Classification of <scp>CEBPA</scp> mutated acute myeloid leukemia by <scp>GATA2</scp> mutations
Authors: Alice Marceau‐Renaut, Soizic Guihard, Sylvie Castaigne, Hervé Dombret, Claude Preudhomme, Meyling Cheok
Journal: American Journal of Hematology (2015)

Acute myeloid leukemia (AML) with mutated CCAAT/enhancer binding protein α (CEBPA) is listed as a provisional subtype by the World Health Organization. AML with CEBPA mutations are classified into the favorable risk group due to their high remission and survival rates. CEBPA mutations are found in ∼10% of AML 1 with two main hotspots i.e., N-terminal out-of-frame mutations abolish translation of the full-length isoform and C-terminal mutations of the basic leucine zipper domain disrupting DNA binding or dimerization 2. The majority of mutations are bi-allelic or double, however, homozygous mutations have been described 3, 4. The GATA2 gene encodes a transcription factor involved in normal hematopoiesis. Recent studies associated GATA2 mutations with CEBPA double mutations and improved overall survival (OS) 5, 6. In contrast, another study failed to show any impact thereof on OS 1. In this study, we investigate the prognostic significance of GATA2 mutations in a cohort of 128 patients with CEBPA-mutated AML. Diagnostic bone marrow samples of 128 patients with CEBPA mutated AML were analyzed. Patients were enrolled between 1999 and 2012 and included in the clinical trials of the ALFA group 9801, 9802, 9803, 0701, and 0702 (ClinicalTrials.gov IDs: NCT 00931138, 00880243, 00363025, 00927498, 00932412). Children were enrolled on the ELAM02 trial (ClinicalTrials.gov ID: NCT 00149162). Studies were approved by the local Institutional Review Boards and conducted after informed consent was obtained in accordance with the Declaration of Helsinki. All patients were treated with intensive chemotherapy. Genomic DNA was analyzed for the entire coding region of CEBPA, WT1 (exon 7 and 9), and NPM1 (exon 12) using PCR and direct Sanger sequencing. FLT3 internal tandem duplications (FLT3-ITD) were detected by PCR and fragment analysis 7. We sequenced GATA2 (exons 4–6, aa291 to 466) according to Greif et al. 8. Additionally, high-quality RNA of 70 CEBPA mutated AML patients was available for gene expression analysis using Affymetrix HG U133 Plus 2.0 microarrays (Santa Clara, USA). Cumulative incidence of relapse (CIR) and OS were estimated by the Kaplan–Meier method after stratification by trials. Death in first complete remission (CR) was taken into account as competing risk. Hazard ratios (HR) are given with 95% confidence intervals (CI). CR rates and minimal residual disease (MRD) response were compared using Fisher's exact test. Patients who received bone marrow transplantation were censored at the date of transplant for CIR and OS analyses (n = 10). Comparison of mutational frequencies between CEBPA double mutated AML and CEBPA simple mutated AML was also performed using Fisher's exact test. All statistical tests were two-tailed and were performed with the Stata/IC 12.1 software (StataCorp, College Station, TX). We evaluated CEPBA mutations as simple N-terminal- (N-ter) or C-terminal- (C-ter), as double-, or as homozygous mutations. We detected all five subgroups of CEBPA mutated AML. Most of them were double-mutated (dm) cases (n = 80; 63%), 32% were simple mutated (sm) cases with either N-ter- (n = 29; 23%), or C-ter mutations (n = 12; 9%). Homozygous mutations were rare with C-ter- (n = 5; 4%) and N-ter- (n = 2; 1%) bi-allelic mutations (Fig. 1A). GATA2 mutations were detected in 29 (23%) of CEBPA mutated patients and were mainly clustered within the first ZF domain of GATA2 (Fig. 1B). Furthermore, we detected three patients with two different mutations. We confirmed that GATA2 mutations were significantly associated with CEBPA dm cases (29%, 4 of 41 sm vs. 25 of 87 dm: Fisher's exact test P = 0.022). In contrast to previous reports 5, 8, we have detected GATA2 mutations