ALBERT

Description: Introduction Next generation sequencing (NGS) studies have documented the highly heterogeneous landscape of somatic mutations in CLL. Beside the mere genetic diversity of the disease, frequency and clinical impact of mutations vary between analyzed cohorts since samples may be collected at variable clinical stages. To address this point, we undertook an integrative systematic genomic study of patients included in a prospective clinical trial. Material and methods We performed conventional karyotype (K), fluorescence in situ hybridization (FISH) and investigated 31 genes and 2 non-coding regions, in pretreatment samples obtained from 146 patients enrolled on a phase 3 trial comparing fludarabine (F)-cyclophosphamide (C)-rituximab vs. F-C-alemtuzumab (Lepretre et al, Blood 2012). All patients were Binet stage B (n=115) or C (n=31) and devoid of 17p deletion. All gave their informed consent according to the declaration of Helsinki. We studied by targeted deep sequencing exonic regions of 31 genes including TP53, SF3B1, NOTCH1, ATM, BIRC3, FBXW7, NFKBIE, MED12, MYD88 and POT1. DNA was extracted from total blood mononuclear cells (fraction of tumor cells determined by flow cytometry 〉80%). Targeted regions were PCR-amplified using an AmpliSeq (Life technologies) approach and PCR products sequenced in a MiSeq (Illumina) instrument with a mean depth of 1000X. We used a variant allele frequency (VAF) minimal threshold of 5% to detect mutation. EGR2, PAX5 enhancer and NOTCH1 3'UTR regions were analyzed by Sanger sequencing. For each identified mutation, clinical and biological variables were compared between mutant and wild-type patients using either Wilcoxon or Fisher exact test. For each mutation present in a sufficient number of samples, prognostic impact on overall survival (OS) and progression free survival (PFS) was assessed using log-rank test. For important clinical and molecular prognostic variables, adjusted prognostic effect was assessed using a multivariate Cox model for OS and PFS with a stepwise-forward selection based on Akaike criteria. Results Chromosomal abnormalities were observed by K in 63% of 126 patients, 11% showing complex K and 23% carrying translocations. FISH revealed 78/144 (54%) 13q deletion, 25/144 (17%) trisomy 12 (tri12), 30/146 (21%) 11q deletion. Unmutated (UM) IGHV status was observed in 84/146 (58%) cases. At least one mutation was observed in 118/146 (81%) patients, with 1, 2, 3 and 4 or 5 mutations in, respectively, 34%, 23%, 17% and 7%. The most frequently mutated genes were SF3B1 (23%), NOTCH1 (16%), ATM (14%), NFKBIE (10%), XPO1 (10%), MED12 (7%), EGR2 (6%), TP53 (5%), BIRC3 (5%), FBXW7 (5%), PAX5 enhancer (5%), CHD2 (5%), PCLO (5%), MYD88 (4%), POT1 (4%), KMTD2 (4%) and NOTCH1 3'UTR (4%). MYD88 and SAMHD1 mutations were always observed as clonal (VAF〉40%) whereas BIRC3 and BRAF mutationsalways observed as sub-clonal. NOTCH1 mutations were associated with high CD38 expression (P

Description: Introduction: CLL is a heterogeneous disease in terms of response to treatment, with some patients reaching complete and prolonged remissions, while others relapsing early and requiring several lines of treatments. This highly variable course is partly explained by the existence of a heterogenic panel of genetic alterations (mutations, chromosomal abnormalities) that allow the development of drug-resistant aggressive CLL subclones. Therefore, a functional characterization of the cytogenetic alterations associated to CLL drug resistance may provide new means of improving the current therapeutic strategies. We and others have already reported that the gain of 2p (2p+) is recurrent in CLL. However, the candidate gained gene(s) on the 2p remain to be identified. Previously data: we have observed that the 2p gain is frequent