Identification of Marker Genes Including RUNX3 (AML2) That

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Identiﬁcation of marker genes including RUNX3 (AML2) that discriminate between different myeloproliferative neoplasms and normal individuals

Leukemia (2008) 22, 1773–1778; doi:10.1038/leu.2008.41; A second objective of this study was to identify genes that published online 6 March 2008 would allow us to discriminate between the different diseases, and from normal individuals (Table 2). As described above, expression of PER1 could distinguish ET from the other MPNs. Myeloproliferative neoplasms (MPN; myeloproliferative dis- Other examples include glycoprotein GP1BB and erythrocyte orders) are clonal stem cell diseases, defined by excessive membrane protein EPB42 genes that are clearly overexpressed production of cells in one or more hematopoietic lineages. in PMF compared to PV and ET, which in turn, had much higher Primary myelofibrosis (PMF), polycythemia vera (PV), essential expression compared to the normal samples. Also, PRV1 was thrombocythemia (ET), as well as chronic myelogenous leuke- overexpressed in PV samples compared to ET or PMF. Our data mia are included in the current 2008 WHO classification for MPNs.1 In 2005, a missense mutation in the JAK2 tyrosine demonstrate that MPN samples can be stratified into specific kinase (JAK2V617F) leading to constitutive activation of JAK2, diseases based upon the level of expression of several genes. was found in about 95% of PV patients, 40% of ET patients and Amongst the dysregulated genes, RUNX3 has a precedent in 2 tumor biology; it and its family members are transcription 40% of PMF patients. This provided insights into common 4 lesions leading to pathogenesis of PV, ET and PMF. factors associated with cell proliferation and differentiation. To investigate other possible lesions present in MPNs, we RUNX1 (AML1) is frequently involved in chromosomal translo- performed microarray analysis on 26 granulocyte samples from cations resulting in oncogenic fusion proteins including PMF (4), ET (5), PV (6) and normal (11) individuals (Table 1). We t(8;21)(q22;q22) (AML1/ETO) in FAB M2 acute myelogenous also determined the JAK2 mutational status of these samples leukemia (AML), one of the most frequent karyotypic abnorm- using an adaptation of the method described by Baxter et al.2 alities in AML. RUNX2 is required for osteoblast differentiation (Supplementary Figure 1S). A total of 246 genes were found to and bone development, and heterozygous loss-of-function of be dysregulated between normal and MPN granulocytes, RUNX2 produces the bone disease cleidocranial dysplasia. including those associated with TGFb and TNFa signaling Expression of RUNX3 parallels the expression pattern of RUNX1 pathways, and cell cycle (Supplementary Table 1S). in the hematopoietic system, but in contrast to RUNX1, no Expression levels of several of these genes were confirmed by inactivating mutations of RUNX3 have been found in AML quantitative real-time (qRT)-PCR (Figure 1). Particularly, over- blasts.5 However, high expression of RUNX3, together with expression of the TGFb-regulated transcription factors, RUNX3 FLT3 mutations in childhood AML, has been associated with and TIEG1, are clearly seen in MPN compared to normal unfavorable outcome. Low levels of RUNX3, on the other hand, granulocytes (Figure 1a). Several studies have recently shown have been found in low-risk AML with inv(16).6 that elevated levels of TGFb can promote myelofibrosis.3 TGFb Immunohistochemical staining for RUNX3 protein was is produced and secreted predominantly by megakaryocytes, performed on bone marrow aspirates of 7 ET, 10 PMF, 1 PV platelets and monocytes, and it stimulates collagen synthesis.3 and 10 normal samples. Two of 10 normal samples (no. 295 and Several other genes that may play a role in development of no. 79) revealed weak staining in some cells (Figure 2). The fibrosis in MPN are also dysregulated. For example, metallo- remaining nine normal samples were clearly negative. All PMF proteinase (ADAM8), integrins (ITGA2B and ITGB3) and anti- samples were negative due to staining being especially difficult metastasis-associated genes (NME2). ITGA2B and ITGB3 by because of predominantly fibrotic tissue and hypocellular bone microarray analysis were much more highly expressed in PMF marrow, as can be seen in PMF no. 534. The single PV sample than in either ET or PV; and in turn, these genes were greatly was also negative for RUNX3 (data not shown). Three of seven overexpressed in ET and PV compared to normal samples. ET samples stained strongly for RUNX3 (two of these are qRT-PCR confirmed higher expression of ITGA2B in ET and PV presented in Figure 2: no. 383 and no. 860). In fact, all three ET samples while PMF and normal samples had similar levels of samples showed very prominent nuclear staining of RUNX3, expression of this gene (Figure 1b). ITGB3 was also over- consistent with its role as a transcription factor. Analysis of expressed in MPNs compared to normal samples, but the levels RUNX3 mRNA expression in AML cell lines revealed that of overexpression was not statistically significant (Figure 1b). RUNX3 levels were highest in the erythroleukemia (AML M6) The disintegrin gene, ADAM8, was significantly downregulated cell line HEL (Figure 2b). The second highest cell line was KG-1 in PMF granulocytes compared to PV, ET and normal samples. and these cells were established from a patient who had TNFa-associated genes, such as TNFAIP3 and CIAS1, were erythroleukemia. The mean relative RUNX3 mRNA expression overexpressed in all types of MPNs compared to normal, of all the myeloid cell lines (640±315) is comparable to the suggesting a disrupted inflammatory response in MPN mean level of RUNX3 mRNA expression in the MPN (Figure 1b). Dysregulation of cell cycle regulatory genes, such granulocytes (693±302). Normal granulocytes show signifi- as PER1 and CCNL2, in MPN samples were also found. This cantly lower expression of RUNX3 than either MPN granulo- is perhaps not surprising as this study compared granulocytes cytes or myeloid leukemia cells. that are derived from the abnormal ‘MPN/proliferative’ clone Since RUNX3 expression was elevated in MPN granulocytes to normal granulocytes. Interestingly, PER1 was highly over- and AML cells, we were interested to find out what happens expressed in ET samples compared to normal samples, and only to RUNX3 during myeloid differentiation. We chose HL-60 cells mildly overexpressed in PV cases. as our model for granulocytic and monocytic differentiation. Leukemia 1774

