Leukemia (2005) 19, 329–334 & 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00 www.nature.com/leu

REVIEW

CEBPA point mutations in hematological malignancies

H Leroy1, C Roumier1, P Huyghe2, V Biggio1, P Fenaux3 and C Preudhomme1

1Laboratoire d’He´matologie A, CHRU Lille, U524 INSERM Lille, France; 2Service des Maladies du sang, CHRU Lille, France; and 3Service d’He´matologie Clinique, Hoˆpital Avicenne-Paris 13 University, France

The CCAAT/enhancer-binding protein-alpha (CEBPA) is a wild-type (wt) CEBPA. The 42-kDa CEBPA protein has four transcription factor strongly implicated in myelopoiesis principal domains: the C-terminal part containing a leucine through control of proliferation and differentiation of myeloid progenitors. Recently, several works have reported the pre- zipper domain mediating homo- or heterodimerization; the sence of CEBPA-acquired mutations in hematological malig- DNA-binding domain (DBD), a basic positively charged domain nancies. In this work, we analyzed characteristics of mutations able to interact with specific DNA sequences; and two and their correlation with disease characteristics described regulatory and transactivating domains TAD1 and TAD2 in previous studies. In the 1175 patients reported, 146 CEBPA (Figure 1). mutations were identified in 96 patients. Mutations were found The 42-kDa normal protein acts as a transcription factor with in the whole gene sequence, but cluster regions were clearly identified. Furthermore, two categories of mutations were a crucial role during differentiation of various cell types reported: out-of-frame ins/del often in the N-terminal region, including hepatocytes, adipocytes, enterocytes, keratinocytes, and in-frame ins/del often in the C-terminal region. CEBPA lung, mammary gland cells and hematopoietic cells. In mutations were reported exclusively in acute myeloid hematopoiesis, CEBPA plays a pivotal role in early stages of leukemia (AML) (according to WHO classification criteria) myeloid differentiation and is particularly expressed in myelo- and mutated patients preferentially belonged to M1, M2 and monocytic cells.4–8 CEBPA has multiple actions such as down- M4 FAB subtypes. All but one case belonged to the ‘inter- mediate’ prognostic subgroup of MRC classification. In the regulation of C-MYC expression allowing differentiation, direct absence of poor prognostic factors, patients with CEBPA upregulation of the expression of granulocytic lineage-specific mutation had favorable outcome, very similar to that of the genes and synergistic action with other key genes in myeloid t(8;21), inv(16), t(15;17) subgroup. Systematic analysis of development including CBF complex genes and PU.1.5,9–11 In CEBPA mutations, in addition to that of alterations in master addition to specific DNA binding, CEBPA could act by protein– genes of hematopoiesis, may be useful to assess the prognosis protein interaction. The principal partners of those interactions of AML particularly in patients belonging to the ‘intermediate’ prognostic subgroup. are p21, CDK2, CDK4 and E2F. Repression of E2F-dependent Leukemia (2005) 19, 329–334. doi:10.1038/sj.leu.2403614 transcription genes by CEBPA had previously been shown to be Published online 13 January 2005 a critical event in suppressing cellular proliferation and inducing Keywords: CEBPA; mutations; AML; prognosis; transcription factor; granulocytic or adipocyte differentiation. CEBPA also inhibits CCAAT/enhancer-binding protein cell proliferation by activating transcription of p21/WAF1,by stabilizing p21 and inhibiting CDK2 and CDK4.12–14 CEBPA expression begins with the commitment of myeloid lineage precursors and is upregulated during granulo- Introduction 7 cytic differentiation. CEBPA-defective mice have no mature granulocytes, whereas cells of the other lineages are not Among the many oncogenes affecting proliferation and cell 15 affected. In addition, CEBPA expression could block mono- death, anomalies of the genes implicated in the closely 16 cytic differentiation. regulated pathways of hematopoietic differentiation are key The strong implication of CEBPA in granulocytic differentia- oncogenic events.1 In acute myeloid leukemia (AML), cell tion points to this gene as a key target in leukemogenesis, as differentiation arrest can occur at different levels by alteration 3 shown now in many studies. Pabst et al found that events of specific genes like those of the CBF complex.2 The CEBPA leading to the loss of CEBPA function observed in AML gene (located on chromosome 19q13.1 band) belongs to the contribute to leukemogenesis by blocking granulocytic differ- CCAAT/enhancer-binding protein family, which is involved entiation. Moreover, myeloid blasts observed in those cases in the balance between cell proliferation and terminal were committed myeloid cells generally classified in the M1 or differentiation. CEBPA gene mRNA can be translated from 17 M2 FAB AML subtypes. Recently, three mechanisms of CEBPA the first AUG encoding the 42-kDa normal isoform and also inactivation have been reported. One is downregulation of from the second AUG (nt 508–510) encoding the 30-kDa CEBPA expression by the AML1-ETO fusion transcript in t(8,21) normal isoform, which has lost the 119 first AA including leukemia cells. In this model, conditional expression of CEBPA the TAD1 functional domain. The functions of CEBPA 30-kDa 3 in those cells is sufficient to trigger granulocytic differentia- protein are not well known. Nevertheless, Pabst et al 18 tion. The second mechanism is inhibition of the translation of demonstrated that this shorter CEBPA protein had lost normal CEBPA mRNA by interaction with hnRNPE2, induced by CEBPA functions and had dominant negative effect on 42-kDa 19 BCR-ABL fusion protein. This mechanism could contribute to the transition from chronic phase to myeloid blast crisis in ´ Correspondence: Dr C Preudhomme, Laboratoire d’Hematologie A – CML, by blocking myeloid differentiation. Finally, inactivating hoˆpital Calmette, CHRU, Place de Verdun, 59000 Lille, France; Fax: þ 33 3 20 44 55 10; E-mail: [email protected] CEBPA mutations have been reported in hematological 3,20–26 Received 19 July 2004; accepted 4 November 2004; Published online malignancies, especially in AML. In this review, we 13 January 2005 focused on CEBPA mutations and how, through inactivation of CEBPA mutations in hematological malignancies H Leroy et al 330 120 2nd ATG