in previously untreated CLL Binet stages B/C (21/132, 15.9%), and is associated with bad prognostic factors, such as 11q deletion (p=0.0008) and unmutated IGHV (p=0.02). Using a SNP-array approach, we have identified a minimally gained region of 1.28Mb on 2p16.1-15. This region included the gene CRM1/XPO1 (Chromosome Region Maintenance 1/Exportin-1), a gene also recurrently mutated in CLL. A qPCR assessment confirmed that XPO1 was overexpressed in the 2p+/CLL patients (1.4-fold increase compared to 2p-/CLL; p=0.02). The objective of our work was to identify the potential role of XPO1 in CLL drug resistance by using the selective XPO1 inhibitor Selinexor (KPT-330, provided by Karyopharm Therapeutics), which is currently in Phase II human clinical trials in hematological and solid cancers. Methods: We have analyzed 36 2p+/CLL and we have searched for XPO1 mutations in 436 CLL samples. CLL drug resistance associated to XPO1 overexpression/mutation was assessed by measuring the rate of programmed cell death (PCD) on cells from 2p- and wildtype (wt) XPO1/CLL (n=20), 2p+/XPO1 wt/CLL (n=8) and on XPO1 mut/CLL (n=6). After 24 hours treatment with Fludarabin + Cyclophosphamid + Rituximab (FCR), Ibrutinib (Ibru), Idelalisib + Rituximab (Ide+R) and Selinexor, cells were stained with Annexin-V and propidium iodide and PCD was assessed by flow cytometry. KPT-301 was used as a negative control. For the inhibition assay, the inhibitor Q-VD-Oph was added 30 min before inducing cell death. Mitochondrial membrane depolarisation was assessed using tetramethyllrhodamine ethyl ester probe and flow cytometry analysis. Results: (i) Using a FISH approach, we fully confirmed the gain of XPO1 in 2p+/CLL samples. Additionally, we found that the XPO1 gain was often subclonal, suggesting that it tends to arise late in leukemic development. Longitudinal FISH analyses, performed on 8 2p+/CLL-treated patients, showed a similar or increasing percentage of cells carrying XPO1 gain at relapse, when compared to diagnosis; (ii) XPO1 was mutated in 23/436 (5.3%) CLL and in 2/30 (6.7%) 2p+/CLL; (iii) Selinexor induced PCD in 2p-/XPO1 wt/CLL (35% of PCD). The results were similar in all tested CLL, independently of prognostic factors (del13q, tri12, del11q, del17p, IGHV status), while sparing the non leukemic cells from patients or B cells from healthy donors; (iv) Selinexor induced CLL PCD through a caspase-dependant apoptotic pathway, as evidenced by inhibition of cell death by Q-VD-Oph, and cleavage of the caspase-3. Selinexor also induced mitochondrial depolarization and was associated with upregulation and activation of the pro-apopototic Bax protein; (v) XPO1 mut/CLL were significantly resistant to PCD induced by Selinexor (p=0.003). In contrast, the mutations in XPO1 had no effect in FCR and Ibru PCD induction; (vi) 2p+/CLL cells were resistant to PCD induced by all tested drugs: FCR (p=0.01), Ibru (p=0.003), Ide+R (p=0.004) and Selinexor (p=0.0001). Conclusion: Our data show that 2p+/CLL is associated to FCR, Ibru and Ide+R drug resistance. Strikingly, Selinexor, a new XPO1 inhibitor, is unable to induce PCD in 2p+ and/or XPO1 mut CLL, which strongly suggests a key role for XPO1 in the CLL drug resistance associated to the 2p gain. Altogether, our work provide substantial progress in the understanding of the role of XPO1 in CLL drug resistance and suggests that the assessment of the 2p gain and the mutations in XPO1 will be considered before to decide a CLL therapy. As 2p gain could be observed in other B malignancies, it is tempting to extend these recommendations to all Selinexor treatments. Disclosures Choquet: Janssen: Consultancy; Roche: Consultancy. Leblond:Janssen: Consultancy, Honoraria, Speakers Bureau; GSK: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Mundipharma: Honoraria.

