High-throughput DNA sequencing significantly contributed to diagnosis and prognostication in patients with myelodysplastic syndromes (MDS). We determined the biological and prognostic significance of genetic aberrations in MDS. In total, 944 patients with various MDS subtypes were screened for known/putative mutations/deletions in 104 genes using targeted deep sequencing and array-based genomic hybridization. In total, 845/944 patients (89.5%) harbored at least one mutation (median, 3 per patient; range, 0–12). Forty-seven genes were significantly mutated with TET2, SF3B1, ASXL1, SRSF2, DNMT3A, and RUNX1 mutated in >10% of cases. Many mutations were associated with higher risk groups and/or blast elevation. Survival was investigated in 875 patients. By univariate analysis, 25/48 genes (resulting from 47 genes tested significantly plus PRPF8) affected survival (P<0.05). The status of 14 genes combined with conventional factors revealed a novel prognostic model (‘Model-1') separating patients into four risk groups (‘low', ‘intermediate', ‘high', ‘very high risk') with 3-year survival of 95.2, 69.3, 32.8, and 5.3% (P<0.001). Subsequently, a ‘gene-only model' (‘Model-2') was constructed based on 14 genes also yielding four significant risk groups (P<0.001). Both models were reproducible in the validation cohort (n=175 patients; P<0.001 each). Thus, large-scale genetic and molecular profiling of multiple target genes is invaluable for subclassification and prognostication in MDS patients.

We investigated ten --eleven translocation 2 (TET2) mutations in acute myeloid leukemia (AML), their correlation with other gene mutations and prognostic value. By deep-sequencing, 131 somatic TET2 mutations were identified in 87/318 (27.4%) patients. Of 87 mutated cases, 44 (50.6%) carried two mutations. TET2 mutations were concomitantly observed with mutations in NPM1, FLT3-ITD, FLT3-TKD, JAK2, RUNX1, CEBPA, CBL and KRAS. However, TET2 mutations rarely concomitantly occurred with IDH1mut or IDH2mut (2/251 or 0/184; P ¼ 0.046 and P ¼ 0.003, respectively). TET2 mutations were associated with normal karyotype AML (CN-AML) (62/206 (30.1%) CN-AML vs 20/107 (18.7%) aberrant karyotype; P ¼ 0.031), higher white blood cell count (mean 65.3 vs 40.3 Â 10 9 /l, P ¼ 0.023), lower platelet count (mean 68.6 vs 92.4 Â 10 9 /l, P ¼ 0.03) and higher age (67.5 vs 65.2 years, Po0.001). Survival analyses were restricted to de novo CN-AML patients (n ¼ 165) and showed inferior event-free survival (EFS) of TET2 mutations compared with TET2wt (median: 6.7 vs 18.7 months, P ¼ 0.009). This negative effect of TET2 mutation on EFS was particularly observed in patients p65 years (median: 8.9 months vs not reached (n.r.), P ¼ 0.027) as well as in patients of the European LeukemiaNet favorable-risk subgroup, that is, patients harboring mutated CEBPA and/or mutated NPM1 without FLT3-ITD (median: 10.3 vs 41.3 months, P ¼ 0.048). These data support a role for TET2 as an important prognostic biomarker in AML.

