Clinical Relevance of Concomitant Gene Mutations in CEBPA Double-Mutated Acute Myeloid Leukemia.

CEBPA mutations occur in 5–14% of patients with acute myeloid leukemia (AML). A difference in clinical outcome between single- (sm) and double-mutated (dm) cases has been reported, whereupon dm cases were shown to be associated with longer overall (OS) and event-free survival (EFS).

1. Determine the frequency and clinical impact of CEBPA sm and dm in a large AML cohort. 2. Evaluate the spectrum of additional molecular mutations in CEBPA dm AML and their prognostic influence.

First, we investigated CEBPA mutations in a cohort of 997 AML cases (462 female, 535 male; median age: 66.8 years) by 454 deep-sequencing (454 Life Sciences, Branford, CT). The cohort included: t(15;17)(q22;q12) n=33; t(8;21)(q22;q22) n=39; inv(16)(p13q22) n=31; normal karyotype (NK) n=447; complex karyotype (CK) (≥4 abnormalities) n=116; other abnormalities n=331. Second, we investigated an additional cohort of 111 AML dm CEBPA cases for mutations in ASXL1, DNMT3A, FLT3-ITD, FLT3-TKD, GATA2, IDH1/2, KRAS, MLL-PTD, NPM1, NRAS, RUNX1, TET2, TP53, and WT1 using 454 sequencing, Sanger sequencing, conventional PCR and melting curve analyses. This cohort was composed of 60 female and 51 male cases; median age: 62.3 years; 76 cases showed NK, 19 aberrant karyotype (n=4 n.a.). Survival data was available in 90/111 (81.1%) cases.

1. In total, CEBPA mutations were detected in 75/997 (7.5%) of cases (t(15;17)(q22;q12) n=2/33; NK n=52/447; CK n=1/116; other abnormalities n=20/331). Of the 75 patients with CEPBA mutations 31 (41.3%) were sm, while 44 (58.7%) were dm. Patients with dm CEPBA showed better outcome compared to sm cases (OS at 3 yrs: 78.9% vs 38.5%, P=0.014; EFS after 3 yrs: 53.9% vs 36.6%, P=0.108). OS and EFS of CEBPA sm cases were comparable to CEPBA wt cases (OS at 3 yrs: 38.5% and 43.6%, P=0.689, EFS at 3 yrs: 36.6% and 29.4%, P=0.678). OS of CEBPA dm cases was comparable to patients with t(15;17)(q22;q12) (OS after 3 yrs: 78.9% vs 86.1%, P=0.597).

2. In the cohort of 111 patients we detected 227 CEBPA mutations. In 106 (95.5%) cases two mutations, and in 5 (4.5%) cases three mutations were detected. The median mutation load was 42% (range: 2–98%). The majority of mutations were frame-shift (n=135) and in-frame (n=66). Further, missense (n=19) and nonsense (n=7) mutations were observed. Most cases showed one N- and one C-terminal mutation (92/111, 82.8%), 10 (9.0%) cases harbored two N-terminal mutations, and 4 (3.6%) cases showed two C-terminal mutations. In addition, two cases showed one N- and two C-terminal mutations, two cases two N- and one C-terminal mutations, and one case harbored three N-terminal mutations.

In 92/111 (82.9%) cases we observed at least one additional mutation (mean: 1.6 mutations; range: 1–4): TET2 39/109 (35.8%), ASXL1 20/111 (18.0%), GATA2 20/111 (18.0%), WT1 14/111 (12.6%), DNMT3A 11/109 (10.1%), IDH1/2 9/111 (8.1%) (IDH1 n=2, IDH2 n=7), NRAS 9/111 (8.1%), RUNX1 7/111 (6.3%), FLT3-ITD 7/111 (6.3%), KRAS 4/109 (3.7%), NPM1 3/111 (2.7%), FLT3-TKD 2/110 (1.8%), MLL-PTD 1/111 (1.0%), and TP53 1/110 (1.0%). With respect to clinical outcome we observed no differences in OS for concomitant mutations in DNMT3A, FLT3-ITD, IDH1/2, NRAS, TET2 and WT1. Cases with additional GATA2 mutations showed longer survival than wt cases (OS at 3 yrs: 100% versus 73.4%, P=0.026, EFS at 3 yrs: 67.5% versus 48.5%, P=0.137). In contrast, cases harboring additional ASXL1 or RUNX1 mutations were associated with worse outcome (ASXL1: OS at 3 yrs: 32.8% versus 85.7%, P<0.001, EFS at 3 yrs: 0% versus 57.9%, P=0.002; RUNX1: OS at 3 yrs: 0% versus 81.8%, P=0.001, EFS at 3 yeaers: 0% versus 53.8%, P=0.003). Since mutations in ASXL1 and RUNX1 frequently occurred concomitantly, they were grouped together (n=21). When then separating cases into (1) GATA2 mut, (2) GATA2 wt, ASXL1wt, RUNX1wt and (3) ASXL1mut and/or RUNX1mut we observed 3 distinct survival curves (OS at 3 yrs: 100% vs 81.2% vs 32.8, P<0.001, EFS at 3 yrs: 67.5% and 55.3% and 0%, P=0.005). No statistical analysis was performed for FLT3-TKD, KRAS, MLL-PTD, NPM1, TP53 due to the low number of mutations.

1. In CEBPA dm cases a high frequency of concomitant mutations (82.9%) was observed. 2. Most common mutated genes were TET2 (35.8%), ASXL1 (18.0%), GATA2 (18.0%), WT1 (12.6%), DNMT3A (10.1%), and RUNX1 (6.3%). 3. In CEBPA dm cases GATA2 mutations were associated with longer OS, whereas OS was poor in ASXL1 and/or RUNX1 mutated cases.

Grossmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Stopp:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.