Concomitant WT1 mutations predict poor prognosis in acute myeloid leukemia patients with double mutant CEBPA

Acute myeloid leukemia (AML) with double mutant CCAAT/enhancer binding protein α (CEBPA) is a new entity in the 2016 World Health Organization (WHO) classification with unique biologic features and prognostic implications. The incidence of CEBPA ranges from 7.5% to 11% in AML. CEBPA AML patients, when treated with standard chemotherapy, achieve a high complete remission (CR) rate. However, relapse occurs in 40% of patients who attain CR. This has raised the clinically relevant question whether concomitant genetic alterations influence the prognosis of CEBPA patients. Apart from GATA2, the prognostic impact of other concomitant gene mutations is largely unsettled because limited patient numbers preclude informative analyses. Given that AML is a heterogeneous disease, risk-adapted treatment may not only improve the prognosis, but also reduce toxicity from the therapy. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) in first CR is not beneficial for cytogenetically normal AML (CN-AML) patients with CEBPA . If any concomitant mutations adversely affect the clinical outcome of CEBPA patients, it will be interesting to know whether allo-HSCT should be performed for these patients. As yet, there is no data to answer this question. In this study, the aim was to identify additional mutations in CEBPA AML patients that conferred prognostic significance. Furthermore, we investigated the role of allo-HSCT in CEBPA patients with concurrent adverserisk mutations. Mutation analyses in CEBPA and 19 other relevant genes, including FLT3-ITD, FLT3-TKD, NRAS, KRAS, KIT, PTPN11, RUNX1, GATA2, MLL/PTD, ASXL1, IDH1, IDH2, TET2, DNMT3A, SF3B1, SRSF2, U2AF1, NPM1, WT1, and TP53 were performed by Sanger sequencing for patients (n=500) diagnosed from 1994 to 2007. For patients (n=256) diagnosed after 2008, Ion Torrent next generation sequencing (NGS) (Thermo Fisher Scientific, MA, USA) was performed. The WT1 mutations detected by NGS were all confirmed by Sanger sequencing. We identified 102 (13.5%) CEBPA-mutated patients from 756 patients with newly diagnosed de novo AML (Online Supplementary Table S1); 33 (4.4%) had CEBPA single mutation (CEBPA) and 69 (9.1%), CEBPA. Sixtynine CEBPA patients were found to have 109 distinct mutations (Figure 1A, Online Supplementary Table S2). All patients had a combination of one N-terminal and one C-terminal mutation. Most (53 of 56, 94.6%) of the N-terminal mutations were frame-shift mutations, while most (42 of 53, 79.2%) of the C-terminal mutations were in-frame mutations with internal tandem duplications clustered in the junction between the basic region and the leucine zipper. CEBPA patients were significantly younger and had higher hemoglobin levels at diagnosis than CEBPA and CEBPA wild-type patients. All except one CEBPA patient had intermediate-risk cytogenetics (P<0.0001) (Figure 1A). The most frequent intermediate-risk cytogenetic change was del(9) (n=4, 5.8%), and CNAML occurred in 81.2% of CEBPA patients (n=56). Fifty (72.5%) of the CEBPA patients had additional genetic alterations (Online Supplementary Table S3). Among them, 29 (58%) had one, 17 (34%) had two, 3 (6%) had three and 1 (2%) had four changes. The most common concurrent molecular event in CEBPA patients was GATA2 mutation (33.8%), followed by FLT3-ITD (14.5%), NRAS (14.5%), TET2 (13.2%), and WT1 (11.8%) mutations. GATA2was more frequently mutated in CEBPA patients than in CEBPA wild-type patients (33.8% vs. 2.8%, P<0.0001). In contrast, CEBPA patients less frequently harbored NPM1, ASXL1, IDH2, DNMT3A and RUNX1 mutations (Figure 1B). Survival analyses were restricted to 530 patients, including 62 CEBPA patients and 468 others (22 with CEBPA and 446 CEBPA wild-type), who received standard intensive chemotherapy. The CR rate was 90.2% for CEBPA patients and 72.2% for others (P=0.003). In multivariate analysis, CEBPA was an independent favorable prognostic factor for OS and DFS (RR 0.420, 95% CI 0.246-0.718, P=0.002 and RR 0.544, 95% CI 0.351-0.842, P=0.006, respectively, Online Supplementary Table S4). Of the 56 CEBPA patients who achieved first CR, 10 received allo-HSCT and 46 had postremission chemotherapy alone. The reasons for frontline alloHSCT were persistent residual leukemia cells in 4 patients, concurrent FLT3-ITD in 3 patients, initial hyperleukocytosis in 2 patients and complex cytogenetics in 1 patient. Intriguingly, the relapse rate was 45.7% in the postremission chemotherapy group and 0% in the alloHSCT group (P=0.009). DFS was significantly better in