The t(8;21) translocation is one of the most frequent cytogenetic abnormalities in acute myeloid leukaemia (AML) and results in the RUNX1/RUNX1T1 rearrangement. Despite the causative role of the RUNX1/RUNX1T1 fusion gene in leukaemia initiation, additional genetic lesions are required for disease development. Here we identify recurring ZBTB7A mutations in 23% (13/56) of AML t(8;21) patients, including missense and truncating mutations resulting in alteration or loss of the C-terminal zinc-finger domain of ZBTB7A. The transcription factor ZBTB7A is important for haematopoietic lineage fate decisions and for regulation of glycolysis. On a functional level, we show that ZBTB7A mutations disrupt the transcriptional repressor potential and the anti-proliferative effect of ZBTB7A. The specific association of ZBTB7A mutations with t(8;21) rearranged AML points towards leukaemogenic cooperativity between mutant ZBTB7A and the RUNX1/RUNX1T1 fusion.

The fusion genes CBFB/MYH11 and RUNX1/RUNX1T1 block differentiation through disruption of the core binding factor (CBF) complex and are found in 10-15% of adult de novo acute myeloid leukemia (AML) cases. This AML subtype is associated with a favorable prognosis; however, nearly half of CBF-rearranged patients cannot be cured with chemotherapy. This divergent outcome might be due to additional mutations, whose spectrum and prognostic relevance remains hardly defined. Here, we identify nonsilent mutations, which may collaborate with CBF-rearrangements during leukemogenesis by targeted sequencing of 129 genes in 292 adult CBF leukemia patients, and thus provide a comprehensive overview of the mutational spectrum ('mutatome') in CBF leukemia. Thereby, we detected fundamental differences between CBFB/MYH11and RUNX1/RUNX1T1-rearranged patients with ASXL2, JAK2, JAK3, RAD21, TET2, and ZBTB7A being strongly correlated with the latter subgroup. We found prognostic relevance of mutations in genes previously known to be AML-associated such as KIT, SMC1A, and DHX15 and identified novel, recurrent mutations in NFE2 (3%), MN1 (4%), HERC1 (3%), and ZFHX4 (5%). Furthermore, age >60 years, nonprimary AML and loss of the Y-chromosomes are important predictors of survival. These findings are important for refinement of treatment stratification and development of targeted therapy approaches in CBF leukemia. Supplementary informationThe online version of this article (https://

Key Points• FLT3 N676K mutations without concurrent internal tandem duplication (ITD) are associated with core-binding factor leukemia.• N676K activates FLT3 and downstream signaling pathways.The t(8;21) and inv(16)/t(16;16) rearrangements affecting the core-binding factors RUNX1 and CBFB, respectively, are found in 15% to 20% of adult de novo acute myeloid leukemia (AML) cases and are associated with a favorable prognosis. Since the expression of the fusion genes CBFB/MYH11 or RUNX1/RUNX1T1 alone is not sufficient to cause leukemia, we performed exome sequencing of an AML sample with an inv(16) to identify mutations, which may collaborate with the CBFB/MYH11 fusion during leukemogenesis. We discovered an N676K mutation in the adenosine triphosphate (ATP)-binding domain (tyrosine kinase domain 1 [TKD1]) of the fms-related tyrosine kinase 3 (FLT3) gene. In a cohort of 84 de novo AML patients with a CBFB/MYH11 rearrangement and in 36 patients with a RUNX1/RUNX1T1 rearrangement, the FLT3 N676K mutation was identified in 5 and 1 patients, respectively (5 [6%] of 84; 1 [3%] of 36). The FLT3-N676K mutant alone leads to factor-independent growth in Ba/F3 cells and, together with a concurrent FLT3-ITD (internal tandem duplication), confers resistance to the FLT3 protein tyrosine kinase inhibitors (PTKIs) PKC412 and AC220. Gene expression analysis of AML patients with CBFB/MYH11 rearrangement and FLT3 N676K mutation showed a trend toward a specific expression profile. Ours is the first report of recurring FLT3 N676 mutations in core-binding factor (CBF) leukemias and suggests a defined subgroup of CBF leukemias. This trial was registered at www.clinicaltrials.gov as #NCT00266136. (Blood.