in CEBPA sm cases at a small frequency of 4% as has Green et al. 9 (Table 1). GATA2- and CEBPA mutations in AML. A: Frequency of five subgroups of CEBPA mutations within 128 cases. B: Genomic location and frequency of GATA2 mutations detected (n = 29). C: Gene expression hierarchical clustering of AML with CEBPA mutations, where gene probe sets are shown in rows (n = 276), and cases in columns (n = 70). Sample dendrogram and sample 3D-principal component plot are given with respective color code. D: Overall survival according to GATA2 mutations and CEBPA bi-allelic mutation. Presence is represented by the orange line and absence by the blue line. We evaluated the mutational status for NPM1 and FLT3-ITD, two other important mutations implicated in AML. NPM1 mutations were detected in 12 (9%) of CEBPA mutated patients. They were specifically associated with CEBPA sm cases (12 of 40 sm vs. 0 of 78 dm, P < 0.0001) and mutually exclusive of GATA2 mutations (12 of 93 GATA2 wild-type vs. 0 of 25 GATA2 mutated, P < 0.0001). Previous reports found no co-occurrence of FLT3-ITD with GATA2 mutations 5, 9. In contrast to our finding using a larger cohort of CEBPA mutated patients, we have detected three GATA2 mutated cases within 10 FLT3-ITD positive patients (30%). Similarly to Wouters et al. 10, we found that CEBPA dm AML harbored a specific gene expression profile distinct from CEBPA sm cases. However, the gene expression profile of all five AML subgroups of CEBPA AML had never been determined. Our results demonstrate that homozygous- and dm CEBPA mutated AML have a similar gene expression signature. Based on these data, we pooled the two groups as CEBPA dm AML for the subsequent prognostic analysis. Finally, we did not detect a specific gene expression signature for GATA2 mutated vs. non-mutated cases. Overall, CR was achieved in 113 patients (88%), of whom 36 relapsed (estimated CIR at 3 and 5 years, 39%). Neither age nor karyotype or gene mutations (including NPM1, FLT3, of GATA2 and type CEBPA mutation) significantly influenced CIR in multivariate analysis. Five-year OS was estimated at 58% in the whole cohort, with longer OS in cases with normal karyotype (P = 0.05) and CEBPA biallelic mutations (dm vs. sm, P = 0.03). In multivariate analysis, younger age (P = 0.020) and normal karyotype (P = 0.029) remained the only factors significantly associated with better overall survival. Of note, CEBPA bi-allelic mutations occur more frequently in younger patients. More specifically, the presence of GATA2 mutations did not affect OS in CEBPA mutated AML in general and not within CEBPA dm mutated AML neither. In conclusion, we confirmed a strong association of GATA2 mutations with CEPBA biallelic-mutations, which was not exclusive. However, we confirmed no prognostic impact of GATA2 mutations (n = 25) compared to wild-type (n = 62) in our cohort of CEBPA dm AML 8, 9 (22 vs. 58 and 10 vs. 16) with a trend similar of better outcome in CEBPA dm with GATA2 mutations compared to wild-type 5, 6 (18 vs. 62). Furthermore, our study provided first evidence that homozygous CEPBA mutations have a similar gene expression signature as CEPBA dm and thus may be considered as equivalent. Authors acknowledge the contribution of Nicolas Duployez PharmD, Olivier Nibourel PharmD PhD, Nathalie Helevaut, Céline Rodriguez, Karine Celli-Lebras, Céline Berthon MD PhD, Guy Leverger MD, Nicolas Boissel MD PhD, Christine Terré PharmD, Xavier Thomas MD, and Aline Renneville PharmD PhD. Alice Marceau-Renaut,1,2 Soizic Guihard,1 Sylvie Castaigne,3 Hervé Dombret,4 Claude Preudhomme,1,2 Meyling Cheok1* 1U837, INSERM, Lille, France; 2Hematology Laboratory, Biology and Pathology Center, Lille University Hospital, Lille, France; 3Hematology Department, Versailles Hospital, Versailles, France; 4ALFA Cooperative Group, Saint-Louis Hospital, Paris, France Contract grant sponsors: Institut National du Cancer (INCa)-DHOS Recherche Translationnelle 2010, SIRIC ONCOLille.