AA : 99 104 183 189 286 306 317 345

nt : 445 462 697 714 1006 1068 1099 1183

Clustering regions R1 R2 R3 R4 R5

nt 1 445 462 732 855 1059 1062 1137 1152 1196 % 60 50 40 Single alteration 30 20 10 0

R1 R2 R3 R4 R5 % 60 50

Multiple 40 alterations 30 20 10 0

R1 R2 R3 R4 R5 Legend:

: Transactivating Domain 1 (Poly-Gly) : Out of frame ins / del mutations

: Transactivating Domain 2 (Poly-Pro) : In frame ins / del mutations : DNA Binding Domain (DBD) : Other type mutations : Leucine Zipper Domain

Figure 1 Schematic representation of CEBPA functional domain; incidence of the different types of mutation and localization of mutation hot spot regions.

transcriptional properties of the CEBPA protein, they could lead Correlation of CEBPA mutations with hematological to leukemogenesis. parameters and prognosis

CEBPA mutations and morphological classification Overview of CEBPA gene mutations reported All but two of the 87 reported patients with CEBPA mutation had In the last 3 years, 1175 patients were screened for CEBPA AML, including 30 M1 AML, 35 M2 AML, 14 M4 AML, three M5 mutations in seven studies.3,21–25,27.Those patients included 962 AML, two unclassified AML and one therapy-related AML AML (Table 1), 156 myelodysplastic syndromes, 23 acute (incidence in AML: 85 on 962 patients (8.8 %) ; see Table 1). lymphoblastic leukemia and 34 non-Hodgkin’s lymphoma. The two remaining patients had RAEB-t (a disorder now Among those 1175 patients, 96 (8.2%) had CEBPA mutations included in AML in the new WHO classification of myeloid including nine with silent mutations and 87 with acquired disorders). nonsilent mutations (described in online additional data, FAB classification of AML and MDS was available in 712 supplementary data). CEBPA mutations were only observed in patients studied for CEBPA mutations. Incidences of mutations myeloid malignancies. were 14.5 % of M1, 6.4% of M2, 4% of M4, 2% of M5, one of