t(8;21)/RUNX1-RUNX1T1-positive acute myeloid leukemia (AML) is prognostically favorable; however, outcome is heterogeneous. We analyzed 139 patients with t(8;21)/RUNX1-RUNX1T1-positive AML (de novo: n=117; therapy-related: n=22) to determine frequency and prognostic impact of additional genetic abnormalities. All patients were investigated for mutations (mut) in ASXL1, FLT3, KIT, NPM1, MLL, IDH1, IDH2, KRAS, NRAS, CBL and JAK2. Sixty-nine of 139 cases (49.6%) had 1 mutation in addition to RUNX1-RUNX1T1, and 23/139 (16.5%) had ⩾2 additional mutations. Most common were KITmut (23/139; 16.5%), NRASmut (18/139; 12.9%) and ASXL1mut (16/139; 11.5%). FLT3-ITD, FLT3-TKDmut, CBLmut, KRASmut, IDH2mut and JAK2mut were found in 2.9-5.0%. Additional chromosomal abnormalities (ACAs) were found in 97/139 (69.8%). Two-year overall survival (OS) was 73.4% in 111 intensively treated patients. KITD816mut negatively impacted on OS in de novo AML (2-year OS: 59.1% vs 82.0%, P=0.03), ASXL1mut on EFS (de novo AML: 20% vs 59.1%, P=0.011; total cohort: 28.6% vs 56.7%, P=0.021). Sex chromosome loss was favorable (2-year EFS: 66.9% vs 43.0%, P=0.031), whereas +8 was adverse on EFS (2-year EFS: 26.7% vs 55.9%, P=0.02). In conclusion, t(8;21)/RUNX1-RUNX1T1-positive AML shows a high frequency of additional genetic alterations. Investigation for KITD816 and ASXL1mut combined with investigation of ACAs is recommended in t(8;21)/RUNX1-RUNX1T1-positive AML because of the prognostic significance of these parameters.

SummaryAcute myeloid leukaemia (AML) with CEBPA mutations is listed as a provisional entity in the current World Health Organization classification. A difference in clinical outcome between single-(sm) and double-mutated (dm) cases has been reported, whereupon CEBPAdm cases were shown to be associated with better overall survival (OS). The occurrence and prognostic impact of concomitant molecular mutations in addition to CEBPAdm has not been assessed until now with exception of GATA2 mutations. Here, we investigated a cohort of 95 AML CEBPAdm cases for concomitant mutations. TET2 was found to be most frequently mutated (34Á0%) gene, followed by GATA2 (21Á0%), WT1 (13Á7%), DNMT3A (9Á6%), ASXL1 (9Á5%), NRAS (8Á4%), KRAS (3Á2%), IDH1/2 (6Á3%), FLT3-internal tandem duplication (6Á3%), FLT3-tyrosine kinase domain (2Á1%), NPM1 (2Á1%), and RUNX1 (1/94). Patients harbouring additional mutations in the TET2 gene showed significantly worse OS than TET2 wild-type cases (P = 0Á035), whereas GATA2-mutated patients showed improved OS (P = 0Á032). Serial analyses were performed for 39 CEBPAdm cases with concomitant mutations. Here, we observed that CEBPA mutations present the primary pathogenetic event in the majority of cases (76Á9%). Further, a distinct gene expression profile (GEP) was confirmed for CEBPAdm versus CEBPAsm or CEBPA wild-type cases while no significant changes in GEP were observed related to additional mutations within the CEBPAdm AML.

marrow was 118 days vs 140 days for Cdkn2a null irradiated (P ¼ .009). In all four groups, the majority of deaths were due to B-cell lymphoma/leukemia. We had hypothesized that irradiation would accelerate the development of disease in at least some of the recipient animals. Although there was substantial overlap of the survival curves of genetically matched irradiated and unirradiated groups, the survival curves of the irradiated groups appeared shifted to the left as compared with the respective unirradiated groups. Using the shortest time to leukemia in the control Cdkn2a null unirradiated group for comparison (97 days) we observed that 9 of 25 mice in the Cre/TA1 Cdkn2a irradiated group developed disease prior to this time, whereas only 2 of 24 mice in the Cre/TA1 Cdkn2a unirradiated group showed early disease (P ¼ .02; see Materials and Methods for statistical discussion). This finding suggests that some mice received cells for which low-dose radiation caused mutations that cooperated with TEL-AML1 expression and Cdkn2a loss to induce leukemia. Our results provide support for a model of TEL-AML1 leukemogenesis, in which environmental exposures may both enable expansion of initially disadvantaged TEL-AML1 expressing cells and cause cooperative leukemogenic mutations.