Table 1 Myeloproliferative and normal granulocyte samples

Patient Disease Site JAK2 status Microarray qPCR Patient Disease Site JAK2 status Microarray qPCR Patient Disease Site JAK2 Microarray qPCR no. no. no. status

1 PMF PB Y 38 PV PB V617F/V617F Y Y E-3 ET BM Y 2 PMF PB Y 39 PV BM Y E-4 ET PB Y 3 PMF PB Y 40 PV PB Y E-5 ET PB Y 4 PMF PB Y 41 PV PB Y Y E-6 ET PB Y 6 PMF WT/WT Y 42 PV PB V617F/V617F Y Y E-7 ET BM Y

7 PMF Y 44 Normal PB WT/WT Y Y E-8 ET BM Y Editor the to Letters 9 PMF PB V617F/V617F Y 45 Normal/ETa PB WT/V617F Y Y E-9 ET BM Y 10 PMF PB V617F/V617F Y Y 46 Normal PB WT/WT Y Y E-10 ET PB Y 11 PMF PB WT/WT Y Y 47 Normal PB WT/WT Y E-11 ET BM WT/WT Y Y 12 PMF PB Y Y 48 Normal PB WT/WT Y Y E-12 ET PB Y 13 PV PB WT/V617F Y Y 49 Normal PB WT/WT Y Y E-13 ET PB Y 14 ET PB Y 50 Normal PB WT/WT Y P-1 PV PB Y 15 ET BM Y 51 Normal PB WT/WT Y Y P-2 PV PB Y 17 ET BM Y 52 Normal PB WT/WT Y Y P-3 PV PB Y 20 ET BM Y 53 Normal PB WT/WT Y Y P-4 PV PB Y 22 ET BM WT/V617F Y 54 Normal PB WT/WT Y P-5 PV PB Y 24 ET WT/WT Y 55 Normal PB WT/WT Y P-6 PV BM Y 26 ET PB WT/WT Y Y 56 Normal PB WT/WT Y Y P-7 PV PB Y 27 ET BM Y 57 ET PB Y P-8 PV BM Y 29 PV BM Y A-1 PMF BM Y P-9 PV BM Y 31 PV BM Y A-2 PMF BM Y P-10 PV PB Y 33 PV BM WT/V617F Y A-3 PMF PB WT/V617F Y Y P-11 PV BM Y 34 PV BM WT/V617F Y A-4 PMF BM Y P-12 PV PB Y 35 PV PB Y E-1 ET BM Y P-13 PV PB Y 36 PV PB Y E-2 ET PB Y P-14 PV PB Y Abbreviations: BM, bone marrow; ET, essential thrombocythemia; PB, peripheral blood; PMF, primary myelofibrosis; PV, polycythemia vera; V617F, JAK2V617F allele; WT, wild-type JAK2 allele; Y, microarray or qPCR analysis was performed on this sample. PMF according to: Mesa RA, Verstovsek S, Cervantes F, Barosi G, Reilly JT, Dupriez B et al. Primary myelofibrosis (PMF), post polycythemia vera myelofibrosis (post-PV MF), post essential thrombocythemia myelofibrosis (post-ET MF), blast phase PMF (PMF-BP): Consensus on terminology by the international working group for myelofibrosis research and treatment (IWG-MRT). Leuk Res 2007; 31:737–740. aThis individual was later found to have ET (from personal communication). Letters to the Editor 1775

