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Acute Leukemias

The role of different genetic subtypes of CEBPA mutated AML

Abstract

The prognostic impact of mutations in the CCAAT/enhancer binding protein α (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% P<0.001). 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=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.

Introduction

The CCAAT/enhancer binding protein α (CEBPA) has gained increasing attention as a favorable prognostic factor in acute myeloid leukemia (AML). CEBPA is a transcription factor with critical roles in tissue-specific gene expression and proliferation arrest. In the hematopoietic system, CEBPA expression is restricted to myelomonocytic cells and is specifically upregulated during granulocyte differentiation.1

CEBPA is an intronless gene that maps to chromosome band 19q13.1 and has a GC-rich coding region. It belongs to the basic leucin zipper (b-ZIP) family of transcription factors and consists of highly homologous C-terminal DNA-binding (basic region) and dimerization (leucin zipper) motifs and two less conserved N-terminal transactivation domains.2

Mutations in the CEBPA gene have been described in 15% of all AML patients.3, 4 Mutations can occur across the whole coding region. However, studies revealed a clustering in two main hot spots. N-terminal frame-shift mutations between the major translational start codon and a second ATG in the same open reading frame lead to a premature stop of translation of the wild-type (wt) p42 CEBPA protein while conserving the translation of a short p30 isoform which inhibits the function of the full-length protein by a dominant negative mechanism.3 C-terminal mutations are generally in-frame insertions/deletions in the DNA-binding or basic leucin zipper domains that disrupt binding to DNA or dimerization.5

Studies revealed three different CEBPA mutant patterns in AML patients: (1) AML carrying one mutation on one allele (single-mutated CEPBA, CEBPAsm), accounting for 50% of CEPBA mutated AML, still expresses wt CEBPA transcribed from the second allele; (2) AML with two CEPBA mutations (double-mutated CEPBA, CEBPAdm) typically shows an N-terminal and a basic leucin zipper gene mutation. As these mutations are usually biallelic, no wt CEBPA is expressed in these cases.6 (3) AML carrying a homozygous CEBPA mutation due to loss of heterozygosity (LOH) also expresses no wt CEBPA.7

In AML, CEBPA mutations previously have been reported to be prognostically favorable in the intermediate risk karyotype.8, 9 They do not occur together with recurrent fusion genes like PML-RARA, CBFB-MYH11 or RUNX1-RUNX1T1.5 Because of this singular biologic and prognostic pattern, CEBPA mutations recently have been included in the WHO classification as a provisional entity. However, more recent data have shown that a favorable prognosis in CEBPA-mutated AML is restricted to those cases that present with CEBPAdm, do not carry cytogenetic aberrations and do not harbor an FLT3-ITD.10 Recently, we have performed a detailed analysis of collaborating mutations in CEBPAdm AML and discovered that the favorable prognosis of CEBPAdm AML is influenced by the presence of additional mutations.11 To further evaluate the role of different types of CEBPA mutations in the context of other molecular mutations and cytogenetic aberrations we have analyzed CEBPA mutations in a cohort of 2296 adult AML cases.

This study confirms the favorable outcome of patients with CEPBAdm compared with AML with CEPBAsm. Further, differences in the frequency and pattern of additional cytogenetic and molecular genetic aberrations and clinical outcome were observed between these two CEPBA mutated subsets.

Materials and methods

Patients

Bone marrow samples (n=2016) or blood samples (n=280) from 2296 AML patients were screened for CEBPA mutations. All 2296 patient samples were referred to our laboratory for first diagnosis of AML between August 2005 and July 2012. AML was diagnosed according to the French–American–British and WHO classifications.2, 12, 13 The patients were selected according to karyotype, excluding cases with t(15;17)/PML-RARA, t(8;21)/RUNX1-RUNX1T1, inv(16)/t(16;16)/CBFB-MYH11, inv(3)/t(3;3)/EVI1, t(6;9)/DEK-CAN, 11(q23)/MLL and complex karyotype.