Leukemia CEBPA mutations in hematological malignancies H Leroy et al 331 the seven therapy-related AML and one of the 12 RAEB-t. No In these four studies, the complete remission rate was not mutations were found in M0, M3, M6 and M7-AML. significantly different in patients with or without CEBPA mutations, but all studies except that of Snaddon et al21 showed better relapse-free survival (RFS) or overall survival (OS) in Cytogenetic findings mutated cases. Discrepancies between Snaddon et al21 and other studies could have been due to some characteristics of Cytogenetic data was available in 83 of the 87 mutated patients their mutated cases, including higher peripheral blood blast (see online additional data). Only one had a complex karyotype count (median of 93 109/l vs o24 109/l in the mutated cases of with more than three abnormalities; 58 cases (70%) had a other studies), the localization of mutations (five of eight normal karyotype and eight (10%) had only one abnormality. In mutated patients had a single alteration of the C-terminal part the remaining 15 mutated cases, the karyotype was classified in of CEBPA protein vs zero of 27 in the other studies), and, the ‘intermediate’ prognostic subgroup without further details; although this is more speculative, the fact that five of their eight no structural or numerical alteration of chromosome 19 and no mutated patients received standard dose AraC instead of high- ‘favorable’ karyotype (t(8;21), inv(16), t(16;16), t(15;17)) was dose AraC in the other studies. reported in those patients. Therefore, all except one of the An analysis of cooperating mutations, made in two studies, mutated cases belonged to the ‘intermediate’ subgroup of the showed no difference in the incidence of FLT3 and MLL MRC classification.28 This may suggest that CEBPA loss of alterations in CEBPA-mutated and -nonmutated patients.24,27 function induced by balanced translocations and CEBPA loss of However, in patients with CEBPA mutation, Preudhomme function by point mutation are mutually exclusive. et al,24 showed that the presence of FLT3 internal tandem duplication (FLT3-ITD) had a negative prognostic influence, whereas, in the study of Fro¨hling et al,27 the presence of FLT3- Other clinical and biological correlations (Table 2) ITD or FLT3 mutations had no prognostic impact. Nevertheless, in the two studies, the size of the population studied was too When they were studied, no correlation was observed between small for definite conclusions (15 patients in Preudhomme et al, CEBPA mutations and age, sex, WBC count and bone marrow study24 and 36 patients in Fro¨hling et al, study27). blast percentage.

Prognostic value of CEBPA mutations CEBPA mutation profile in mutated cases (Table 3)

Four studies have evaluated the prognostic impact of CEBPA A total of 45 patients had a single CEBPA alteration, whereas 42 mutations in 71 mutated patients, by comparison to 670 patients cases had multiple alterations resulting from biallelic mutations without CEBPA mutation.21,22,24,27 or from several mutations on the same allele. There was no

Table 1 Prevalence of nonsilent CEBPA mutations in acute myeloid leukemia (AML)

Study 1 2 3 4 5 6 No of patients studied 236 78 137 99 135 277 Cumulative incidence (%)

M0 AML FAB subtypes not available 0/1 NC 0/1 0/10 0 M1 AML 2/8 6/56 8/33 7/62 14.5 M2 AML 5/62 2/43 2/34 4/64 6.4 M3 AML 0/22 NC NC 0/22 0 M4 AML 0/32 NC 3/19 1/50 4 M5 AML 0/5 NC 2/32 0/61 2 M6 AML NC NC 0/5 0/3 0 M7 AML NC NC 0/4 NC 0 Sec, AML 1/7 NC NC NC 14.3 Uncl, AML NC NC 0/7 0/5 0 Incidence of mutations 36/236 6/78 8/137 8/99 15/135 12/277 8.8 (85/962) Sec. AML ¼ secondary AML; Uncl. AML ¼ unclassified AML; NC ¼ no case. Study 1 ¼ Fro¨ hling et al; Study 2 ¼ Gombart et al; Study 3 ¼ Pabst et al; Study 4 ¼ Snaddon et al; Study 5 ¼ Preudhomme et al; Study 6 ¼ Khosrovani et al.