The t(10;11)(p13-14;q14-21) translocation, giving rise to the CALM-AF10 fusion gene, is a recurrent chromosomal rearrangement observed in patients with poor prognosis acute myeloid leukemia (AML). Although splicing of the CALM-AF10 fusion transcripts has been described in AML patients, the contribution of different CALM and AF10 domains to in vivo leukemogenesis remains to be defined. We therefore performed detailed structure-function studies of the CALM-AF10 fusion protein. We demonstrate that fusion of the C-terminal 248 amino acids of CALM, which include the clathrin-binding domain, to the octapeptide motif-leucine-zipper (OM-LZ) domain of AF10 generated a fusion protein (termed CALM-AF10 minimal fusion (MF)), with strikingly enhanced transformation capabilities in colony assays, providing an efficient system for the expeditious assessment of CALM-AF10-mediated transformation. Leukemias induced by the CALM-AF10 (MF) mutant recapitulated multiple aspects of full-length CALM-AF10-induced leukemia, including aberrant Hoxa cluster upregulation, a characteristic molecular lesion of CALM-AF10 leukemias. In summary, this study indicates that collaboration of the clathrin-binding and the OM-LZ domains of CALM-AF10 is sufficient to induce AML. These findings further suggest that future approaches to antagonize CALM-AF10-induced transformation should incorporate strategies, which aim at blocking these key domains.

404 Inversion inv(16) or translocation t(16;16) and translocation t(8;21) are recurring rearrangements in acute myeloid leukemia (AML), which result in the fusion genes CBFB/MYH11 or RUNX1/RUNX1T1, respectively. These rearrangements are found in 15–20% of adult de novo AML cases and are associated with favourable prognosis. CBFB and RUNX1 form the core binding factor (CBF), which is a transcription factor essential for normal hematopoiesis and myeloid development. By disrupting the physiological transcription factor activity of CBF the fusion proteins causes repression of the CBF-target genes resulting in a block of differentiation. Since expression of CBFB/MYH11 or RUNX1/RUNX1T1 on their own is not sufficient to cause leukemia it is likely that additional mutations are required for malignant transformation. To systematically identify mutations which may collaborate with CBFB/MYH11 during leukemogenesis we performed exome sequencing of an AML sample with an inv(16). The sample was selected based on availability and absence of known additional genetic alterations. By comparing the AML exome sequence with the exome sequence of a remission sample from the same patient we were able to identify leukemia-specific sequence variants as described previously (Greif et al., 2012, Blood). Using this approach we found an N676K mutation in the ATP-binding domain (TKD1) of the fms-related tyrosine kinase 3 (FLT3) gene. Mutations affecting N676 resulting in variable amino acid changes (N676D or N676S) were initially discovered in a screen for resistance to tyrosine kinase inhibitors (TKI) in FLT3 internal tandem duplication (ITD) expressing Ba/F3 cells (Cools et al., 2004, Cancer Res). An N676K point mutation has been reported in a cytogenetically normal (CN) AML patient with FLT3-ITD and TKI-resistance (Heidel et al., 2006, Blood). In contrast, our patient with inv(16) and the FLT3 N676K did not carry an additional FLT3-ITD. In a cohort of 69 patients with inv(16) we found a total of 4 patients with FLT3 N676K mutations (4/69, 6%). In 14 patients with t(16;16) and in 36 patients with t(8;21) we identified one patient each with FLT3 N676K (1/14, 7% and 1/36, 3%). Thus, the overall mutation frequency for patients with CBFB/MYH11 rearrangement was 6% (5/83). None of the CBF AML patients with FLT3 N676K mutation had an additional FLT3-ITD. In 90 CN-AML patients we detected only a single FLT3 N676K mutation and the affected patient had a concurrent FLT3-ITD. We are currently testing the frequency of FLT3 N676K mutations in independent CBF-AML cohorts. To test the transforming potential we expressed the FLT3 N676K mutant in Ba/F3 cells. As controls we expressed FLT3 wild type (WT), FLT3 mutants D835Y or ITD in parallel. Cell surface expression of N676K was similar to WT, but increased compared to D835Y and ITD. Cell proliferation assays were done in presence and absence of IL-3 or FLT3 ligand (FL). FLT3 N676K leads to IL-3 and FL independent cell growth reaching 25% of IL-3 mediated growth. FLT3 inhibition by AC220 or PKC412 abrogates this proliferation, but N676K is slightly more resistant to inhibition than ITD. In contrast to ITD expression that results in STAT5 phosphorylation, N676K expression leads to phosphorylation of MAPK and AKT. Gene expression profiling of patients with inv(16) revealed that patients with additional FLT3 N676K mutation show a significant enrichment of gene sets including ubiquitin mediated proteolysis, adherens junction and JAK/STAT signaling pathway. According to a structural model, the N676K mutation stabilizes the fold of the kinase domain between the juxtamembrane domain (JMD) and a hydrophobic pocket that is the target of FLT3 inhibitors. N676K mutations could therefore reduce the autoinhibition of FLT3 by the JMD and also negatively affect the binding affinity of FLT3 inhibitors. Ours is the first report of recurring FLT3 N676 mutations in the absence of FLT3-ITD. Our findings point towards a specific association of FLT3 N676K mutations with CBF leukemia. Based on our functional assays N676K acts as a gain-of-function mutation. Although FLT3 has been known for more than a decade to be mutated in one third of AML patients, it appears that the spectrum of FLT3 mutations is still not fully understood, in particular, in defined cytogenetic subgroups of AML. Unbiased mutation screening by exome sequencing allows the detection of novel sequence variations even in extensively studied genes. Disclosures: Krebs: Illumina: Honoraria. Greif:Illumina: Honoraria.