One thousand fifty two patients were female and 1244 male. Median age was 68.4 years (range 15.7–100.4 years). Bone marrow blast percentages ranged from 20.0 to 99.5% (median 67.5%) in 1931 patients with non-FAB M6 AML. The 81 patients with AML M6 subtype had bone marrow blast percentages below 20% (0.5–19.5%, median 10.5%), as characteristic for the AML FAB M6 subtype. In 274 patients only peripheral blood was available. Peripheral blood blast percentages in these patients ranged from 20–100% (median 71.0%). Data on other molecular markers was available in: NPM1: n=2296, FLT3-ITD: n=2296, FLT3-TKD: n=2115, MLL-PTD: n=2287, RUNX1: n=1884, ASXL1: n=1402, IDH1 and IDH2: n=1515, WT1: n=2112, TET2: n=452 and GATA2: n=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 design adhered to the tenets of the Declaration of Helsinki and was approved by the institutional review board before its initiation.

Cytomorphology, cytogenetics, immunophenotyping

Cytomorphologic assessment was based on May–Grünwald–Giemsa stains, myeloperoxidase reaction and non-specific esterase using alpha-naphtyl-acetate as described before, and was performed according to the criteria defined in the FAB and the WHO classification.13, 14, 15 Cytogenetic studies were performed after short-term culture. Karyotypes, analyzed after G-banding, were described according to the International System for Human Cytogenetic Nomenclature.16 Prognostic classification into ‘favorable’, ‘intermediate’ and ‘adverse’ groups was performed according to the refined MRC (Medical Research Counsil) classification.17 Cytogenetic results were available for all patients in this study. Immunophenotyping was performed as described previously in a subset of 1227 cases.18

Molecular analysis

Isolation of mononuclear cells, mRNA extraction and random primed cDNA synthesis was performed as described previously.19 The entire CEBPA coding sequence was amplified by PCR in four overlapping fragments using the following primers (Ensembl Transcript ID ENS00000498907): Frag1—F 5′-IndexTermTCGCCATGCCGGGAGAACTCTAAC-3′, Frag1—R 5′-IndexTermGTCCAGGTAGCCGGCG-3′ (amino acid (aa) 1–103), Frag2—F 5′-IndexTermCTTCAACGACGAGTTCCTGGCCGA-3′, Frag2-R 5′-IndexTermAGCTGCTTGGCTTCATCCTCCT-3′ (aa 79–176), Frag3—F 5′-IndexTermCCGCTGGTGATCAAGCAGGA-3′, Frag3—R 5′-IndexTermCCGGTACTCGTTGCTGTTCT-3′ (aa 173–277), Frag4—F 5′-IndexTermGAGTGGCGGCAG CGGCGCGGGCA-3′, Frag4—R 5′-IndexTermCACGGCTCGGGCAAGCCTCGAGAT-3′ (aa 255–359). The PCR was carried out in a 50 μl reaction volume. PCR assays for fragments 1 and 2 contained 0.25 μM of both forward and reverse primers, 1.5 M GC-Rich resolution solution, 10 μl 5 × GC-Rich PCR reaction buffer with dimethyl sulfoxide and 2 U GC-Rich system enzyme mix (Roche Diagnostics GmbH, Penzberg, Germany) Each 50 μl-reaction contained 1 μl of cDNA or DNA. PCR reactions for fragments 3 and 4 contained 25 μl Qiagen Taq PCR master mix (Qiagen, Hilden, Germany), 2.5 μl dimethyl sulfoxide and 0.125 μl of each forward and reverse primer. Amplification for fragments 1, 2 and 4 was performed with 40 cycles using 60 °C annealing temperature. Fragment 3 was amplified with touchdown PCR with 27 cycles using 60 and 72 °C annealing temperatures. Products were analyzed by WAVE denaturing high-performance liquid chromatography. All samples were also screened for homozygous mutations by mixing PCR products from patient samples and wt controls. All samples with an abnormal chromatogram were subsequently further characterized by direct Sanger sequencing.

Additionally, 849/2296 samples were analyzed by a sensitive next-generation amplicon deep-sequencing assay (454 Life Sciences, Branford, CT, USA).20, 21 For deep-sequencing, CEBPA-oligonucleotide primer plates were provided as part of the IRON-II study (Roche Diagnostics GmbH).22

Next generation sequencing data was analyzed using both the GS variant analyzer software 2.5.3 (454 Life Sciences) and Sequence Pilot version 3.5.2 (JSI Medical Systems, Kippenheim, Germany).