Figure 1 Quantitative real-time (qRT)-PCR analysis of dysregulated genes in myeloproliferative neoplasms (MPNs). qRT-PCR measurement of expressions of (a) TGFb-regulated genes and (b) ﬁbrosis-associated genes, inﬂammatory genes and cell cycle-regulated genes. Relative expression of each gene is normalized to b-actin values. Relative gene expression values are given (mean±s.e.). NL, normal granulocyte samples.

HL-60 cells had a median level of RUNX3 expression shown that overexpression of RUNX3 increased the proportion compared to other AML cell lines. RUNX3 mRNA levels in of mature CD8 thymocytes, pointing to the role of RUNX3 in HL-60 cells cultured with all-trans retinoic acid (ATRA; 0.1 and differentiation of these cells also. Furthermore, neutrophils 1.0 mM) markedly and maximally increased by day 5 of culture isolated from MPN patients are functionally impaired in their (15–20 fold). Levels decreased to control values by day 7 ability to ﬁght infection compared to those from normal (Figure 2c). In addition, induction of differentiation towards individuals. Thus, the high level of RUNX3 in MPN granulocytes neutrophils with 1.5% (v/v) dimethyl sulfoxide (DMSO) showed might indicate that these cells are not completely and a slight delay in induction of RUNX3 expression; but functionally mature granulocytes and are arrested at an nonetheless, upregulation of RUNX3 (8–9 fold) was observed earlier stage of maturation/differentiationFcomparable to day at day 6, which rapidly returned to basal levels by day 7. 5 ATRA-treated, or day 6 DMSO-treated/Vitamin D3-treated Similarly, when treated with 0.5 mM 1,25-dihydroxyvitamin D3 HL-60 cells. to differentiate HL-60 cells towards monocytes, we observed a Runx3, in mice, has also been shown to regulate TGFb- similar induction of RUNX3 expression at day 6 that returned to mediated cell function in dendritic cells, as well as integrin basal levels at day 7 (Figure 2c). Similar experiments were expression in T cells. Thus, the role of RUNX3 in MPN appears performed in NB4 and U937 cells resulting in similar regulation to be associated with both TGFb signaling/myeloﬁbrosis and of RUNX3 (data not shown). Our data suggest that RUNX3 leukocyte development. In other studies, Runx3 (murine may be initially involved in myeloid differentiation but returns homolog) knockout mice have hyperplasia of the epithelial to basal levels as the cells undergo terminal differentiation cells of the glandular stomach.7 Gastric epithelial cells were less toward granulocytes or monocytes. It had been experimentally sensitive to the growth-inhibitory effect of TGFb and apoptosis

Leukemia Letters to the Editor 1776 Table 2 Genes that could differentiate PV, ET and PMF from each other

Common Description GenBank PV ET PMF

A CIAS1 Cold autoinﬂammatory syndrome 1 AK027194 mmm m m OSM Oncostatin M BG437034 mm m 2 BIA2 Bia2 AL080170 mm mmm mmm FOSL2 FOS-like antigen 2 N36408 mm m m PRV1 Polycythemia rubra vera 1 NM_020406 mm 2 m RANBP2 RAN binding protein 2 D42063 mm m m UP Uridine phosphorylase NM_003364 mm m m