Table 2 Clinical and biological characteristics of AML patients with and without CEBPA mutation (median values)

Age % Female WBC (giga/l) PB blasts BM blasts (%) Platelets (giga/l) WT Mutated WT Mutated WT Mutated WT Mutated WT Mutated WT Mutated

Fro¨ hling et al 47 47 55 50 19.9 28.9 40% 62% 80 80 93 45 Preudhomme et al 45 45 49 33 13 20 NA NA NA NA NA NA Snaddon et al 46.5 55.5 36 62 NA NA 9.2 G/l 9.2 G/l 75 80 NA NA Khosrovani et al NA 44 NA 33 NA 34.5 NA NA NA 64.5 NA 41 WT ¼wild-type; WBC ¼ white blood count; PB ¼ peripherical blood; Na ¼ not available.

Leukemia CEBPA mutations in hematological malignancies H Leroy et al 332 significant difference in location and type of mutations between multiple mutations, patients had preferentially M1-AML (45%), patients with single or multiple alterations (Figure 1). whereas, in case of single mutation, patients had preferentially M2-AML (44%) (Table 3).

Characteristics of CEBPA mutations Pathogenetic role of CEBPA mutations Until now, 146 CEBPA mutations have been described in 96 patients,3,21–25,27 including nine isolated silent mutations with- How CEBPA mutations can contribute to leukemogenesis in AML out transcriptional effect in nine patients and 137 nonsilent and to prognosis remains uncertain. It has been shown that CEBPA mutations in 87 patients (five missense mutations, 54 in- CEBPA mutations induced modifications in the gene expression frame ins/del mutations, 78 nonsense mutations including five profile obtained from blast cells, and that, if AML patients were STOP codon point mutations and 73 out-of-frame ins/del classified according to the gene expression profile, CEBPA mutations inducing a STOP codon downstream). Reported mutated patients segregated in two homogeneous clusters.29 A mutations were nonrandomly distributed all over the CEBPA review of the currently published CEBPA mutations suggests two gene sequence and occurred in five regions of the protein main types of mutated cases. The first group contains patients (regions R1 to R5) (Figure 1): R1 corresponding to the AA before with at least one mutation in R1 or R2 clustering regions leading the Poly-Gly domain, R2 between the Poly-Pro and the Poly-Gly to increased translation of the 30-kDa isoform from the second domain, R3 corresponding to the N-terminal part of the DBD AUG downstream of the mutation, inducing the indirect loss of and the region just before the DBD domain, R4 corresponding to CEBPA main function in a dominant negative manner. The other the C-terminal part of the DBD and to the N-terminal part of the group contains patients where CEBPA gene mutations result in b-Zip domain (fork region), R5 corresponding to the C-terminal loss of the main transregulatory function of CEBPA, mainly due to part of the B-zip domain. multiple alterations of the gene; absence of wt CEBPA protein in R1 and R4 were the two main cluster regions as 76% of the blast cells could explain the differentiation arrest of these cells. 137 nonsilent mutations belonged to these regions. Moreover, How can mutations contributing to the loss of function of a the distribution of the type of mutations between these two master gene in early myeloid differentiation result in a myeloid regions was heterogeneous. Nearly all out-of-frame ins/del type of leukemia? It has previously been reported that biallelic nonsense mutations occurred in R1 region, whereas most 54 in- mutations of another early myeloid differentiation gene (AML1/ frame ins/del mutations occurred in R4 region. Functional study RUNX1) could induce M0 AML subtype.2 Furthermore, mice of the mutated protein showed important loss of transactivation models of CML blast crisis with CEBPA knockout result in properties of CEBPA in almost all cases of mutation25 immature erythroid