Analyses for mutations in NPM1, FLT3, MLL-PTD, NRAS, IDH1 IDH2, ASXL1 RUNX1, TET2 and GATA2 were described previously.23, 24, 25, 26, 27, 28, 29

Statistics

Survival curves were calculated for overall survival (OS) and event-free survival (EFS) according to Kaplan–Meier and compared using the two-sided log rank test. OS was the time from diagnosis to death or the last follow-up. EFS was the time from diagnosis to treatment failure, relapse, death or the last follow-up in complete remission. Relapse was defined according to Cheson et al.30 Cox regression analysis was performed for OS and EFS with different parameters as covariates. Median follow-up was calculated using the Kaplan–Meier analysis. Parameters which were significant in univariate analyses were included into multivariate analyses. Dichotomous variables were compared between different groups using the χ2 test and continuous variables by Student’s t-test. Results were considered significant at P<0.05. All reported P-values are two-sided. No adjustments for multiple comparisons were performed. SPSS software version 19.0.1 (IBM Corporation, Armonk, NY, USA) was used for statistical analysis.

Results

Characterization of CEBPA mutations

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 (p.Thr230Thr and p.His195_Pro196dup) was detected as the only change. A total of 13 cases had synonymous coding changes, leaving 244 patients (10.6%) with somatic mutations (Figure 1).

Figure 1
figure1

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.

The majority (201/358; 56.1%) of mutations comprised frame-shift insertions or deletions. 81/358 (23.5%) were in-frame insertions or deletions, 46/358 (12.8%) were missense mutations, 26/358 (7.2%) were nonsense mutations and one was a splice-site mutation (0.3%) (Figure 1).

One hundred forty cases (57.4%) were CEBPAsm with 21 cases (15.0%) showing loss of the wt allele (equivalent to ‘LOH’ of this 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 N-terminal mutation (aa 1–120), 38 patients (27.1%) had C-terminal changes (aa 278–385) and 45 patients (32.1%) had mutations between these two regions (aa 121–277). The majority of CEBPAdm patients (78/104; 75%) showed a combination of mutations in the N-terminal and C-terminal hotspots, mostly combining an N-terminal frame-shift and a C-terminal in-frame mutation (60/104; 57.7%) (Figure 2, Table 1). Mutations other than the N-terminal frame-shift and C-terminal in-frame were more frequent in the CEBPAsm than in the CEBPAdm group (79/140 mutations in CEBPAsm, 56.4% vs 39/208 mutations in CEBPAdm, 18.8%; P<0.001).

Figure 2
figure2

Circos plot showing the combinations of CEBPAdm cases. The left part of the circle indicates CEBPA mutation 1 and the right part indicates CEBPA mutation 2. Each part is divided in C-terminal-, middle- and N-terminal mutation and further subdivided into specific mutation subtypes (frameshift, missense, nonsense, inframe). The width of the arches indicates the percentage of samples with the respective combination of CEBPA mutations. Table 2 depicts the detailed case numbers.

Table 1 Summary of CEBPA mutations detected in 2296 AML cases

Buccal swabs or samples taken in the complete remission stage were available in 56/104 CEBPAdm cases. In 2/56 cases CEBPA germline mutations were detected (case 1: p.Tyr181* and case 2 (with three diagnostic mutations): p.His18Gln and p.Gly53Alafs*107). In both cases, the mutational burden of one CEBPA mutation declined and disappeared during therapy while the other one or two, respectively, were detected in each examination with an unchanged mutational burden of 50%.

Clinical parameters

Comparison of the clinical and laboratory features of CEBPAmut and CEBPAwt cases is summarized in Table 2. Clinical parameters of CEBPA LOH patients did not differ from CEBPAsm cases (data not shown), and thus CEBPA LOH were grouped together with CEBPAsm cases.

