in of Haematopoietic and Lymphoid Tissues. IARC press: Lyon, 6 France, 2001, pp. 75–108. particular microgranular APL may also express CD2, thus, this 4 Xu Y, McKenna RW, Karandikar NJ, Pildain AJ, Kroft SH. Flow finding alone lacks specificity. cytometric analysis of monocytes as a tool for distinguishing chronic We found that all 11 cases of AMLs with 11q23 rearrange- myelomonocytic leukemia from reactive monocytosis. Am J Clin ments in this series fell into the FAB M4 or M5 categories: nine Pathol 2005; 124: 799–806. AMoLs (eight monoblastic and 1 monocytic) and two AMMLs. 5 Adriaansen HJ, te Boekhorst PA, Hagemeijer AM, van der Schoot Blasts in 10 of the 11 cases coexpressed CD36 and CD64; the CE, Delwel HR, van Dongen JJ. Acute myeloid leukemia M4 with bone marrow eosinophilia (M4Eo) and inv(16)(p13q22) exhibits a remaining one was a group 2 AMML with 25% aberrant specific immunophenotype with CD2 expression. Blood 1993; 81: monocytes. In agreement with previous studies, no immuno- 3043–3051. phenotypic features (other than monocytic differentiation) 6 Claxton DF, Reading CL, Nagarajan L, Tsujimoto Y, Andersson BS, predicted the presence of an 11q23 rearrangement.7 Estey E et al. Correlation of CD2 expression with PML gene In summary, blasts of AMoL can be identified by coexpression breakpoints in patients with acute promyelocytic leukemia. Blood of CD36 and CD64 with 96% sensitivity. Blasts of also AMML 1992; 80: 582–586. 7 Baer MR, Stewart CC, Lawrence D, Arthur DC, Mrozek K, group 1 are CD36/CD64 positive. Therefore, it should be Strout MP et al. Acute myeloid leukemia with 11q23 translocations: searched for more differentiated cells in order to separate AMML myelomonocytic immunophenotype by multiparameter flow from AMoL when making the diagnosis upon immunological cytometry. Leukemia 1998; 12: 317–325.

Presence of JAK2 V617F tyrosine kinase mutation as a myeloid-lineage-specific mutation in chronic neutrophilic leukaemia

Leukemia (2006) 20, 1324–1326. doi:10.1038/sj.leu.2404240; mia is of essence a diagnosis of exclusion, and affected patients published online 4 May 2006 have to be distinguished from other CMPD, such as chronic myelogenous leukaemia (CML), chronic myelomonocytic leu- kaemia and atypical CML. Causes of reactive neutrophilia need Chronic neutrophilic leukaemia (CNL) is a rare haematological to be ruled out, and there should be no cytogenetic or molecular disease of uncertain pathological aetiology. It has been recently presence of the . defined as a chronic myeloproliferative disorder (CMPD) under In the absence of a defining biological or molecular the World Health Organization (WHO) classification, and is characteristic, the diagnosis of CNL is often challenging, and characterized by a persistent neutrophilia, splenomegaly and there have been approximately 150 reported cases in the bone marrow (BM) hyperplasia.1 Chronic neutrophilic leukae- literature. The overall outcome of these patients is poor, with the

Leukemia