leukemia30,31 and expression of a dominant Interestingly, among the 78 nonsense mutations, 65 occurred negative 30-kDa CEBPA protein inhibits differentiation of myeloid at the 50 of the second ATG codon of CEBPA (R1 region) leading and erythroid progenitors.32 On the other hand, all CEBPA- to increased translation of the alternative 30-kDa form of the mutated patients reviewed in this work had M1, M2, M4 or M5 protein. As demonstrated by Pabst et al, this shorter protein has a AML subtypes, clearly showing persistence of myeloid differentia- dominant negative effect on 42-kDa wt CEBPA. Conversely, tion in blast cells. However, in mutated cases with multiple none of the 54 in-frame ins/del mutations lead to increased alterations of CEBPA, preventing expression of wt protein, or in synthesis of the dominant negative 30-kDa isoform. single alterations with expression of p30 dominant negative form, A minor cluster region (R2 region) has been reported by the persistence of CEBPA residual cellular functions has been Fro¨hling et al,27 who found 14 recurring mutations in the TAD2 demonstrated.31,33,34 Therefore, those cases are not identical to domain. Those alterations were 6 bp duplications or 3 bp KO models since the loss of transactivating properties is deletions inducing the addition of H-P AA in a motif of three dependent of on cis-regulating elements of the CEBPA target H-P repeats, or deletion of one P in a motif of seven P repeats. genes. Moreover, other members of the CEBPA family, including Of the nine cases with TAD2 mutation, seven had no 30-kDa CEBPB and CEBPE, could partially prevent the effects of the loss of dominant negative form. The significance of such recurrent function of mutated CEBPA and allow incomplete myeloid alterations (ie repeats of GCC nucleotide triplets) remains to be differentiation resulting in M1, M2, M4 and M5 blasts. understood. Pabst et al18 demonstrated that, when CEBPA nonsense Overall, in 55 of the 87 mutated cases, mutations lead to the mutations occurred before the second AUG, the 30-kDa CEBPA synthesis of the dominant negative 30-kDa form, which inhibits isoform was increased by 4.2–8.6-fold compared with unmu- CEBPA normal function. No significant correlation between tated cases. This shorter form of CEBPA lacks antiproliferative localization, number of mutations and FAB subtypes could be activity35 and most of the transactivation properties of the established (Figure 1). Of note, however, is the fact thatin case of normal protein,36,37 and acts dominantly on wt CEBPA by inhibiting DNA binding of CEBPA on targeted genes.3 Indeed, in Table 3 Expression of 30-kDa dominant negative isoform accord- the presence of equal amounts of 42-kDa and 30-kDa in ing to FAB subtype and number of CEBPA mutations vitro, the 30-kDa isoform totally abolished transactivation of a plasmid reporter by the full-length CEBPA protein.18 The Single alteration Multiple alteration 30-kDa protein binds DNA with only 14% affinity compared with the full-length protein and can inhibit DNA binding of wt 30-kDa 30-kDa 30-kDa 30-kDa CEBPA.3 However, the 30-kDa isoform does not inhibit 42-kDa isoformÀ isoform+ isoformÀ isoform+ normal function on each CEBPA-transregulated genes. For M1 AML 5 (45%) 6 (55%) 5 (26%) 14 (74%) example, MPO gene expression is not abrogated by the M2 AML 11 (55%) 9 (45%) 2 (13%) 13 (87%) 30-kDa form.31,34 The predominance of M1 and M2 FAB M3 AML 8 (89%) 1 (11%) 1 (20%) 4 (80%) subtypes in CEBPA-mutated AML, with partial myeloid differ- Other 2 (40%) 3 (60%) 1 (33%) 2 (66%) entiation block in blast cells, is compatible with this effect and the higher incidence of CEBPA mutations in M1, M2 and M4 Total 26 (75%) 19 (25%) 9 (21%) 33 (%) FAB subtypes supports a critical role of CEBPA gene function in

