Leukemia (2014) 28, 794–803 & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu

ORIGINAL ARTICLE The role of different genetic subtypes of CEBPA mutated AML

A Fasan, C Haferlach, T Alpermann, S Jeromin, V Grossmann, C Eder, S Weissmann, F Dicker, A Kohlmann, S Schindela, W Kern, T Haferlach and S Schnittger

The prognostic impact of mutations in the CCAAT/enhancer binding protein a (CEBPA) gene was evaluated in the context of concomitant molecular mutations and cytogenetic aberrations in acute myeloid leukemia (AML). CEBPA was screened in a cohort of 2296 adult AML cases. Of 244 patients (10.6%) with CEBPA mutations, 140 cases (6.1%) were single-mutated (CEBPAsm) and 104 cases (4.5%) were double-mutated (CEBPAdm). Cytogenetic analysis revealed normal karyotype in 172/244 (70.5%) of CEBPAmut cases, whereas in 72/244 cases (29.5%) at least one cytogenetic aberration was detected. Concurrent molecular mutations were seen less frequently in CEBPAdm than in CEBPAsm AML cases (69.2% vs 88.6% Po0.001). In detail, the spectrum of concurrent mutations was different in both groups with the frequent occurrence of GATA1 and WT1 mutations in CEBPAdm patients. In contrast, FLT3-ITD, NPM1, ASXL1 and RUNX1 mutations were detected more frequently in CEBPAsm cases. Favorable outcome was restricted to CEBPAdm cases and remained an independent prognostic factor for a favorable outcome in multivariate analysis (hazard ratio: 0.438, P ¼ 0.020). Outcome in CEBPAsm cases strongly depended on concurrent FLT3-ITD. In conclusion, we propose that only CEBPAdm should be considered as an entity in the WHO classification of AML and should be clearly distinguished from CEBPAsm AML.

Leukemia (2014) 28, 794–803; doi:10.1038/leu.2013.273 Keywords: CEBPA; AML; mutation; pronosis

INTRODUCTION zipper gene mutation. As these mutations are usually biallelic, no 6 The CCAAT/enhancer binding protein a (CEBPA) has gained wt CEBPA is expressed in these cases. (3) AML carrying a increasing attention as a favorable prognostic factor in acute homozygous CEBPA mutation due to loss of heterozygosity (LOH) 7 myeloid leukemia (AML). CEBPA is a transcription factor with also expresses no wt CEBPA. critical roles in tissue-specific gene expression and proliferation In AML, CEBPA mutations previously have been reported to be 8,9 arrest. In the hematopoietic system, CEBPA expression is restricted prognostically favorable in the intermediate risk karyotype. They to myelomonocytic cells and is specifically upregulated during do not occur together with recurrent fusion genes like PML-RARA, 5 granulocyte differentiation.1 CBFB-MYH11 or RUNX1-RUNX1T1. Because of this singular biologic CEBPA is an intronless gene that maps to chromosome band and prognostic pattern, CEBPA mutations recently have been 19q13.1 and has a GC-rich coding region. It belongs to the basic included in the WHO classification as a provisional entity. leucin zipper (b-ZIP) family of transcription factors and consists of However, more recent data have shown that a favorable highly homologous C-terminal DNA-binding (basic region) and prognosis in CEBPA-mutated AML is restricted to those cases dimerization (leucin zipper) motifs and two less conserved that present with CEBPAdm, do not carry cytogenetic aberrations 10 N-terminal transactivation domains.2 and do not harbor an FLT3-ITD. Recently, we have performed a Mutations in the CEBPA gene have been described in B15% of detailed analysis of collaborating mutations in CEBPAdm AML and all AML patients.3,4 Mutations can occur across the whole coding discovered that the favorable prognosis of CEBPAdm AML is 11 region. However, studies revealed a clustering in two main hot influenced by the presence of additional mutations. To further spots. N-terminal frame-shift mutations between the major evaluate the role of different types of CEBPA mutations in the translational start codon and a second ATG in the same open context of other molecular mutations and cytogenetic aberrations reading frame lead to a premature stop of translation of the wild- we have analyzed CEBPA mutations in a cohort of 2296 adult AML type (wt) p42 CEBPA protein while conserving the translation of a cases. short p30 isoform which inhibits the function of the full-length This study confirms the favorable outcome of patients with protein by a dominant negative mechanism.3 C-terminal CEPBAdm compared with AML with CEPBAsm. Further, differences mutations are generally in-frame insertions/deletions in the in the frequency and pattern of additional cytogenetic and DNA-binding or basic leucin zipper domains that disrupt binding molecular genetic aberrations and clinical outcome were observed to DNA or dimerization.5 between these two CEPBA mutated subsets. Studies revealed three different CEBPA mutant patterns in AML patients: (1) AML carrying one mutation on one allele (single- mutated CEPBA, CEBPAsm), accounting for B50% of CEPBA MATERIALS AND METHODS mutated AML, still expresses wt CEBPA transcribed from the Patients second allele; (2) AML with two CEPBA mutations (double-mutated Bone marrow samples (n ¼ 2016) or blood samples (n ¼ 280) from 2296 CEPBA, CEBPAdm) typically shows an N-terminal and a basic leucin AML patients were screened for CEBPA mutations. All 2296 patient samples