Description: Background Intensive efforts of genome sequencing studies during the past decade identified 〉100 driver genes recurrently mutated in one or more subtypes of myeloid neoplasms, which collectively account for the pathogenesis of 〉90% of the cases. However, approximately 10% of the cases have no alterations in known drivers and their pathogenesis is still unclear. A possible explanation might be the presence of alterations in non-coding regions that are not detected by conventional exome/panel sequencing; mutations and complex structural variations (SVs) affecting these regions have been shown to deregulate expression of relevant genes in a variety of solid cancers. Unfortunately, however, no large studies have ever been performed, in which a large cohort of myeloid malignancies were analyzed using whole genome sequencing (WGS) in an attempt to identify a full spectrum of non-coding alterations, even though its efficacy have been demonstrated in many solid cancers. In this study, we performed WGS in a large cohort of pan-myeloid cancers, in which both coding and non-coding lesions were comprehensively analyzed. Patients and methods A total of 338 cases of myeloid malignancies, including 212 with MDS, 70 with AML, 17 with MDS/MPN, 23 with t-AML/MDS, and 16 with MPN were analyzed with WGS, of which 173 were also analyzed by transcriptome sequencing. Tumor samples were obtained from patients' bone marrow (N=269) or peripheral blood (N=69), while normal controls were derived from buccal smear (N=263) or peripheral T cells (N=75). Sequencing of target panel of 86 genes were performed for all samples. Sequencing data were processed using in-house pipelines, which were optimized for detection of complex structural variations (SVs) and abnormalities in non-coding sequences. Results WGS identified a median of 586,612 single nucleotide variants (SNVs) and 124,863 short indels per genome. NMF-based decomposition of the variants disclosed three major mutational signatures, which were characterized by age-related C〉T transitions at CpG sites (Sig. A), C〉T transitions at CpT sites (Sig. B), and T〉C transitions at ApTpN context (Sig. C). Among these, Sig. C showed a prominent strand bias and corresponds to COSMIC signature 16, which has recently been implicated in alcohol drinking. Significant clustering of SNVs and short indels were interrogated across the genome divided into different window sizes (1Kbp, 10Kbp, 100Kbp) or confining the targets to coding exons and known regulatory regions, such as promoters, enhancers/super enhances, and DNase I hypersensitive sites. Recapitulating previous findings, SNVs in the coding exons were significantly enriched in known drivers, including TP53, TET2, ASXL1, DNMT3A, SF3B1, RUNX1, EZH2, and STAG2. We detected significant enrichment of SNVs in CpG islands, and promoters/enhancers. We also detected a total of 8,242 SVs with a median of 15 SVs/sample, which is more prevalent than expected from conventional karyotype analysis. Focal clusters of complex rearrangements compatible with chromothripsis were found in 8 cases, of which 7 carried biallelic TP53 alterations. NMF-based signature analysis of SVs revealed that large (〉1Mb) deletions, inversions, and tandem duplications and translocations are clustered together and were strongly associated with TP53 mutations, while smaller deletions and tandem duplications, but not inversions, constitute another cluster. As expected, FLT3-ITD (N=15) and MLL-PTD (N=12) were among the most frequent SVs. Unexpectedly, in addition to known SVs associated with t(8;21) (RUNX1-RUNX1T1) (N=6) and t(3;21) (RUNX1-MECOM) (n=1) as well as non-synonymous SNVs within the coding exons (N=30), we detected frequent non-coding alterations affecting RUNX1, including SVs (N=15) and SNVs around splicing acceptor sites (N=5), suggesting that RUNX1 was affected by multiple mechanism, where as many as 38% of RUNX1 lesions were explained by non-coding alterations. Other recurrent targets of non-coding lesions included ASXL1, NF1, and ETV6. Conclusions WGS was successfully used to reveal a comprehensive registry of genetic alterations in pan-myeloid cancers. Non-coding alterations affecting known driver genes were more common than expected, suggesting the importance of detecting non-coding abnormalities in diagnostic sequencing. Disclosures Nakagawa: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Usuki:Mochida Pharmaceutical: Speakers Bureau; Astellas Pharma Inc.: Research Funding; Sanofi K.K.: Research Funding; GlaxoSmithKline K.K.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Daiichi Sankyo: Research Funding; Celgene Corporation: Research Funding, Speakers Bureau; SymBio Pharmaceuticals Limited.: Research Funding; Shire Japan: Research Funding; Janssen Pharmaceutical K.K: Research Funding; Boehringer-Ingelheim Japan: Research Funding; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Pfizer Japan: Research Funding, Speakers Bureau; Novartis: Speakers Bureau; Nippon Shinyaku: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Takeda Pharmaceutical: Speakers Bureau; Ono Pharmaceutical: Speakers Bureau; MSD K.K.: Speakers Bureau. Chiba:Bristol Myers Squibb, Astellas Pharma, Kyowa Hakko Kirin: Research Funding. Miyawaki:Otsuka Pharmaceutical Co., Ltd.: Consultancy; Novartis Pharma KK: Consultancy; Astellas Pharma Inc.: Consultancy.