Leukemia CEBPA mutations in hematological malignancies H Leroy et al 333 the intermediate stages of granulocytic differentiation. One may Supplementary Information hypothesize that events inducing loss of CEBPA gene function in transformed myeloid precursor cells could lead to M1, M2 or Supplementary Information accompanies the paper on the M4 FAB AML. Leukemia website (http://www.nature.com/leu). In mutated cases where no 30-kDa is synthetized, other mechanisms are required to explain the contribution of CEBPA mutations to leukemogenesis. Since as few as three-fold increases References in CEBPA 42-kDa protein are sufficient to induce differentiation 8 of myeloid cell lines, it is possible that only moderate decreases 1 Tenen DG. Disruption of differentiation in human cancer: AML of CEBPA protein in vivo are sufficient to explain the differentia- shows the way. Nat Rev Cancer 2003; 3: 89–101. tion block in those AML. This could be achieved by haploinsuffi- 2 Roumier C, Fenaux P, Lafage M, Imbert M, Eclache V, ciency resulting from single C-terminal mutations. Preudhomme C. New mechanisms of AML1 gene alteration in It has been shown that patients with CEBPA mutations had hematological malignancies. Leukemia 2003; 17: 9–16. similar prognosis as patients belonging to the ‘favorable’ group 3 Pabst T, Mueller BU, Zhang P, Radomska HS, Narravula S, Schnittger S et al. Dominant-negative mutations of CEBPA, according to MRC classification (including t(8;21), inv(16) and encoding CCAAT/enhancer binding protein-alpha (C/EBPalpha), t(15;17) AML). It is difficult to speculate on the reasons of this in acute myeloid leukemia. Nat Genet 2001; 27: 263–270. favorable prognosis of AML with CEBPA mutations. However, 4 Tenen DG, Hromas R, Licht JD, Zhang DE. Transcription factors, there are some similarities between the cellular effects of the normal myeloid development, and leukemia. Blood 1997; 90: chimeric oncoproteins observed in AML with ‘favorable’ 489–519. karyotype and the cellular effects of CEBPA mutations found 5 Reddy VA, Iwama A, Iotzova G, Schulz M, Elsasser A, Vangala RK et al. Granulocyte inducer C/EBPalpha inactivates the myeloid in AML with normal karyotype (belonging to the ‘intermediate’ master regulator PU.1: possible role in lineage commitment karyotype subgroup of MRC classification). In both situations, decisions. Blood 2002; 100: 483–490. CEBPA function is repressed. Indeed, chimeric proteins induce 6 Zhang P, Nelson E, Radomska HS, Iwasaki-Arai J, Akashi K, the disruption of CEBPA normal functions: in t(8;21), AML1-ETO Friedman AD et al Induction of granulocytic differentiation by 2 is able to suppress CEBPA protein expression by decreasing the pathways. Blood 2002; 99: 4406–4412. level of CEBPA mRNA18,38; in inv(16), CBFb-MYH11 is also able 7 Keeshan K, Santilli G, Corradini F, Perrotti D, Calabretta B. 38 39 Transcription activation function of C/EBPalpha is required to disturb CEBPA functions and in t(15;17), Truong et al, for induction of granulocytic differentiation. Blood 2003; 102: showed that PML-RARa blocked CEBPA activity. Those simila- 1267–1275. rities could contribute to explain why AML with CEBPA 8 Radomska HS, Huettner CS, Zhang P, Cheng T, Scadden DT, mutations have favorable prognosis in AML. Tenen DG. CCAAT/enhancer binding protein alpha is a regulatory This favorable prognostic value and the predominance of switch sufficient for induction of granulocytic development CEBPA mutations in M1, M2 and M4 AML justifies, in our opinion, from bipotential myeloid progenitors. Mol Cell Biol 1998; 18: 4301–4314. systematic screening for CEBPA mutations in