MLL Munich Leukemia Laboratory, Munich, Germany. Correspondence: Dr S Schnittger, MLL Munich Leukemia Laboratory, Max-Lebsche-Platz 31, Munich 81377, Germany. E-mail: [email protected] Received 15 August 2013; accepted 6 September 2013; accepted article preview online 23 September 2013; advance online publication, 18 October 2013 CEBPA mutations in AML A Fasan et al 795 were referred to our laboratory for first diagnosis of AML between Statistics August 2005 and July 2012. AML was diagnosed according to the Survival curves were calculated for overall survival (OS) and event-free 2,12,13 French–American–British and WHO classifications. The patients were survival (EFS) according to Kaplan–Meier and compared using the two- selected according to karyotype, excluding cases with t(15;17)/PML-RARA, sided log rank test. OS was the time from diagnosis to death or the last t(8;21)/RUNX1-RUNX1T1, inv(16)/t(16;16)/CBFB-MYH11, inv(3)/t(3;3)/EVI1, follow-up. EFS was the time from diagnosis to treatment failure, relapse, t(6;9)/DEK-CAN, 11(q23)/MLL and complex karyotype. death or the last follow-up in complete remission. Relapse was defined One thousand fifty two patients were female and 1244 male. Median according to Cheson et al.30 Cox regression analysis was performed for OS age was 68.4 years (range 15.7–100.4 years). Bone marrow blast and EFS with different parameters as covariates. Median follow-up was percentages ranged from 20.0 to 99.5% (median 67.5%) in 1931 patients calculated using the Kaplan–Meier analysis. Parameters which were with non-FAB M6 AML. The 81 patients with AML M6 subtype had bone significant in univariate analyses were included into multivariate analyses. marrow blast percentages below 20% (0.5–19.5%, median 10.5%), as Dichotomous variables were compared between different groups using characteristic for the AML FAB M6 subtype. In 274 patients only peripheral the w2 test and continuous variables by Student’s t-test. Results were blood was available. Peripheral blood blast percentages in these patients considered significant at Po0.05. All reported P-values are two-sided. No ranged from 20–100% (median 71.0%). Data on other molecular markers adjustments for multiple comparisons were performed. SPSS software was available in: NPM1: n ¼ 2296, FLT3-ITD: n ¼ 2296, FLT3-TKD: n ¼ 2115, version 19.0.1 (IBM Corporation, Armonk, NY, USA) was used for statistical MLL-PTD: n ¼ 2287, RUNX1: n ¼ 1884, ASXL1: n ¼ 1402, IDH1 and IDH2: analysis. n ¼ 1515, WT1: n ¼ 2112, TET2: n ¼ 452 and GATA2: n ¼ 316, respectively. The patients received different treatment schedules and were in part included into controlled trials of German study groups. Prior to therapy all patients gave their informed consent for scientific evaluations. The study RESULTS design adhered to the tenets of the Declaration of Helsinki and was Characterization of CEBPA mutations approved by the institutional review board before its initiation. A CEBPA sequence abnormality was observed in one or more fragments of 471/2296 patients (20.5%). However, in 214 cases (9.3%) one or both of the two most common polymorphisms Cytomorphology, cytogenetics, immunophenotyping (p.Thr230Thr and p.His195_Pro196dup) was detected as the only Cytomorphologic assessment was based on May–Gru¨nwald–Giemsa stains, change. A total of 13 cases had synonymous coding changes, myeloperoxidase reaction and non-specific esterase using alpha-naphtyl- leaving 244 patients (10.6%) with somatic mutations (Figure 1). acetate as described before, and was performed according to the criteria 13–15 The majority (201/358; 56.1%) of mutations comprised frame- defined in the FAB and the WHO classification. Cytogenetic studies shift insertions or deletions. 81/358 (23.5%) were in-frame were performed after short-term culture. Karyotypes, analyzed after insertions or deletions, 46/358 (12.8%) were missense mutations, G-banding, were described according to the International System for Human Cytogenetic Nomenclature.16 Prognostic classification into 26/358 (7.2%) were nonsense mutations and one was a splice-site ‘favorable’, ‘intermediate’ and ‘adverse’ groups was performed according mutation (0.3%) (Figure 1). to the refined MRC (Medical Research Counsil) classification.17 Cytogenetic One hundred forty cases (57.4%) were CEBPAsm with 21 cases results were available for all patients in this study. Immunophenotyping (15.0%) showing loss of the wt allele (equivalent to ‘LOH’ of this was performed as described previously in a subset of 1227 cases.18 chromosomal region) and 104 patients (42.6%) were CEBPAdm. The location of the CEBPA mutations in patients with CEBPAsm was distributed across the entire gene: 57 patients (40.7%) had an Molecular analysis N-terminal mutation (aa 1–120), 38 patients (27.1%) had Isolation of mononuclear cells, mRNA extraction and random primed cDNA C-terminal changes (aa 278–385) and 45 patients (32.1%) had synthesis was performed as described previously.19 The entire CEBPA mutations between these two regions (aa 121–277). The majority coding sequence was amplified by PCR in four overlapping fragments of CEBPAdm patients (78/104; 75%) showed a combination of using the following primers (Ensembl Transcript ID ENS00000498907): mutations in the N-terminal and C-terminal hotspots, mostly Frag1—F 50-TCGCCATGCCGGGAGAACTCTAAC-30, Frag1—R 50-GTCCAGG combining an N-terminal frame-shift and a C-terminal in-frame TAGCCGGCG-30 (amino acid (aa) 1–103), Frag2—F 50-CTTCAACGAC 0 0 0 mutation (60/104; 57.7%) (Figure 2, Table 1). Mutations other than GAGTTCCTGGCCGA-3 ,Frag2-R5-AGCTGCTTGGCTTCATCCTCCT-3 (aa 79–176), the N-terminal frame-shift and C-terminal in-frame were more Frag3—F 50-CCGCTGGTGATCAAGCAGGA-30, Frag3—R 50-CCGGTACTC GTTGCTGTTCT-30 (aa 173–277), Frag4—F 50-GAGTGGCGGCAG CGGCGCGG frequent in the CEBPAsm than in the CEBPAdm group (79/140 GCA-30, Frag4—R 50-CACGGCTCGGGCAAGCCTCGAGAT-30 (aa 255–359). mutations in CEBPAsm, 56.4% vs 39/208 mutations in CEBPAdm, The PCR was carried out in a 50 ml reaction volume. PCR assays for 18.8%; Po0.001). fragments 1 and 2 contained 0.25 mM of both forward and reverse primers, Buccal swabs or samples taken in the complete remission stage 1.5 M GC-Rich resolution solution, 10 ml5Â GC-Rich PCR reaction buffer were available in 56/104 CEBPAdm cases. In 2/56 cases CEBPA with dimethyl sulfoxide and 2 U GC-Rich system enzyme mix (Roche germline mutations were detected (case 1: p.Tyr181* and case 2 Diagnostics GmbH, Penzberg, Germany) Each 50 ml-reaction contained 1 ml (with three diagnostic mutations): p.His18Gln and p.Gly53A- of cDNA or DNA. PCR reactions for fragments 3 and 4 contained 25 ml lafs*107). In both cases, the mutational burden of one CEBPA Qiagen Taq PCR master mix (Qiagen, Hilden, Germany), 2.5 ml dimethyl mutation declined and disappeared during therapy while the sulfoxide and 0.125 ml of each forward and reverse primer. Amplification 1 other one or two, respectively, were detected in each examination for fragments 1, 2 and 4 was performed with 40 cycles using 60 C B annealing temperature. Fragment 3 was amplified with touchdown PCR with an unchanged mutational burden of 50%. with 27 cycles using 60 and 72 1C annealing temperatures. Products were analyzed by WAVE denaturing high-performance liquid chromatography. All samples were also screened for homozygous mutations by mixing PCR Clinical parameters products from patient samples and wt controls. All samples with an Comparison of the clinical and laboratory features of CEBPAmut abnormal chromatogram were subsequently further characterized by and CEBPAwt cases is summarized in Table 2. Clinical parameters direct Sanger sequencing. of CEBPA LOH patients did not differ from CEBPAsm cases (data Additionally, 849/2296 samples were analyzed by a sensitive next- not shown), and thus CEBPA LOH were grouped together with generation amplicon deep-sequencing assay (454 Life Sciences, Branford, 20,21 CEBPAsm cases. CT, USA). For deep-sequencing, CEBPA-oligonucleotide primer plates With regard to age, CEBPAdm patients were significantly were provided as part of the IRON-II study (Roche Diagnostics GmbH).22 Next generation sequencing data was analyzed using both the GS younger (median age 56.3 years, range 15.7–87.6 years) compared variant analyzer software 2.5.3 (454 Life Sciences) and Sequence Pilot with CEBPAwt (median age 65.4 years, range 16.8–100.4 years; version 3.5.2 (JSI Medical Systems, Kippenheim, Germany). Po0.001) and CEBPAsm cases (median age 65.8 years, range 20.4– Analyses for mutations in NPM1, FLT3, MLL-PTD, NRAS, IDH1 IDH2, ASXL1 87.0 years; Po0.001). CEBPAdm patients had significantly lower RUNX1, TET2 and GATA2 were described previously.23–29 platelet counts compared with CEBPAwt (median 34.0 vs