High throughput sequencing approaches, including the analysis of exomes or gene panels, are widely used and established to detect tumor-specific sequence variants such as point mutations or small insertions/deletions. Beyond single nucleotide resolution, sequencing data also contain information on changes in sequence coverage between samples and thus allow the detection of somatic copy number alterations (CNAs) representing gain or loss of genomic material in tumor cells arising from aneuploidy, amplifications, or deletions. To test the feasibility of CNA detection in sequencing data we analyzed the exomes of 25 paired leukemia/remission samples from acute myeloid leukemia (AML) patients with well-defined chromosomal aberrations, detected by conventional chromosomal analysis and/or molecular cytogenetics assays. Thereby, we were able to confirm chromosomal aberrations including trisomies, monosomies, and partial chromosomal deletions in 20 out of 25 samples. Comparison of CNA detection using exome, custom gene panel, and SNP array analysis showed equivalent results in five patients with variable clone size. Gene panel analysis of AML samples without matched germline control samples resulted in confirmation of cytogenetic findings in 18 out of 22 cases. In all cases with discordant findings, small clone size (<33%) was limiting for CNA detection. We detected CNAs consistent with cytogenetics in 83% of AML samples including highly correlated clone size estimation (R = 0.85), while six out of 65 cytogenetically normal AML samples exhibited CNAs apparently missed by routine cytogenetics. Overall, our results show that high throughput targeted sequencing data can be reliably used to detect copy number changes in the dominant AML clone. © 2016 Wiley Periodicals, Inc.

Deletions of the long arm of chromosome 9 [del(9q)] are a rare but recurring aberration in acute myeloid leukemia (AML). Del(9q) can be found as the sole abnormality or in combination with other cytogenetic aberrations such as t(8;21) and t(15;17). TLE1 and TLE4 were identified to be critical genes contained in the 9q region. We performed whole exome sequencing of 5 patients with del(9q) as the sole abnormality followed by targeted amplicon sequencing of 137 genes of 26 patients with del(9q) as sole or combined with other aberrations. We detected frequent mutations in NPM1 (10/26; 38%), DNMT3A (8/26; 31%), and WT1 (8/26; 31%) but only few FLT3-ITDs (2/26; 8%). All mutations affecting NPM1 and DNMT3A were exclusively identified in patients with del(9q) as the sole abnormality and were significantly more frequent compared to 111 patients classified as intermediate-II according to the European LeukemiaNet (10/14, 71% vs. 22/111, 20%; P < 0.001, 8/14, 57% vs. 26/111, 23%; P = 0.02). Furthermore, we identified DNMT3B to be rarely but recurrently targeted by truncating mutations in AML. Gene expression analysis of 13 patients with del(9q) and 454 patients with normal karyotype or various cytogenetic aberrations showed significant down regulation of TLE4 in patients with del(9q) (P = 0.02). Interestingly, downregulation of TLE4 was not limited to AML with del(9q), potentially representing a common mechanism in AML pathogenesis. Our comprehensive genetic analysis of the del(9q) subgroup reveals a unique mutational profile with the frequency of DNMT3A mutations in the del(9q) only subset being the highest reported so far in AML, indicating oncogenic cooperativity. © 2016 Wiley Periodicals, Inc.