B PER1 Period homolog 1 (Drosophila) NM_002616 m mmm mm NKG7 Natural killer cell group 7 NM_005601 mmm mm mmm MCP Membrane cofactor protein (CD46) D84105 k2kk

C AOP2 Antioxidant protein 2 BE869583 m m mmm CA2 Carbonic anhydrase II M36532 2 m mmm CLU Clusterin M25915 m m mmm ELA2 Elastase 2, neutrophil NM_001972 m m mmm EPB42 Erythrocyte membrane protein band 4.2 M30646 m mm mmm ERAF Erythroid associated factor NM_016633 m m mmm GP1BB Glycoprotein Ib (platelet), beta polypeptide NM_000407 mm mm mmm GRIM19 Cell death-regulatory protein GRIM19 NM_015965 m m mmm GYPA Glycophorin A U00178 m m mmm ITGA2B Integrin, alpha 2b (antigen CD41B) NM_000419 mm m mmm MPO Myeloperoxidase J02694 m m mmm NRGN Neurogranin NM_006176 m m mmm PPBP Pro-platelet basic protein R64130 mm m mmm RAN RAN, member RAS oncogene family AF054183 m m mmm ANK1 Ankyrin 1 erythrocytic NM_020478 mmmm CA1 Carbonic anhydrase I NM_001738 mmmm CPVL Carboxypeptidase, vitellogenic-like NM_031311 mmmm GPX1 Glutathione peroxidase 1 NM_000581 mmmm H1F0 H1 histone family, member 0 BC000145 mmmm HK1 Hexokinase 1 NM_000188 mmmm IFI27 Interferon, alpha-inducible protein 27 NM_005532 m2mm ITGB3 Integrin, beta 3 M35999 mmmm NME2 Non-metastatic cells 2, protein (NM23B) NM_002512 mmmm SOD1 Superoxide dismutase 1 NM_000454 mmmm TFRC Transferrin receptor (p90, CD71) NM_003234 mmmm MME Membrane metalloendopeptidase (CALLA, CD10) AI433463 k2kk PECAM1 Platelet/endothelial cell adhesion molecule (CD31 antigen) M37780 22kk ADAM8 A disintegrin and metalloproteinase domain 8 AI814527 k 2 kkk GLIPR1 GLI pathogenesis-related 1 (glioma) NM_006851 k 2 kkk IL8RB Interleukin 8 receptor, beta NM_001557 2 2 kkk SIAT8D Sialyltransferase 8D NM_005668 k 2 kkk TLR8 Toll-like receptor 8 NM_016610 2 2 kkk Abbreviations: ET, essential thrombocythemia; PMF, primary myeloﬁbrosis; PV, polycythemia vera. A, genes differentiating PV from ET, PMF and normal. B, genes differentiating ET from PV, PMF and Normal. C, genes differentiating PMF from PV, ET and Normal. All mean gene expression levels in experimental samples have been compared to the mean level of expression in normal samples:mmm, 420-fold overexpression; mm, 10- to 20-fold overexpression; m, 2- to 9-fold overexpression; k, 2- to 5-fold downregulation; kk, 6- to 10-fold downregulation; kkk, 410-fold downregulation; 2, similar expression level to normal samples.

was suppressed in these cells compared to those in wild-type Of the MPN samples, 5 had wild-type JAK2 products only, while mice. Furthermore, inactivation of Runx3 by hemizygote 10 samples (including no. 45) had at least one JAK2V617F allele deletion and DNA methylation of the promoter region was (Table 1). Re-analysis of our microarray data (where available), found in 90% of stage IV gastric cancers. These data suggest that comparing MPNs with JAK2V617F mutation to those that had Runx3 is a tumor suppressor gene in the TGFb signaling only wild-type JAK2 and to normal samples revealed that pathway. However, recently Salto-Tellez et al.8 showed over- approximately 100 genes were differentially expressed between expression of RUNX3 protein in human basal cell carcinomas, the wild-type JAK2 and JAK2V617F MPN groups. A subset of which suggests that RUNX3 may have a dual role, as a tumor genes showing the highest levels of dysregulated expression is suppressor and as an oncogene. presented in Supplementary Table 2S. Despite reports suggest- Lastly, in light of the discovery of the JAK2V617F mutation in ing that the JAK2V617F mutation ultimately leads to the MPN, we examined if this mutation had an impact on the genes upregulation of JAK2 itself, we did not ﬁnd JAK2 to be expressed in MPNs. In total, 12 of 13 normal samples had wild- upregulated in our analysis. In addition, other genes associated type JAK2 products only. The remaining sample (patient no. 45) with JAK2 activity, for example STAT3, SHP2 and SOCS3 were had both wild-type and mutant bands; and later, we discovered not increased in their expression in MPN samples with that this individual did in fact have ET (personal communication). JAK2V617F mutation compared to those without the abnormality.