& 2014 Macmillan Publishers Limited Leukemia (2014) 794 – 803 CEBPA mutations in AML A Fasan et al 796

Figure 1. Molecular mutations in CEBPA. The numbers of mutations are reflected by the numbers of colored dots, which also indicate the mutation effect on the amino acid level.

66.0 Â 109/l; Po0.001) and CEBPAsm cases (median 56.0 Â 109/l; Po0.001). Furthermore, CEBPAdm cases had significantly higher bone marrow blast percentages compared with CEBPAwt cases (mean 65.0% vs 56.5%; P ¼ 0.006). There was no difference in sex, white blood cell (WBC) count and hemoglobin levels between CEBPAwt, CEBPAsm and CEBPAdm cases.

Distribution of CEBPA mutations according to FAB subtypes As previously described,31,32 presence of CEBPA mutations was generally associated with FAB subtypes M1 and M2 of AML. In detail, 100/532 cases (18.8%) with AML M1 subtype and 106/756 (14.0%) cases with M2 subtype showed CEBPA mutations in comparison with only 17/704 cases (2.4%) with other FAB subtypes (Po0.001). There was no difference in FAB distribution between CEBPAsm and CEBPAdm.

Distribution of CEBPA mutations according to immunophenotyping Immunophenotyping was done in 1227 patients. As previously described,33 patients with CEBPAmut had significantly higher expression of CD7 (mean±s.d. positive cells 45±31% vs 27±22%; Po0.001), CD15 (49±27% vs 42±26%; P ¼ 0.007), CD33 (73±23% vs 63±27%; P 0.001), CD34 (47±31% vs Figure 2. Circos plot showing the combinations of CEBPAdm cases. o The left part of the circle indicates CEBPA mutation 1 and the right 30±26%; Po0.001), CD65 (45±28% vs 39±25%; P ¼ 0.02) and ± ± part indicates CEBPA mutation 2. Each part is divided in C-terminal-, HLA-DR (41 24% vs 34 22%; P ¼ 0.002) compared with middle- and N-terminal mutation and further subdivided into CEBPAwt cases. Furthermore, we observed a significantly lower specific mutation subtypes (frameshift, missense, nonsense, expression of CD14 in CEBPAmut cases (13±12% vs 22±18%; inframe). The width of the arches indicates the percentage of Po0.001) compared with CEBPAwt cases. Also, the expression of samples with the respective combination of CEBPA mutations. CD7 (59±27% vs 35±30%; Po0.001), CD15 (57±27% vs Table 2 depicts the detailed case numbers. 45±27%; P ¼ 0.047), CD34 (63±24% vs 36±30%; Po0.001), HLA-DR (50±21% vs 37±24%; P ¼ 0.003) and CD65 (52±30% vs Correlation of CEBPA mutation status to cytogenetics 41±26%; P ¼ 0.035) was significantly higher in CEBPAdm cases Analysis of cytogenetic results revealed cytogenetically normal compared with CEBPAsm cases. AML (CN-AML) in 70.5% of CEBPAmut cases. Within the subset of

Leukemia (2014) 794 – 803 & 2014 Macmillan Publishers Limited CEBPA mutations in AML A Fasan et al 797 Table 1. Summary of CEBPA mutations detected in 2296 AML cases

Mutation Mutation 1 Mutation 2 status N-terminal Middle AA C-terminal N-terminal Middle AA C-terminal Number of AA 1–120 121–277 AA 278–358 AA 1–120 121–277 AA 278–358 patients

CEBPAsm Frameshift ins/del 46 Inframe ins/del 1 Missense 5 Nonsense 5 Frameshift ins/del 32 Inframe ins/del 3 Missense 5 Nonsense 5 Frameshift ins/del 13 Inframe ins/del 10 Missense 13 Nonsense 2 Total 140

CEBPAdm Frameshift ins/del Inframe ins/del 60 Frameshift ins/del frameshift ins/del 3 Frameshift ins/del Missense 9 Nonsense Missense 1 Nonsense Inframe 5 Frameshift ins/del frameshift ins/del 1 Frameshift ins/del Missense 2 Frameshift ins/del Nonsense 2 Frameshift ins/del Frameshift 9 ins/del Frameshift ins/del Missense 1 Frameshift ins/del Nonsense 4 Frameshift ins/del Nonsense 2 Frameshift ins/del Frameshift ins/del 1 Frameshift ins/del Inframe ins/del 2 Frameshift ins/del Missense 1 Missense Missense 1 Total 104 Abbreviations: AA, amino acid number; del, deletion; dm, double-mutated; ins, insertion; sm, single-mutated.