those AML groups, 9 Johansen LM, Iwama A, Lodie TA, Sasaki K, Felsher DW, Golub TR especially in patients belonging to the intermediate prognosis et al. c-Myc is a critical target for c/EBPalpha in granulopoiesis. cytogenetic group. Analysis of the currently reported CEBPA Mol Cell Biol 2001; 21: 3789–3806. mutations has shown a nonrandom distribution of mutations in the 10 Smith LT, Hohaus S, Gonzalez DA, Dziennis SE, Tenen DG. PU.1 coding sequence of the gene, with two principal mutation cluster (Spi-1) and C/EBP alpha regulate the granulocyte colony-stimulat- regions (R1 and R4). Mutation types are not equally distributed ing factor receptor promoter in myeloid cells. Blood 1996; 88: 1234–1247. between the two regions as more than 87% of in-frame ins/del 11 Klempt M, Melkonyan H, Hofmann HA, Eue I, Sorg C. The mutations occurred in the R4 region (if we exclude the TAD2 in- transcription factors c-myb and C/EBP alpha regulate the mono- frame ins/del) and nonsense out-of-frame mutation localized in the cytic/myeloic gene MRP14. Immunobiology 1998; 199: 148–151. R1 region in 82% of the cases. As the size of ins/del mutations is 12 D’Alo F, Johansen LM, Nelson EA, Radomska HS, Evans EK, Zhang shorter in out-of-frame ins/del mutations, the best screening P et al. The amino terminal and E2F interaction domains are methods may differ for each region: gene scan method after critical for C/EBP alpha-mediated induction of granulopoietic development of hematopoietic cells. Blood 2003; 102: 3163– amplification may be preferable for the C-terminal region (R3 and 3171. R4 regions) and direct sequencing for the N-terminal region 13 Wang QF, Cleaves R, Kummalue T, Nerlov C, Friedman AD. Cell (genomic sequence coding for AA 1–200, including R1 and R2 cycle inhibition mediated by the outer surface of the C/EBPalpha region). By these methods, 92% of the 137 CEBPA mutations and basic region is required but not sufficient for granulopoiesis. 100% of the mutations inducing the synthesis of dominant Oncogene 2003; 22: 2548–2557. negative 30-kDa isoform would have been identified. 14 Timchenko NA, Wilde M, Nakanishi M, Smith JR, Darlington GJ. CCAAT/enhancer-binding protein alpha (C/EBP alpha) inhibits cell Systematic screening of CEBPA mutations in association with proliferation through the p21 (WAF-1/CIP-1/SDI-1) protein. Genes the search of other alterations in master genes implicated in Dev 1996; 10: 804–815. leukemogenesis such as FLT3, MLL or RAS should probably be 15 Zhang DE, Zhang P, Wang ND, Hetherington CJ, Darlington GJ, performed systematically in AML, particularly in patients Tenen DG. Absence of granulocyte colony-stimulating factor belonging to the ‘intermediate’ prognostic group, to determine signaling and neutrophil development in CCAAT enhancer binding more precisely the impact of the association between genetic protein alpha-deficient mice. Proc Natl Acad Sci USA 1997; 94: 569–574. events on overall survival and therapeutic response in large 16 Friedman AD. Transcriptional regulation of granulocyte and cohorts of patients. monocyte development. Oncogene 2002; 21: 3377–3390. 17 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR et al. Proposals for the classification of the acute Acknowledgements leukaemias. French–American–British (FAB) co-operative group. Br J Haematol 1976; 33: 451–458. 18 Pabst T, Mueller BU, Harakawa N, Schoch C, Haferlach T, Behre This work was supported by the ‘Fondation de France’ and by the G et al. AML1-ETO downregulates the granulocytic differentiation ‘Ligue contre le cancer, Comite´ Nord’. 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