Leukemia Letters to the Editor 1777

Figure 2 RUNX3 expression in myeloproliferative neoplasms (MPN) and normal samples. (a) Immunohistochemical staining for RUNX3 protein in normal, PMF and ET bone marrow biopsies. (b) RUNX3 expression in individual myeloid leukemia cell lines was measured by quantitative real-time (qRT)-PCR. The level of expression of RUNX3 was normalized to b-actin expression. RUNX3 expression for myeloid leukemia cells was calculated from the individual values for each cell line. Mean RUNX3 expression level for MPN samples, AML cell lines and normal samples are compared. (c) HL-60 AML cells were induced to differentiate with ATRA (0.1 or 1.0 mM), 1,25-dihydroxyvitamin D3 (0.5 mM)or DMSO (1.5% v/v) over several days. RUNX3 expression levels were determined by qRT-PCR.

Thus, changes in the level of phosphorylation of these target Chair of Oncology Research at Cedars-Sinai Medical Center/ proteins are more likely to be important. UCLA School of Medicine. This work was also supported, in part, In summary, we have identiﬁed several genes that may give us by a fellowship from the Deutsche Forschungsgemeinschaft to valuable insight into the pathophysiological abnormalities of CIM (MU 1809/1), and by an International Visiting Fellowship myeloproliferative diseases and could provide potential markers from Leukaemia Research Fund (UK) to QTL (no. 0340). that may be useful for diagnosis of these diseases.

Acknowledgements CI Mu¨ller1,2,10, QT Luong1,3,10, L-Y Shih4, LC Jones1, JC Desmond1, N Kawamata1, O Tcherniantchouk5, Q Liu6, HPK is a member of the Johnsson Cancer Center, the Molecular K Ito6,7, M Osato6,7, Y Ito6,7, A Tefferi8, S de Vos9 and Biology Institute of UCLA and has the endowed Mark Goodson HP Koefﬂer1

Leukemia Letters to the Editor 1778 1 Division of Hematology/Oncology, Cedars-Sinai Medical criteria and point-of-care diagnostic algorithms. Leukemia 2008; 22: Center, UCLA, Los Angeles, CA, USA; 14–22. 2Division of Hematology/Oncology, University of Freiburg 2 Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Medical Center, Freiburg, Germany; Swanton S et al. Acquired mutation of the tyrosine kinase 3School of Biosciences, University of Birmingham, JAK2 in human myeloproliferative disorders. Lancet 2005; 365: Birmingham, UK; 1054–1061. 4Division of Hematology/Oncology, Department of Internal 3 Dong M, Blobe GC. Role of transforming growth factor-b in Medicine, Chang Gung Memorial Hospital, Taipei, Taiwan; hematological malignancies. Blood 2006; 107: 4586–4596. 5Division of Pathology and Laboratory Medicine, Cedars-Sinai 4 Coffman JA. Runx transcription factors and the developmental Medical Center, Los Angeles, CA, USA; balance between cell proliferation and differentiation. Cell Biol Int 6Oncology Research Institute, National University of 2003; 27: 315–324. Singapore, Singapore, Singapore; 5 Otto F, Stock M, Fliegauf M, Fenaux P, Preudhomme C, Lu¨bbert M. Absence of somatic mutations within the Runt domain of 7Institute of Molecular and Cell Biology, Singapore, Singapore; 8 AML2/RUNX3 in acute myeloid leukaemia. Leukemia 2003; 17: Division of Hematology, Department of Internal Medicine, 1677–1678. Mayo Clinic, Rochester, MN, USA and 9 6 Gutierrez NC, Lopez-Perez R, Hernandez JM, Isidro I, Gonzalez B, Division of Hematology/Oncology, Department of Pathology Delgado M et al. Gene expression proﬁle reveals deregulation of and Laboratory Medicine, UCLA, Los Angeles, CA, USA genes with relevant functions in the different subclasses of acute E-mail: [email protected] myeloid leukemia. Leukemia 2005; 19: 402–409. 10 These authors contributed equally to this work. 7 Li QL, Ito K, Sakakura C, Fukamachi H, Inoue K, Chi XZ et al. Causal relationship between the loss of RUNX3 expression and References gastric cancer. Cell 2002; 109: 113–124. 8 Salto-Tellez M, Peh BK, Ito K, Tan SH, Chong PY, Han HC et al. 1 Tefferi A, Vardiman JW. Classiﬁcation and diagnosis of RUNX3 protein is overexpressed in human basal cell carcinomas. myeloproliferative neoplasms: the 2008 World Health Organization Oncogene 2006; 25: 7646–7649.