CN-AML 94/1742 (4.0%) and 78/1742 (3.4%) had CEBPAsm and hadtwoand11patients(8.9%)hadthreeadditionalmolecular CEBPAdm, respectively. A total of 72/244 CEBPA mutated patients mutations. Of 72 CEBPAdm patients with additional molecular (29.5%) had cytogenetic aberrations (intermediate: n ¼ 60, un- mutations, 49 (68.0%) had one, 20 patients (27.8%) had two, two favorable n ¼ 12). A similar proportion of intermediate-risk abnormal patients (2.8%) had three and one patient (1.4%) had four karyotype patients were CEBPAsm (38/432, 8.8%) and CEBPAdm additional mutations. (22/432, 5.1%). The most frequent intermediate-risk aberration WT1 (14/104, 13.6% vs 2/140, 1.5%; Po0.001) and GATA2 was trisomy 8 (11/60, 18.3%). Interestingly, all 11 patients with (21/102, 9.1% vs 2/130, 0.9%; Po0.001) were significantly more trisomy 8 were CEBPAsm. Furthermore, of 122 patients with frequently mutated in CEBPAdm cases compared with CEBPAsm adverse karyotype, eight (6.5%) were CEBPAsm and four (3.3%) cases. In contrast, NPM1 mutations (44/140, 31.4% vs 2/104, 1.9%; were CEBPAdm (Table 2, Figure 3). Po0.001), FLT3-ITD (14/140, 10.0% vs 1/104, 1.0%; P ¼ 0.003), ASXL1 mutations (41/140, 30.6% vs 14/104, 13.7%; P ¼ 0.003) and RUNX1 mutations (25/140, 18.0% vs 6/104, 5.8%; P ¼ 0.006) were Correlation of the CEBPA mutation status to other molecular seen more frequently in CEBPAsm cases compared with CEBPAdm mutations cases. To determine, whether CEBPA mutations correlate with other Comparing CEBPAsm and CEBPAwt AML, the total number of mutations frequently reported in AML, we additionally analyzed mutations was comparable in both groups (Table 2). The FLT3-ITD, MLL-PTD and NPM1, FLT3-TKD, ASXL1, GATA2, IDH1, frequency of ASXL1 mutations was higher in the CEBPAsm group IDH2, RUNX1, TET2 and WT1 mutation status. Cases with low compared with CEBPAwt cases (41/134, 30.6% vs 210/1167, mutation burden of FLT3-ITD with mutation/wt ratio o0.5 were 18.0%; P ¼ 0.001). grouped together with the FLT3-ITD negative cases, as it has been shown that only an FLT3-ITD ratio X0.5 has a significant adverse prognostic impact.34 Thus, FLT3-ITD negative patients Prognostic significance of CEBPA mutations and patients with FLT3-ITD ratio o0.5 are combined and Survival analysis was restricted to 1117 patients with intermediate- designated as FLT3-ITD/FLT3wtratioo0.5. Generally, concurrent risk karyotype having received intensive therapy. Numbers of mutations were seen less frequently in CEBPAdm than in CEBPAmut cases in the adverse karyotype risk group were too CEBPAsm AML cases (72/104, 69.2% vs 124/140, 88.6%; small for valid analysis. CEBPAdm patients had significantly longer Po0.001) (Table 2, Figure 4). Of 124 CEBPAsm patients with OS compared with CEBPAsm and CEBPAwt cases (median n.r. vs additional molecular mutations, 59 (47.6%) had one, 54 (43.5%) 26.2 months and 45.6 months; Po0.001 and P ¼ 0.002,

& 2014 Macmillan Publishers Limited Leukemia (2014) 794 – 803 CEBPA mutations in AML A Fasan et al 798 Table 2. Demographics, clinical and molecular characteristics of AML patients according to CEBPA mutations

Characteristic Total no. CEBPAwt CEBPAmut P-value CEBPAsm CEBPAdm P-value P-value P-value

No. % of No. % of No. % of No. % of CEBPAwt vs CEBPAwt vs CEBPAsm vs total total total total CEBPAsm CEBPAdm CEBPAdm

Number of patients 2296 2052 89.4 244 10.6 140 6.0 104 4.5

Age (years) Median 68.4 65.4 61.8 NS 65.8 56.3 NS o0.001 Po0.001 Range 15.7–100.4 16.8–100.4 15.7–87.6 20.4–87.0 15.7–87.6

Sex Female 1052 935 40.7 117 5.1 NS 54 2.3 63 2.7 NS NS NS Male 1244 1117 48.6 127 5.5 86 3.7 41 1.8

Disease state de novo 2041 1815 79.1 226 9.8 125 5.4 101 4.4 NS NS NS Secondary 254 236 10.3 18 0.8 15 0.7 3 0.1

WBC count (1 Â 109/l) Median 1.3 28.9 15.9 NS 12.7 17.1 NS NS NS Range 0.1–600 0.1–600 0.9–365.0 1.4–365.0 0.9–182.0

Hb level (g/dl) Median 9.2 9.2 9.1 NS 8.9 9.6 NS NS NS Range 3.0–18.0 3.0–18.0 3.0–14.0 3.0–14.0 4.0–14.0

Platelet count (1 Â 109/l) Median 64.0 66.0 45.0 NS 56.0 34.0 NS o0.001 o0.001 Range 1.0–950 1.0–950 5.0–540 5.0–540.0 5.0–183.0