Table 2 Demographics, clinical and molecular characteristics of AML patients according to CEBPA mutations

With regard to age, CEBPAdm patients were significantly younger (median age 56.3 years, range 15.7–87.6 years) compared with CEBPAwt (median age 65.4 years, range 16.8–100.4 years; P<0.001) and CEBPAsm cases (median age 65.8 years, range 20.4–87.0 years; P<0.001). CEBPAdm patients had significantly lower platelet counts compared with CEBPAwt (median 34.0 vs 66.0 × 109/l; P<0.001) and CEBPAsm cases (median 56.0 × 109/l; P<0.001). Furthermore, CEBPAdm cases had significantly higher bone marrow blast percentages compared with CEBPAwt cases (mean 65.0% vs 56.5%; P=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 (P<0.001). 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%; P<0.001), CD15 (49±27% vs 42±26%; P=0.007), CD33 (73±23% vs 63±27%; P<0.001), CD34 (47±31% vs 30±26%; P<0.001), CD65 (45±28% vs 39±25%; P=0.02) and HLA-DR (41±24% vs 34±22%; P=0.002) compared with CEBPAwt cases. Furthermore, we observed a significantly lower expression of CD14 in CEBPAmut cases (13±12% vs 22±18%; P<0.001) compared with CEBPAwt cases. Also, the expression of CD7 (59±27% vs 35±30%; P<0.001), CD15 (57±27% vs 45±27%; P=0.047), CD34 (63±24% vs 36±30%; P<0.001), HLA-DR (50±21% vs 37±24%; P=0.003) and CD65 (52±30% vs 41±26%; P=0.035) was significantly higher in CEBPAdm cases compared with CEBPAsm cases.

Correlation of CEBPA mutation status to cytogenetics

Analysis of cytogenetic results revealed cytogenetically normal AML (CN-AML) in 70.5% of CEBPAmut cases. Within the subset of CN-AML 94/1742 (4.0%) and 78/1742 (3.4%) had CEBPAsm and CEBPAdm, respectively. A total of 72/244 CEBPA mutated patients (29.5%) had cytogenetic aberrations (intermediate: n=60, unfavorable n=12). A similar proportion of intermediate-risk abnormal karyotype patients were CEBPAsm (38/432, 8.8%) and CEBPAdm (22/432, 5.1%). The most frequent intermediate-risk aberration was trisomy 8 (11/60, 18.3%). Interestingly, all 11 patients with trisomy 8 were CEBPAsm. Furthermore, of 122 patients with adverse karyotype, eight (6.5%) were CEBPAsm and four (3.3%) were CEBPAdm (Table 2, Figure 3).

Figure 3
figure3

Alignment of underlying karyotype of 244 CEBPAmut patients. Each column represents one of the 244 analyzed samples. The eight cytogenetic subgroups are shown by colored bars. A red bar indicates the respective cytogenetic aberration. CEBPAsm cases are indicated in dark gray, CEBPAdm cases in light gray.

Correlation of the CEBPA mutation status to other molecular mutations

To determine, whether CEBPA mutations correlate with other mutations frequently reported in AML, we additionally analyzed FLT3-ITD, MLL-PTD and NPM1, FLT3-TKD, ASXL1, GATA2, IDH1, IDH2, RUNX1, TET2 and WT1 mutation status. Cases with low mutation burden of FLT3-ITD with mutation/wt ratio <0.5 were grouped together with the FLT3-ITD negative cases, as it has been shown that only an FLT3-ITD ratio 0.5 has a significant adverse prognostic impact.34 Thus, FLT3-ITD negative patients and patients with FLT3-ITD ratio <0.5 are combined and designated as FLT3-ITD/FLT3wtratio<0.5. Generally, concurrent mutations were seen less frequently in CEBPAdm than in CEBPAsm AML cases (72/104, 69.2% vs 124/140, 88.6%; P<0.001) (Table 2, Figure 4). Of 124 CEBPAsm patients with additional molecular mutations, 59 (47.6%) had one, 54 (43.5%) had two and 11 patients (8.9%) had three additional molecular mutations. Of 72 CEBPAdm patients with additional molecular mutations, 49 (68.0%) had one, 20 patients (27.8%) had two, two patients (2.8%) had three and one patient (1.4%) had four additional mutations.

Figure 4
figure4

Alignment of gene mutations for 244 CEBPAmut patients. 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 indicates that no data were available. CEBPAsm cases are indicated in dark gray, CEBPAdm cases in light gray.