The leukemia-associated fusion gene CBFB/MYH11 results from a pericentric inversion of chromosome 16, inv(16)(p13.1q22), or less commonly from a t(16;16)(p13.1;q22). Although this cytogenetic aberration is associated with a rather favorable prognosis in acute myeloid leukemia (AML), nearly half of patients eventually relapse after standard chemotherapy. To systematically analyze the clonal evolution in this AML-subgroup, we performed whole exome sequencing (WES) of 13 adult CBFB/MYH11-rearranged AML patients using matched diagnostic, remission and relapse samples ('triplets'). Thereby, we found 2-12 (median: 8) somatic sequence variants per patient at diagnosis and 2-13 (median: 4) mutations at relapse. These included mutations in genes known to cooperate with CBFB/MYH11 (e.g. RAS, FLT3, KIT) as well as in genes, which had not been associated with AML previously (MYO15A, EVPL, ROS1, FTCD and ASL). Next, we designed a custom targeted sequencing assay (Haloplex, Agilent), including the candidate genes from exome sequencing, as well as genes known to be recurrently mutated in AML (455 genes, 1.86 Mbp total target sequence) and performed targeted sequencing of 32 CBFB/MYH11-rearranged AML triplet samples (including the 13 triplets initially analyzed by WES) with a median read depth of 500. The results are summarized in Figure 1. Fourteen genes were found mutated in at least two patients at diagnosis and 9 genes at the timepoint of relapse. In all CBFB/MYH11-rearranged patients, more than one additional mutation was identified, each of them at a distinct variant allele-frequency, indicating clonal heterogeneity. All but one FLT3 TKD (D835 or N676) mutation were lost at relapse, whereas FLT3 ITDs were stable in 3 out of 7 patients. One FLT3 ITD was gained at relapse. The majority of RAS,KIT and CBL mutations were lost and none was acquired at relapse. Particularly, the loss of 6 out of 7 KIT exon 8 frameshift mutations was surprising since KIT exon 8 frameshift mutations were negative prognostic markers in a cohort of 162 patients with CBFB/MYH11 rearranged leukemia (OS: HR= 3.12, p= 0.001; Opatz et al. submitted). In contrast, mutations in WT1 and DNMT3A were all stable during relapse evolution and four patients gained mutations in these two genes. Furthermore, aberrations in CSF3R, BCORL1 and ZBTB7A were acquired at relapse. Of note, WT1 mutations causing a frameshift in exon 6 were found in 9% of adult de novo AML with CBFB/MYH11-rearrangement and have recently been characterized by our research group as negative prognostic marker for overall survival (HR: 2.93, p= 0.011) (Opatz et al. submitted). These findings are in line with the observed gain of WT1 mutations in 10% of relapsed cytogenetically normal AML patients (Greif et al., 2018, Clin Cancer Res) suggesting a common mechanism of disease progress across cytogenetic subgroups. Surprisingly, a mutation in ZBTB7A, a gene frequently altered in RUNX1/RUNX1T1 positiv leukemia (23%) but rarely in CBFB/MYH11 positiv leukemia (2%), was gained at relapse in one patient. Mutations in epigenetic modifiers, cohesion complex components and janus kinases are known cooperating events in RUNX1/RUNX1T1 rearranged leukemia and were, except for DNMT3A, not found in our CBFB/MYH11-positive cohort. The new recurring mutations in MYO15A (n=3), ROS1 (n=2), FTCD (n=2) and ASL (n=2) were partially lost at relapse, whereas EVPL (n=2) mutations were stable during the course of disease. In addition, we identified mutations in APC2, TP53 and ZFHX4 (gained at relapse), PTPN11, MECOM, BCOR, NPM1 and IDH2 (stable) as well as in ABL1 (lost at relapse) in individual patients. Taken together, our findings suggest that mutations in signaling pathway genes seem to be unstable during disease progression and may thus not be required for the evolution of relapse. The frequent loss of signaling gene mutations indicates that relapse might evolve from an early ancestral clone carrying the CBFB/MYH11 rearrangement only. Figure 1: Mutation profile of 32 patients with CBFB/MYH11-rearranged AML. The stability of recurrently mutated genes during the evolution of relapse is shown. Diagonal lines indicate two mutations in the respective gene. Figure 1 Figure 1. Disclosures Thiede: AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding. Middeke:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees. Stoelzel:Neovii: Speakers Bureau. Metzeler:Novartis: Consultancy; Celgene: Consultancy, Research Funding.