BM blasts (%) Median 67.5 56.5 63.0 NS 57.8 65.0 NS 0.006 NS Range 20–99.5 0.5–99.5 7.5–99.0 7.5–99.0 8.5–95.0

Cytogeneticsa Intermediate CN 1742 1570 68.4 172 7.5 o0.001b 94 4.0 78 3.4 NS NS NS þ 8 194 183 8.0 11 0.5 11 0.5 0 — 9q- 23 19 0.8 4 0.2 1 0.04 3 0.1 11q- 9 6 0.3 3 0.1 2 0.1 1 0.04 Other intermediate 206 164 7.1 42 1.8 24 1.0 18 0.8 Adverse 5q- 48 47 2.1 1 0.04 1 0.04 0 —— À 7 59 51 2.3 8 0.3 6 0.3 2 0.1 Other adverse 15 12 0.5 3 0.1 1 0.04 2 0.1

FAB subtype M0 142 140 6.1 2 0.1 2 0.1 0 — NS NS NS M1 632 532 22.9 100 4.4 o0.001c 48 2.1 52 2.3 M2 862 756 33.2 106 4.6 0.009d 63 2.7 43 1.9 M4 397 387 16.8 10 0.4 9 0.4 1 0.04 M5 79 78 3.4 1 0.04 0 — 1 0.04 M6 83 79 3.4 4 0.2 2 0.1 2 0.1 M7 3 3 0.2 0 0 0 — 0 — Unknown 98 77 3.4 21 0.9 16 0.7 5 0.2

Molecular markers FLT3-ITD (n ¼ 2296) neg and ratioo0.5 1990 1761 88.5 229 93.9 o0.001 126 90.0 103 99.0 NS o0.001 0.003 RatioX0.5 306 291 14.2 15 6.1 14 10.0 1 1.0

FLT3-TKD (n ¼ 2115) wt 1986 1,48 93.4 238 97.5 0.010 136 97.1 102 98.1 NS NS NS mut 129 123 6.6 6 2.5 4 2.9 2 1.9

NPM1 (n ¼ 2296) wt 1453 1255 61.2 198 81.1 o0.001 96 68.6 102 98.1 NS o0.001 o0.001 mut 843 797 38.8 46 18.9 44 31.4 2 1.9

MLL-PTD (n ¼ 2287) neg 2089 1853 90.7 236 96.7 0.001 134 95.7 102 98.1 0.046 0.007 NS pos 198 190 9.3 8 3.3 6 4.3 2 1.9

RUNX1 (n ¼ 1884) wt 1443 1231 75.0 212 87.2 o0.001 114 82.0 98 94.2 NS o0.001 0.006 mut 441 410 25.0 31 12.8 25 18.0 6 5.8

ASXL1 (n ¼ 1402) wt 1137 956 82.0 181 76.6 NS 93 69.4 88 86.3 0.001 NS 0.003 mut 265 210 18.0 55 23.3 41 30.6 14 13.7

IDH1 and IDH2 (n ¼ 1515) wt 1098 891 58.8 207 13.7 o0.001 110 45.5 97 40.1 0.018 o0.001 0.003 mut 417 382 25.2 35 2.3 28 11.6 7 2.9

WT1 (n ¼ 2112) wt 1998 1,78 94.7 220 93.2 NS 131 98.5 89 86.4 NS 0.002 o0.001 mut 114 99 5.3 16 6.8 2 1.5 14 13.6

GATA2 (n ¼ 316) wt 290 81 25.6 209 66.1 NS 128 55.2 81 34.9 NS 0.001 o0.001 mut 26 3 0.9 23 7.3 2 0.9 21 9.1

TET2 (n ¼ 452) wt 294 176 38.9 118 26.1 0.047 60 30.5 58 29.4 0.016 NS NS mut 158 79 17.5 79 17.5 48 24.4 31 15.7 Abbreviations: CN, cytogenetically normal; Hb, hemoglobin; NS, not significant. aclassification according to Medical Research Counsil.17 bCN vs intermediate- risk aberrant karyotype. cby the Fisher exact test comparing M1 subtype versus all others. dby the Fisher exact test comparing M2 subtype versus all others. Significant P-values are written in bold.

Leukemia (2014) 794 – 803 & 2014 Macmillan Publishers Limited CEBPA mutations in AML A Fasan et al 799 Prognostic impact of karyotype We furthermore analyzed the prognosis of CEBPAsm and CEBPAdm cases with regard to the underlying karyotype in the intermediate- risk karyotype group of patients. There was no difference in outcome between CN-AML cases and cases with intermediate risk cytogenetic aberrations, neither in the CEBPAsm nor in the CEBPAdm group of patients (data not shown).