WT1 (14/104, 13.6% vs 2/140, 1.5%; P<0.001) and GATA2 (21/102, 9.1% vs 2/130, 0.9%; P<0.001) were significantly more frequently mutated in CEBPAdm cases compared with CEBPAsm cases. In contrast, NPM1 mutations (44/140, 31.4% vs 2/104, 1.9%; P<0.001), FLT3-ITD (14/140, 10.0% vs 1/104, 1.0%; P=0.003), ASXL1 mutations (41/140, 30.6% vs 14/104, 13.7%; P=0.003) and RUNX1 mutations (25/140, 18.0% vs 6/104, 5.8%; P=0.006) were seen more frequently in CEBPAsm cases compared with CEBPAdm cases.

Comparing CEBPAsm and CEBPAwt AML, the total number of mutations was comparable in both groups (Table 2). The frequency of ASXL1 mutations was higher in the CEBPAsm group compared with CEBPAwt cases (41/134, 30.6% vs 210/1167, 18.0%; P=0.001).

Prognostic significance of CEBPA mutations

Survival analysis was restricted to 1117 patients with intermediate-risk karyotype having received intensive therapy. Numbers of CEBPAmut cases in the adverse karyotype risk group were too small for valid analysis. CEBPAdm patients had significantly longer OS compared with CEBPAsm and CEBPAwt cases (median n.r. vs 26.2 months and 45.6 months; P<0.001 and P=0.002, respectively) (Figure 5a). EFS was also significantly longer for CEBPAdm patients compared with CEBPAsm and CEBPAwt cases (median 45.9 months vs 10.6 months and 15.9 months; P=0.008 and P=0.012, respectively) (Figure 5a). Outcome of CEBPA LOH patients did not differ from CEBPAsm and CEBPAwt cases (Supplementary Figure 1). Therefore, we did not subdivide CEBPA LOH and CEBPAsm cases for further survival analysis. Interestingly, OS of CEBPAsm cases without CEBPA LOH patients was significantly worse compared with CEBPAwt cases (median 23.6 months vs 45.6 months; P=0.024).

Figure 5
figure5

(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/FLT3wtratio<0.5 (gray), CEBPAsm NPM1mut/FLT3-ITD/FLT3wtratio<0.5 (black), CEBPAsm FLT3-ITDpos (blue), CEBPAwt NPM1wt/FLT3-ITD/FLT3wtratio<0.5 (red), CEBPAwt NPM1mut/FLT3-ITD/FLT3wtratio<0.5 (purple) and CEBPAwt FLT3-ITDpos (green).

Prognostic impact of CEBPA mutation type

The impact of CEBPA mutation location was also investigated. Only patients with mutations predicted to cause p30 isoform translation and/or disruption or loss of the C-terminal basic leucin zipper domain were included in this analysis. OS for N-terminal CEBPAsm patients tended to be inferior compared with C-terminal CEBPAsm patients (median 19.2 months vs median n.r.; P=0.089) and was significantly worse compared with CEBPAdm patients (median 19.2 months vs n.r.; P<0.001) (Figure 5b). Regarding EFS, N-terminal CEBPAsm patients had significantly worse prognosis compared with CEBPAdm patients (median 9.7 months vs 45.9 months; P=0.009) (Figure 5b).

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/FLT3wtratio<0.5, NPM1mut/FLT3-ITD/FLT3wtratio<0.5 and FLT3-ITDpos. Strikingly, patients with CEBPAsm and additional FLT3-ITD had significantly impaired OS and EFS (median 2.9 months and 2.0 months, respectively; P<0.001) compared with all other groups (Figure 5c). There was evidence that the presence of CEBPAsm influenced OS of NPM1mut/FLT3-ITD/FLT3wtratio<0.5 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/FLT3wtratio<0.5 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/FLT3wtratio<0.5 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=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 impact on CEBPA mutated AML (data not shown).