Supplementary information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

Association between high expression of natural killer related-genes (NCAM/CD94) and early death during induction in children with acute myeloid leukemia

Leukemia (2008) 22, 1778–1781; doi:10.1038/leu.2008.46; gically diagnosed as having an M3v (Figures 1a and b). She was, published online 6 March 2008 however, found to have an NK cell leukemia, as shown by lack of expression of HLA-DR, CD2 and CD9 antigens and by the coexpression of various myeloid (CD33 and CD13) and NK cell- Early death (ED) during the induction phase is a rare but associated (CD56) surface antigens. To confirm our diagnosis of 1 dramatic event in children with acute myeloid leukemia (AML). NK leukemia, we performed a reverse transcription-PCR (RT- Recent reports emphasized that ED in patients with leukemia is PCR) assay to detect NCAM and CD94 transcripts, as described related to the aggressive disease, signaled by hyperleukocytosis, in Supplementary text. CD56 þ NK lymphoma cells were used 1 and to the early administration of intensive chemotherapy. as positive control. We found a strong expression of both NCAM Many efforts have been made to better characterize specific and CD94 genes (Figure 1c: Lanes 1 and 2). Because the child subgroups of patients with AML by defining biological markers died of complications during the early days of induction, we 2 associated with aggressive disease. Scott et al. have identified a decided to screen our AML population to find a correlation subset of adult patients with myeloid/natural killer (NK) acute between the expression of NK-related genes and ED (before day leukemia misdiagnosed as M3v. Other reports have shown a 15: dead of complications). To evaluate cytoplasmatic expres- strong correlation between CD56 expression and a poor clinical 3 sion of NCAM and CD94 counterparts, we developed an RT- prognosis in adults with AML. In addition, even if little is PCR semiquantitative methodology, using specific primers from known about the molecular mechanisms that regulate blast NCAM, CD94 and b-actin as internal quality and quantity migration to tissues, adhesion receptors are likely to play an 4 control. The sequences of the primers related to CD94 are listed important role in this process. We have attempted to identify in Supplementary text. We amplified a serial dilution of cDNA the molecular characteristics of an aggressive subset of pediatric þ derived from case AC ranging from 100 ng to 0.01 ng, assigning patients with AML through a prospective evaluation of CD56 a score reflecting high ( þþþþor þþþ), low ( þþor þ ) neural cell adhesion molecule (NCAM) and CD94 expression. or absent level of NCAM (Figure 1c: Lanes 5–8) and CD94 We analyzed a total population of 44 children with AML, expression (Figure 1d). We used only specimens containing distributed as follows: five with M3v (group A), nine with acute more than 95% of blasts to exclude contamination from normal promyelocytic leukemic (APL) (group B) and 30 with non-APL NK cells. We found that 9 out of 14 cases, presenting with a high AML (group C). Characteristics of patients in group A, B and C level of NCAM/CD94 expression, died of disease-related events are listed in Table 1. All cases were diagnosed and treated in our during the early days of induction. They mainly died of institution from January 1997 to June 2006, using ongoing hemorrhagic complication (six cases) as well as disseminated protocols (see Table 1). Peripheral blood and bone marrow intravascular coagulation (three cases). Potential risk factors for leukemia specimens were collected at the time of AML death in induction were identified by univariate analysis. The diagnosis and evaluated according to the French–American– following characteristics of patients were examined as risk British criteria.5 The patient (AC) from group A was morpholo- features: hepatomegaly, splenomegaly, WBC count (cut off

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