Prognostic impact of concomitant molecular mutations We were also interested if the inferior prognosis of CEBPAsm patients was influenced by the additional presence of NPM1 mutations and FLT3-ITD. Therefore, we evaluated the impact of three FLT3-ITD/NPM1 genotype subgroups in CEBPAsm and CEBPAwt cases, respectively: NPM1wt/FLT3-ITD/FLT3wtratioo0.5, Figure 3. Alignment of underlying karyotype of 244 CEBPAmut NPM1mut/FLT3-ITD/FLT3wtratioo0.5 and FLT3-ITDpos. Strikingly, patients. Each column represents one of the 244 analyzed samples. patients with CEBPAsm and additional FLT3-ITD had significantly The eight cytogenetic subgroups are shown by colored bars. A red bar indicates the respective cytogenetic aberration. CEBPAsm cases impaired OS and EFS (median 2.9 months and 2.0 months, are indicated in dark gray, CEBPAdm cases in light gray. respectively; Po0.001) compared with all other groups (Figure 5c). There was evidence that the presence of CEBPAsm influenced OS of NPM1mut/FLT3-ITD/FLT3wtratioo0.5 cases. However, because of limited case numbers this difference in outcome was not significant (median n.r. vs 73.4 months; n.s.). EFS of NPM1mut/ FLT3-ITD/FLT3wtratioo0.5 cases was not influenced by an additional CEBPAsm (median 20.8 months vs 22.8 months; n.s.) (Figure 5c). Furthermore, the presence of CEBPAsm had no impact on the outcome in NPM1wt/FLT3-ITD/FLT3wtratioo0.5 genotype cases. Only two patients were both CEBPAdm and NPM1mut, and only one CEBPAdm patient had an additional FLT3-ITD, therefore these subgroups were not analyzed. In CEBPAdm cases, prognosis was positively influenced by the additional presence of GATA2 mutations as previously shown by our group.11,27 In contrast, a concomitant TET2 mutation was associated with an impaired 2 year-OS (23.8% vs 59.6%; P ¼ 0.043; data not shown), as described before.11 Other markers associated with impaired prognosis in AML such as ASXL1, TET2, WT1 and RUNX1 had no additional prognostic Figure 4. Alignment of gene mutations for 244 CEBPAmut patients. impact on CEBPA mutated AML (data not shown). Each column represents one of the 244 analyzed samples. Mutations in the 11 investigated genes are shown by colored bars. A red bar indicates a mutation; a gray bar no mutation, whereas a white bar Univariate and multivariate analysis indicates that no data were available. CEBPAsm cases are indicated In univariate Cox regression analysis of 1117 intensively treated in dark gray, CEBPAdm cases in light gray. AML patients, cases with CEBPAdm (P ¼ 0.026) and NPM1 mutations (P ¼ 0.004) were associated with longer EFS. Higher age (Po0.001), higher WBC count (Po0.001), higher bone marrow respectively) (Figure 5a). EFS was also significantly longer for blasts count (Po0.001), FLT3-ITD/wt ratio X0.5 (Po0.001), MLL- CEBPAdm patients compared with CEBPAsm and CEBPAwt cases PTD (P ¼ 0.016), ASXL1 mutations (P ¼ 0.002) and RUNX1 mutations (median 45.9 months vs 10.6 months and 15.9 months; P ¼ 0.008 (P ¼ 0.043) were associated with worse EFS. In multivariate and P ¼ 0.012, respectively) (Figure 5a). Outcome of CEBPA LOH analysis, age (Po0.001), WBC count (Po0.001), FLT3-ITD/wt ratio patients did not differ from CEBPAsm and CEBPAwt cases o0.5 (P ¼ 0.001) and NPM1 mutations (P ¼ 0.024) had indepen- (Supplementary Figure 1). Therefore, we did not subdivide CEBPA dent relevance for EFS. Investigating OS in univariate analysis, LOH and CEBPAsm cases for further survival analysis. Interestingly, CEBPAdm (P ¼ 0.002) and NPM1 mutations (Po0.001) were OS of CEBPAsm cases without CEBPA LOH patients was signifi- associated with better prognosis, whereas age (Po0.001), male cantly worse compared with CEBPAwt cases (median 23.6 months sex (P ¼ 0.015), WBC count (Po0.001), the FLT3-ITD ratio X0.5 vs 45.6 months; P ¼ 0.024). (Po0.001), MLL-PTD (P ¼ 0.008), ASXL1 mutations (Po0.001) and RUNX1 mutations (P ¼ 0.007) were associated with inferior out- come. In multivariate analysis, age (Po0.001), FLT3-ITD/wt ratio Prognostic impact of CEBPA mutation type X0.5 (Po0.001), CEBPAdm (P ¼ 0.020) and NPM1 mutations The impact of CEBPA mutation location was also investigated. Only (P ¼ 0.022) had independent prognostic impact (Table 3). patients with mutations predicted to cause p30 isoform transla- tion and/or disruption or loss of the C-terminal basic leucin zipper domain were included in this analysis. OS for N-terminal CEBPAsm DISCUSSION patients tended to be inferior compared with C-terminal CEBPAsm In this large cohort of 2296 AML patients the incidence, patients (median 19.2 months vs median n.r.; P ¼ 0.089) and was distribution and outcome of CEBPA mutations were similar to significantly worse compared with CEBPAdm patients (median that reported by others.3,8,35–37 Overall, 10.6% of patients had one 19.2 months vs n.r.; Po0.001) (Figure 5b). Regarding EFS, or more CEBPA mutations. Two mutations were detected in 4.5% N-terminal CEBPAsm patients had significantly worse prognosis of all patients investigated and the favorable outcome was compared with CEBPAdm patients (median 9.7 months vs 45.9 restricted to these CEBPAdm patients. We also were able to further months; P ¼ 0.009) (Figure 5b). confirm the value of CEBPAdm as an independent favorable

& 2014 Macmillan Publishers Limited Leukemia (2014) 794 – 803 CEBPA mutations in AML A Fasan et al 800

CEBPAdm vs. CEBPAsm: p =0.008

CEBPAdm vs. CEBPAwt: p =0.012

CEBPAdm (n=82; median: n.r.)

CEBPAwt (n=963; median: 45.6 CEBPAdm (n=82; median: 45.9 months) months)

CEBPAsm CEBPAsm (n=72; median: 26.2 months) (n=72; median: 10.6 months)

CEBPAdm vs.CEBPAsm p<0.001 CEBPAwt (n=963; median: 15.9 months) CEBPAdm vs. CEBPAwt p=0.002

CEBPAsm N-term vs. CEBPAdm: p<0.001 CEBPAsm N-term vs. CEBPAdm: p=0.009 CEBPAsm N-term vs. CEBPAsm C-term: p=0.089

CEBPAdm (n=82, median n.r.)

CEBPAsm C-term (n=20, median n.r.)