Description: We evaluated concurrent gene mutations, clinical outcome, and gene expression signatures of CCAAT/enhancer binding protein alpha (CEBPA) double mutations (CEBPAdm) versus single mutations (CEBPAsm) in 1182 cytogenetically normal acute myeloid leukemia (AML) patients (16-60 years of age). We identified 151 (12.8%) patients with CEBPA mutations (91 CEBPAdm and 60 CEBPAsm). The incidence of germline mutations was 7% (5 of 71), including 3 C-terminal mutations. CEBPAdm patients had a lower frequency of concurrent mutations than CEBPAsm patients (P 〈 .0001). Both, groups were associated with a favorable outcome compared with CEBPAwt (5-year overall survival [OS] 63% and 56% vs 39%; P 〈 .0001 and P = .05, respectively). However, in multivariable analysis only CEBPAdm was a prognostic factor for favorable OS outcome (hazard ratio [HR] 0.36, P 〈 .0001; event-free survival, HR 0.41, P 〈 .0001; relapse-free survival, HR 0.55, P = .001). Outcome in CEBPAsm is dominated by concurrent NPM1 and/or FLT3 internal tandem duplication mutations. Unsupervised and supervised GEP analyses showed that CEBPAdm AML (n = 42), but not CEBPAsm AML (n = 18), expressed a unique gene signature. A 25-probe set prediction signature for CEBPAdm AML showed 100% sensitivity and specificity. Based on these findings, we propose that CEBPAdm should be clearly defined from CEBPAsm AML and considered as a separate entity in the classification of AML.

Description: Abstract 1697 Background Abnormal and clonal hematopoiesis resulting in peripheral blood cytopenias, and risk of progression to acute myeloid leukemia (AML) are the main characteristics of myelodysplastic syndromes (MDS) which present a high diversity of somatically mutated genes. Recently, mutations targeting genes whose products participate to the early steps of RNA splicing (SF3B1, SRSF2, ZRSR2, and U2AF35) have been reported. Mutations in ASXL1, DNMT3, EZH2, IDH1/2 and TET2, suggest deregulation of the epigenetic control of transcription. Additional genes known to be mutated in MDS include RUNX1, TEL/ETV6, TP53 and NRAS The alteration of some of these genes may carry prognostic value. Next-generation sequencing analyses of AML and related disorders recently identified mutations in two chromosome X genes, BCOR and BCORL1, that code for related transcriptional co-repressors interacting with histone deacetylases and presenting specific properties. BCOR interacts with BCL6 and constitutional inactivating mutations have been described in the Oculo-Facial-Cardio-Dental syndrome. BCOR is also affected by mutations or translocations in retinoblastoma and sarcoma. BCORL1 has been implicated in chromosomal rearrangements in hepatocellular carcinoma. In this study, we have investigated BCOR and BCORL1 gene by Sanger sequencing in a cohort of 221 MDS samples. Methods The 221 MDS samples were collected at diagnosis in multicenter clinical trials in France between 1999 and 2011. The coding sequences of BCOR (ENST00000378444) and BCORL1 (ENST00000218147) were analyzed by Sanger sequencing. Mutational analyses of ASXL1, CBL, DNMT3A, ETV6, EZH2 IDH1/2, JAK2, NRAS, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF35, and ZRSR2 were previously reported. Non tumoral material (buccal swab or CD3+ cells) was analyzed for the presence of the identified variations of BCOR or BCORL1 when available (n=9). The prognostic impact of BCOR mutations was evaluated in MDS patients with available follow-up information (n=203). Results BCOR and BCORL1 coding sequences were analyzed in the cohort of 221 MDS patients and we found mutations in 8.6% (n=19) and 2.3% (n=5) respectively. Alterations were distributed all over the coding regions. Strikingly, two patients presented concomitant inactivating mutations of BCOR and BCORL1. Among the 19 BCOR alterations, 9 were missense, 3 nonsense, 5 frameshift and 2 splice site mutations. No significant difference in age, sex, karyotype, blood counts or bone marrow blasts between BCORmut and BCORwt patients was observed. Mutations were found in patients of all IPSS risk-groups and WHO subtypes. Comparison of cytological bone marrow reports revealed a trend for a higher rate of dysgranulopoiesis in BCORmut patients (P=0.06). Because only truncating mutations (splice, frameshift and nonsense mutations) are unambiguously expected to affect BCOR function, statistical analyses were restricted to truncating BCOR mutations (n=10/221, 4.5%). Truncating BCOR mutations were frequent in RUNX1mut patients (19% versus 3% in RUNX1wt patients; P=0.027). They were by trend associated with SRSF2 and DNMT3A mutations (P=0.09) and were exclusive with IDH1/2, JAK2, NRAS, TP53, and ZRSR2 mutations. In univariate analysis, Overall Survival (OS) and AML transformation rate did not differ between patients with either missense or truncating BCOR mutations and BCORwt patients (Figure A&B). However, inferior OS (P=0.034) and higher AML transformation rate by trend (P=0.051) were observed for patients with truncating BCOR mutations (Figure C&D). Multivariate analysis demonstrated that a truncating BCOR mutation was an independent unfavourable prognostic factor for OS (HR 3.3; 95%CI 1.4 – 8.1; P=.008). The low number of BCORL1 mutated patients precludes any statistical analyses but clinical follow-up was available for 4 out of 5 patients. Three patients died 7, 17 and 27 months after MDS diagnosis. The fourth patient received allogeneic bone marrow transplantation after AML transformation and is in complete remission 5 years after transplantation. Conclusion In summary, truncating mutations of BCOR were independently associated with a worse OS in MDS. These data support the idea that BCOR mutations appear as associated with RUNX1 and DNMT3A mutations as reported in AML. Moreover, and despite a relatively low mutation frequency in MDS, BCOR might be considered as a key gene in risk stratification. Disclosures: No relevant conflicts of interest to declare.