Univariate and multivariate analysis

In univariate Cox regression analysis of 1117 intensively treated AML patients, cases with CEBPAdm (P=0.026) and NPM1 mutations (P=0.004) were associated with longer EFS. Higher age (P<0.001), higher WBC count (P<0.001), higher bone marrow blasts count (P<0.001), FLT3-ITD/wt ratio 0.5 (P<0.001), MLL-PTD (P=0.016), ASXL1 mutations (P=0.002) and RUNX1 mutations (P=0.043) were associated with worse EFS. In multivariate analysis, age (P<0.001), WBC count (P<0.001), FLT3-ITD/wt ratio <0.5 (P=0.001) and NPM1 mutations (P=0.024) had independent relevance for EFS. Investigating OS in univariate analysis, CEBPAdm (P=0.002) and NPM1 mutations (P<0.001) were associated with better prognosis, whereas age (P<0.001), male sex (P=0.015), WBC count (P<0.001), the FLT3-ITD ratio 0.5 (P<0.001), MLL-PTD (P=0.008), ASXL1 mutations (P<0.001) and RUNX1 mutations (P=0.007) were associated with inferior outcome. In multivariate analysis, age (P<0.001), FLT3-ITD/wt ratio 0.5 (P<0.001), CEBPAdm (P=0.020) and NPM1 mutations (P=0.022) had independent prognostic impact (Table 3).

Table 3 Influence of different biological and leukemia-associated parameters on OS and EFS in 1117 AML patients in uni- and multivariate analysis

Discussion

In this large cohort of 2296 AML patients the incidence, distribution and outcome of CEBPA mutations were similar to that reported by others.3, 8, 35, 36, 37 Overall, 10.6% of patients had one or more CEBPA mutations. Two mutations were detected in 4.5% of all patients investigated and the favorable outcome was restricted to these CEBPAdm patients. We also were able to further confirm the value of CEBPAdm as an independent favorable prognostic factor by multivariate analysis, which is in line with previous studies.6, 10, 38, 39

Concurrent mutations were significantly less frequent in CEBPAdm compared with CEBPAsm patients. FLT3-ITD and NPM1 mutations were even almost mutually exclusive of CEBPAdm, a fact, which is consistent with previously published data.31, 39 Conversely, there were also mutations that were associated with CEBPAdm and almost mutually exclusive of CEBPAsm, namely GATA2 and WT1.27, 40, 41 Interestingly, TET2 mutations were found with a frequency of 17.5% in CEBPAdm and CEBPAsm cases, which is in line with the overall mutation rate of TET2 in AML of about 20%.28, 42

However, thus far, detailed investigation of CEBPA mutations in the context of underlying karyotype are rare and most studies restricted their cohorts on CN-AML. One study described 12 CEBPA mutations in a cohort of 277 patients with intermediate-risk karyotype. Eight patients showed normal karyotype, whereas 4 of these 12 patients had chromosomal aberrations [del(9q), n=2; del(11q), n=1; del(12p), n=1].36 Although in our cohort the majority of CEBPA mutated cases were cytogenetically normal, 29.5% of CEBPA mutated cases showed an aberrant karyotype. Trisomy 8 was the most common intermediate risk alteration with an incidence of 8.4% and, interestingly, all 11 CEBPA mutated cases harboring trisomy 8 were CEBPAsm. Trisomy 8 has recently been described to be associated with the concomitant presence of adverse molecular markers, particularly ASXL1 and RUNX1 mutations or both, and to have inferior outcome compared with CN-AML patients.43 In our cohort, we were not able to show that the presence of trisomy 8 further impairs the unfavorable prognosis of CEBPAsm patients, but this may be explained by the small number of CEBPAsm cases with concurrent trisomy 8.

Owing to the large number of cases included in our study, we were able to evaluate the prognostic impact of CEBPA mutational status in the context of FLT3-ITD and NPM1 mutations. Among CEBPAsm cases, NPM1 mutations had no effect on prognosis (Figure 5c). However, among NPM1 mutated cases, CEBPAsm seems to have prognostic influence. NPM1mut/FLT3-ITD/FLT3wtratio<0.5 cases with an additional CEBPA single mutation showed a trend to worse OS compared with NPM1mut/FLT3-ITD/FLT3wtratio<0.5 cases with CEBPAwt. This difference, however, did not reach statistical significance because of limited case numbers and is a subject for further studies. This finding is in contrast to the large study of Dufour et al.32 on 663 CN-AML cases, who reported a positive influence of single CEBPA mutations in patients harboring NPM1 mutations.