CEBPAdm (n=82, median 45.9 months)

CEBPAwt (n=963, median 45.6 CEBPAsm C-term (n=20, median months) 10.3 months)

CEBPAsm N-term (n=27, median 9.7 months) CEBPAsm N-term (n=27, median 19.2 months) CEBPAwt (n=963, median 15.9 months)

vs. p =0.007 vs. p =0.007 vs. p =0.003 vs. p =0.003 vs. p <0.001 vs. p <0.001 vs. p =0.001 vs. p =0.008 vs. p <0.001 vs. p <0.001 vs. p =0.002 vs. p =0.024 vs. p <0.001 vs. p <0.001 vs. p <0.001 vs. p <0.001

p <0.001 p <0.001

CEBPAsm NPM1mut/FLT3-ITDneg (n=24; median n.r.) CEBPAsm NPM1mut/FLT3-ITDneg (n=24; median 20.8 months)

CEBPAsm FLT3-ITDpos (n=10; median 2.9 months) CEBPAsm FLT3-ITDpos (n=10; median 2.0 months)

CEBPAsm NPM1wt/FLT3-ITDneg (n=38; median 26.2 months) CEBPAsm NPM1wt/FLT3-ITDneg (n=38; median 12.0 months)

CEBPAwt NPM1mut/FLT3-ITDneg (n=377; median 73.4) CEBPAwt NPM1mut/FLT3-ITDneg (n=377; median 22.8 months)

CEBPAwt FLT3-ITDpos (n=163; median 33.9 months) CEBPAwt FLT3-ITDpos (n=163; median 8.9 months)

CEBPAwt NPM1wtFLT3-ITDneg (n=423; median 27.7 months) CEBPAwt NPM1wt/FLT3-ITDneg (n=423; median 13.9 months) Figure 5. (a) Kaplan–Meier survival analysis according to CEBPA mutation status. Survival within the total cohort of 2296 patients. Kaplan Meier plot showing (left) overall and (right) event-free survival of CEBPAwt (gray) compared with CEBPAsm (black) and CEBPAdm cases (red). (b) Kaplan–Meier survival analysis of CEBPAwt, CEBPAsm N-terminal, CEBPAsm C-terminal and CEBPAdm cases. Kaplan–Meier plot showing (left) overall and (right) event- free survival of CEBPAwt (gray) compared with CEBPAsm N-terminal (black), CEBPAsm C-terminal (green) and CEBPAdm cases (red). (c) Kaplan–Meier survival analysis of the three FLT3-ITD/NPM1 genotype subgroups in CEBPAsm and CEBPAwt cases. Kaplan–Meier plot showing (left) overall and (right) event-free survival of CEBPAsm NPM1wt/FLT3-ITD/FLT3wtratioo0.5 (gray), CEBPAsm NPM1mut/FLT3-ITD/FLT3wtratioo0.5 (black), CEBPAsm FLT3-ITDpos (blue), CEBPAwt NPM1wt/FLT3-ITD/FLT3wtratioo0.5 (red), CEBPAwt NPM1mut/FLT3-ITD/FLT3wtratioo0.5 (purple) and CEBPAwt FLT3-ITDpos (green).

Leukemia (2014) 794 – 803 & 2014 Macmillan Publishers Limited CEBPA mutations in AML A Fasan et al 801 Table 3. Influence of different biological and leukemia-associated parameters on OS and EFS in 1117 AML patients in uni- and multivariate analysis