Description: To integrate available clinical and molecular information for cytogenetically normal acute myeloid leukemia (CN-AML) patients into one risk score, 275 CN-AML patients from multicenter treatment trials AML SHG Hannover 0199 and 0295 and 131 patients from HOVON/SAKK protocols as external controls were evaluated for mutations/polymorphisms in NPM1, FLT3, CEBPA, MLL, NRAS, IDH1/2, and WT1. Transcript levels were quantified for BAALC, ERG, EVI1, ID1, MN1, PRAME, and WT1. Integrative prognostic risk score (IPRS) was modeled in 181 patients based on age, white blood cell count, mutation status of NPM1, FLT3-ITD, CEBPA, single nucleotide polymorphism rs16754, and expression levels of BAALC, ERG, MN1, and WT1 to represent low, intermediate, and high risk of death. Complete remission (P = .005), relapse-free survival (RFS, P 〈 .001), and overall survival (OS, P 〈 .001) were significantly different for the 3 risk groups. In 2 independent validation cohorts of 94 and 131 patients, the IPRS predicted different OS (P 〈 .001) and RFS (P 〈 .001). High-risk patients with related donors had longer OS (P = .016) and RFS (P = .026) compared with patients without related donors. In contrast, intermediate-risk group patients with related donors had shorter OS (P = .003) and RFS (P = .05). Donor availability had no impact on outcome of patients in the low-risk group. Thus, the IPRS may improve consolidation treatment stratification in CN-AML patients. Study registered at www.clinicaltrials.gov as #NCT00209833.

Description: *Contributed equally as first authors. **Contributed equally as senior authors. Recurrent mutations within EGR2, a versatile transcription factor involved in differentiation of hematopoietic cells, were recently reported in 8% of advanced-stage chronic lymphocytic leukemia (CLL) patients, where they appear to be associated with a worse outcome. EGR2 is activated through ERK phosphorylation upon B-cell receptor (BcR) stimulation, and we have previously shown that EGR2 -mutated CLL patients display altered expression of EGR2 down-stream target genes compared to wildtype (wt) patients, thereby pointing to a pathogenic role for EGR2 mutations in dysregulating BcR signaling. To gain further insight into the incidence and prognostic impact of EGR2 mutations in CLL, we screened samples from a well-characterized series of 1430 patients, either by Sanger sequencing (n=1019) or targeted deep-sequencing (n=370), both covering the recently reported EGR2 hotspot in exon 2. In addition, whole-exome data was available for an additional 43 patients. Different cohorts were included in our analysis ranging from 'general practice' CLL (33% IGHV-unmutated (U-CLL), 6% TP53 -aberrant (TP53abn), n=693), to adverse-prognostic CLL (89% U-CLL, 26% TP53abn, n=325), patients belonging to clinically aggressive stereotyped subsets #1-3 & #5-8 (n=342), patients relapsing after FCR therapy (n=41) and Richter transformed cases (n=31), thus reflecting the heterogeneous nature of CLL. Nineteen EGR2 mutations were detected by Sanger sequencing, while 22 additional mutations were identified with deep-sequencing using a 5% variant allele frequency (VAF) cutoff (median 39%, range 5.6-63.9%, median coverage 43,000X). With the exception of one in-frame deletion, all mutations were missense alterations located within the three zinc-finger domains. Significant enrichment of EGR2 mutations was observed in adverse-prognostic (18/325, 5.5%) and FCR-relapsing (4/41, 9.8%) CLL compared to the 'general practice' cohort (18/693, 2.6%, Figure 1A). A surprisingly low frequency was observed among clinically aggressive stereotyped subsets (5/342, 1.5%), although the cause for this observation is currently unknown. Finally, 2/31 (6.5%) cases with Richter transformation carried an EGR2 mutation. Of the 4 FCR-relapsing, EGR2 -mutated cases with available overtime samples, all demonstrated a significant expansion of the EGR2 -mutated clone at relapse (VAF-increase between 15-41%). In addition, subclonal levels