Regarding FLT3-ITD, we found a significant modification of the prognostic effect for CEBPAsm by FLT3-ITD. Outcome of CEBPAsm patients is drastically reduced by the additional presence of FLT3-ITD (Figure 5c). This is in line with the findings of Taskesen et al.39 who showed that prognosis of CEBPAsm cases (n=60) is impaired by concurrent FLT3-ITD in a cohort of 1182 CN-AML cases. In contrast, Dufour et al. found no prognostic effect of FLT3-ITD in CEBPAsm patients (n=28) in a cohort of 663 CN-AML cases.32 Furthermore, Green et al.31 investigated CEBPA mutations in a cohort of 1427 adult AML cases. They showed that the presence of FLT3-ITD was associated with a significantly worse outcome, irrespective of the CEBPA mutation status.

The positive impact on prognosis of CEBPAdm cases is also modified by the presence of additional mutations. TET2 mutations significantly impair the outcome of CEBPAdm patients, whereas patients harboring an additional GATA2 mutation show significantly better OS. These modifying effects are well substantiated by now and based on a growing amount of studies.11, 27, 41

Expression profiling of a previous study had suggested that C-terminal CEBPAsm patients were less distinct from CEBPAdm patients than N-terminal CEBPAsm cases.6 Therefore, we investigated whether the mutation location had any influence on outcome in CEBPAsm cases. We found that N-terminal CEBPAsm cases tended to have a poorer OS compared with C-terminal CEBPAsm cases. However, this difference in outcome was not significant and has to be validated in further studies. Interestingly, only N-terminal CEBPAsm and not C-terminal CEBPAsm cases showed a significant impaired OS compared with CEBPAdm patients. This would support the theory of a similar gene expression profile of C-terminal CEBPAsm AML and CEBPAdm AML.

On the basis of our data, we recommend screening for CEBPA mutations for all intermediate-risk karyotype AML and not restrict it to the CN group only. Screening of CEBPA mutations should be accompanied by FLT3-ITD quantification to identify the extremely unfavorable subgroup of CEBPAsm/FLT3-ITDpos. Furthermore, we recommend screening for CEBPA mutations in NPM1mut/FLT3-ITD/FLT3wtratio<0.5 cases, as we showed that the positive prognosis of patients harboring NPM1 mutations is negatively influenced by an additional CEBPA single mutation.

In conclusion, on the basis of differences in the frequency and pattern of additional cytogenetic and molecular genetic aberrations and clinical outcome, our data suggests to separate CEPBA mutated AML into 2 subsets: (1) CEBPA double-mutated AML and (2) CEPBA single-mutated AML. In the current WHO classification of AML, AML with mutated CEBPA has been designated as a provisional entity among ‘AML with recurrent genetic abnormalities’. On the basis of the current knowledge supported by several studies, we propose that only CEBPAdm AML in the intermediate-risk cytogenetic group should be designated as a distinct entity in the WHO classification, and should be clearly distinguished from CEBPAsm AML. For CEBPAsm, AML concomitant mutations clearly have to be taken into account for improved risk stratification.

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Acknowledgements

We thank all clinicians for sending samples to our laboratory for diagnostic purposes, and for providing clinical information and follow-up data. We thank Roche Diagnostics GmbH, for providing CEBPA oligonucleotide primer plates as part of the IRON-II research study framework. In addition, we would like to thank all co-workers at the MLL (Munich Leukemia Laboratory) for bringing together many aspects in the field of leukemia diagnostics and research. In addition, we thank Elke Roos and Dominic Rose for preparing and revising figures and submitting the manuscript.

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Correspondence to S Schnittger.

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CH, WK, TH and SuS have equity ownership of MLL Munich Leukemia Laboratory GmbH. AF, TA, CE, SW, FD, AK, SJ and SoS are employed by MLL Munich Leukemia Laboratory GmbH.

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AF investigated the mutation status of CEBPA, analyzed the data and wrote the manuscript; CH was responsible for chromosome analysis; TA collected and documented clinical data and compiled statistical analyses; VG, CE, SW, FD, AK, SJ and SoS performed molecular analyses; WK was responsible for immunophenotyping and was involved in statistical analyses; TH was responsible for cytomorphologic analysis and was involved in the collection of clinical data. SS was the principle investigator of the study and wrote the manuscript. All authors read and contributed to the final version of the manuscript.

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Fasan, A., Haferlach, C., Alpermann, T. et al. The role of different genetic subtypes of CEBPA mutated AML. Leukemia 28, 794–803 (2014). https://doi.org/10.1038/leu.2013.273

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Keywords

  • CEBPA
  • AML
  • mutation
  • pronosis

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