Parameter EFS univariate EFS multivariate OS univariate OS multivariate

P-value RR P-value RR P-value RR P-value RR

Agea o0.001 1.279 o0.001 1.282 o0.001 1.149 o0.001 1.393 Male sex NS — — — 0.015 1.295 NS — WBC countb o0.001 1.042 o0.001 1.041 o0.001 1.055 NS — Blast count o0.001 1.068 NS 0.009 1.062 NS — FLT3-ITD/wt ratio (X0.5) o0.001 1.531 o0.001 1.930 o0.001 1.643 o0.001 2.028 NPM1mut 0.004 0.791 0.024 0.724 o0.001 0.687 0.022 0.667 NPM1mut/ FLT3-ITD/wt ratio (X0.5) o0.001 0.678 NA NA o0.001 0.544 NA NA CEBPAdm 0.026 0.674 NS — 0.002 0.426 0.020 0.438 ASXL1mut 0.002 1.484 NS — o0.001 1.901 NS — MLL-PTD 0.016 1.392 NS — 0.008 1.554 NS — RUNX1mut 0.043 1.239 NS — 0.007 1.417 NS — Abbreviations: EFS, event-free survival; NA, not applicable; NS, not significant; OS, overall survival; RR, relative risk. Age, peripheral blood cell counts were considered as continuous parameters. aPer 10 years of increase. bPer 10 Â 109/l. Significant P-values are written in bold. prognostic factor by multivariate analysis, which is in line with FLT3-ITD (Figure 5c). This is in line with the findings of Taskesen previous studies.6,10,38,39 et al.39 who showed that prognosis of CEBPAsm cases (n ¼ 60) is Concurrent mutations were significantly less frequent in impaired by concurrent FLT3-ITD in a cohort of 1182 CN-AML CEBPAdm compared with CEBPAsm patients. FLT3-ITD and NPM1 cases. In contrast, Dufour et al. found no prognostic effect of FLT3- mutations were even almost mutually exclusive of CEBPAdm, a ITD in CEBPAsm patients (n ¼ 28) in a cohort of 663 CN-AML fact, which is consistent with previously published data.31,39 cases.32 Furthermore, Green et al.31 investigated CEBPA mutations Conversely, there were also mutations that were associated with in a cohort of 1427 adult AML cases. They showed that the CEBPAdm and almost mutually exclusive of CEBPAsm, namely presence of FLT3-ITD was associated with a significantly worse GATA2 and WT1.27,40,41 Interestingly, TET2 mutations were found outcome, irrespective of the CEBPA mutation status. with a frequency of 17.5% in CEBPAdm and CEBPAsm cases, which The positive impact on prognosis of CEBPAdm cases is also is in line with the overall mutation rate of TET2 in AML of about modified by the presence of additional mutations. TET2 mutations 20%.28,42 significantly impair the outcome of CEBPAdm patients, whereas However, thus far, detailed investigation of CEBPA mutations in patients harboring an additional GATA2 mutation show signifi- the context of underlying karyotype are rare and most studies cantly better OS. These modifying effects are well substantiated by restricted their cohorts on CN-AML. One study described 12 CEBPA now and based on a growing amount of studies.11,27,41 mutations in a cohort of 277 patients with intermediate-risk Expression profiling of a previous study had suggested that karyotype. Eight patients showed normal karyotype, whereas 4 of C-terminal CEBPAsm patients were less distinct from CEBPAdm these 12 patients had chromosomal aberrations [del(9q), n ¼ 2; patients than N-terminal CEBPAsm cases.6 Therefore, we del(11q), n ¼ 1; del(12p), n ¼ 1].36 Although in our cohort the investigated whether the mutation location had any influence majority of CEBPA mutated cases were cytogenetically normal, on outcome in CEBPAsm cases. We found that N-terminal 29.5% of CEBPA mutated cases showed an aberrant karyotype. CEBPAsm cases tended to have a poorer OS compared with Trisomy 8 was the most common intermediate risk alteration with C-terminal CEBPAsm cases. However, this difference in outcome an incidence of 8.4% and, interestingly, all 11 CEBPA mutated was not significant and has to be validated in further studies. cases harboring trisomy 8 were CEBPAsm. Trisomy 8 has recently Interestingly, only N-terminal CEBPAsm and not C-terminal been described to be associated with the concomitant presence CEBPAsm cases showed a significant impaired OS compared of adverse molecular markers, particularly ASXL1 and RUNX1 with CEBPAdm patients. This would support the theory of a similar mutations or both, and to have inferior outcome compared with gene expression profile of C-terminal CEBPAsm AML and CEBPAdm CN-AML patients.43 In our cohort, we were not able to show that AML. the presence of trisomy 8 further impairs the unfavorable On the basis of our data, we recommend screening for CEBPA prognosis of CEBPAsm patients, but this may be explained by mutations for all intermediate-risk karyotype AML and not restrict the small number of CEBPAsm cases with concurrent trisomy 8. it to the CN group only. Screening of CEBPA mutations should be Owing to the large number of cases included in our study, we accompanied by FLT3-ITD quantification to identify the extremely were able to evaluate the prognostic impact of CEBPA mutational unfavorable subgroup of CEBPAsm/FLT3-ITDpos. Furthermore, we status in the context of FLT3-ITD and NPM1 mutations. Among recommend screening for CEBPA mutations in NPM1mut/FLT3-ITD/ CEBPAsm cases, NPM1 mutations had no effect on prognosis FLT3wtratioo0.5 cases, as we showed that the positive prognosis of (Figure 5c). However, among NPM1 mutated cases, CEBPAsm patients harboring NPM1 mutations is negatively influenced by an seems to have prognostic influence. NPM1mut/FLT3-ITD/FLT3wtratioo0.5 additional CEBPA single mutation. cases with an additional CEBPA single mutation showed a trend to In conclusion, on the basis of differences in the frequency and worse OS compared with NPM1mut/FLT3-ITD/FLT3wtratioo0.5 cases pattern of additional cytogenetic and molecular genetic aberra- with CEBPAwt. This difference, however, did not reach statistical tions and clinical outcome, our data suggests to separate CEPBA significance because of limited case numbers and is a subject for mutated AML into 2 subsets: (1) CEBPA double-mutated AML and further studies. This finding is in contrast to the large study of Dufour (2) CEPBA single-mutated AML. In the current WHO classification of et al.32 on 663 CN-AML cases, who reported a positive influence of AML, AML with mutated CEBPA has been designated as a single CEBPA mutations in patients harboring NPM1 mutations. provisional entity among ‘AML with recurrent genetic abnormal- Regarding FLT3-ITD, we found a significant modification of the ities’. On the basis of the current knowledge supported by several prognostic effect for CEBPAsm by FLT3-ITD. Outcome of CEBPAsm studies, we propose that only CEBPAdm AML in the intermediate- patients is drastically reduced by the additional presence of risk cytogenetic group should be designated as a distinct entity in

& 2014 Macmillan Publishers Limited Leukemia (2014) 794 – 803 CEBPA mutations in AML A Fasan et al 802 the WHO classification, and should be clearly distinguished from of the French-American-British Cooperative Group. Ann Intern Med 1985; 103: CEBPAsm AML. For CEBPAsm, AML concomitant mutations clearly 620–625. have to be taken into account for improved risk stratification. 13 Arber DA, Brunning RD, Le Beau MM. Acute Myeloid Leukemia (AML) snd Related Precursor Neoplasms. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H et al. (eds). WHO Classification of Tumours of Haematopoietic and CONFLICT OF INTEREST Lymphoid Tissues. International Agency for Research on Cancer (IARC): Lyon, France, 2008, pp 110–123. CH, WK, TH and SuS have equity ownership of MLL Munich Leukemia Laboratory 14 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR et al. GmbH. AF, TA, CE, SW, FD, AK, SJ and SoS are employed by MLL Munich Leukemia Proposals for the classification of the acute leukaemias. French-American-British Laboratory GmbH. (FAB) co-operative group. Br J Haematol 1976; 33: 451–458. 15 Haferlach T, Kern W, Schoch C, Hiddemann W, Sauerland MC. Morphologic dysplasia in acute myeloid leukemia: importance of granulocytic dysplasia. J Clin ACKNOWLEDGEMENTS Oncol 2003; 21: 3004–3005. We thank all clinicians for sending samples to our laboratory for diagnostic purposes, 16 ISCN (1995). Guidelines for Cancer Cytogenetics, Supplement to: an International and for providing clinical information and follow-up data. We thank Roche System for Human Cytogenetic Nomenclature. Mitelman F, Karger S (eds). Karger: Diagnostics GmbH, for providing CEBPA oligonucleotide primer plates as part of Basel, New York, 1995 the IRON-II research study framework. In addition, we would like to thank all co- 17 Grimwade D, Hills RK, Moorman AV, Walker H, Chatters S, Goldstone AH et al. workers at the MLL (Munich Leukemia Laboratory) for bringing together many Refinement of cytogenetic classification in acute myeloid leukemia: determi- aspects in the field of leukemia diagnostics and research. In addition, we thank Elke nation of prognostic significance of rare recurring chromosomal abnormalities Roos and Dominic Rose for preparing and revising figures and submitting the among 5876 younger adult patients treated in the United Kingdom Medical manuscript. Research Council trials. Blood 2010; 116: 354–365. 18 Kern W, Voskova D, Schoch C, Hiddemann W, Schnittger S, Haferlach T. 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