of EGR2 hotspot mutations (VAF 0.5-5%) were detected in an additional 13/370 (3.5%) cases by deep-sequencing. The majority of EGR2 -mutated CLL patients (32/39, 82%) concerned U-CLL and the following aberrations co-occurred: 11q-deletions (n=10), TP53abn (n=6), NOTCH1 (n=3)or SF3B1 (n=3) mutations. EGR2 -mutated patients displayed a significantly worse overall survival compared to wt patients (median survival 59 vs. 141 months, p=0.003, using a conservative 10% VAF cutoff), and a poor outcome similar to cases with TP53abn (Figure 1B). In multivariate analysis (n=583), EGR2 status remained an independent factor (p=0.038), along with stage (p=0.048) and IGHV status (p

Description: Background The cohesin complex presents a ring structure that regulates chromosome segregation during meiosis and mitosis and is thus essential for cell division. Genes that belong to the cohesin complex in somatic vertebrate cells are SMC1A, SMC3, RAD21 (SCC1), STAG2 (SA-2) and STAG1 (SA-1). Recently, mutations in the cohesin complex have been reported to occur in AML. The aim of this study was to evaluate the clinical and prognostic implications of cohesin mutations in 389 uniformly treated younger AML patients. Patients and Methods Diagnostic bone marrow or peripheral blood samples were analyzed from 389 younger adult patients with de novo (n=348) or secondary AML (n=41) with French-American-British (FAB) classification M0-M2 or M4-M7, who were entered into the multicenter treatment trials AML SHG 0199 (n=276) or AML SHG 0295 (n=113). Patient samples were assessed for frequently occurring mutations including FLT3-ITD, NPM1, DNMT3A, IDH1, IDH2. In the subgroup of cytogenetically normal AML (CN-AML), additional mutation analyses were performed for CEBPA, MLL-PTD, WT1 and WT1 SNP rs16754, NRAS, and expression levels of BAALC, ERG, EVI1, MN1, MLL and WT1. Sequencing of all coding exons of the cohesin genes was performed with the SOLiDTM system (Life Technologie, Darmstadt, Germany). Sequences were analyzed twice separately using the DNAnexus software and a separate pipeline of bioinformatics software. Identified mutations were validated by Sanger sequencing. Results 27 patients (7%) were identified to carry a mutation in one of the cohesin genes. 10 patients harboured a mutation in STAG1. The second most frequently mutated gene in the cohesin complex was SMC3 with 7 patients showing this mutation. 5 patients harboured a mutation in RAD21, 3 patients in STAG2 and 2 patients in SMC1A. All mutations were mutually exclusive. The majority of mutations were missense mutations, apart from 2 frameshift mutations and 3 nonsense mutations. All evaluable patients lost their cohesin mutation during remission and with the exception of one patient regained their mutation during relapse. Patients with mutations in the cohesin genes were older (47 vs 44 years, P=.04), had a higher white blood cell count (WBC) (P=.046) and a higher peripheral blast count (P=.048). We observed a strong correlation between cohesin gene and NPM1 mutations (P=.007). BAALC expression was lower in patients with a mutation compared to patients without a mutation in the cohesin genes (P=.026). In order to get a better understanding of when mutations in the cohesin complex occur during clonal evolution we evaluated the allelic burden of mutations in the cohesin complex. Because of the strong association between NPM1 and cohesin mutations, the allelic ratio of mutated and wildtype NPM1 was compared to the allelic ratio of mutated and wildtype cohesin genes. Interestingly, we found a similar mutation burden between NPM1 and genes of the cohesin complex (Pearson correlation coefficient R=0.67), suggesting that cohesin gene mutations occurred in the same clone as NPM1 mutations. When considering all mutations in the cohesin complex as one group, overall survival (OS), relapse free survival (RFS) and complete remission rate (CR) were not influenced by the presence of cohesin mutations (OS: HR 1.23; 95%CI 0.77-1.99; P=.4; RFS: HR 0.92; 95%CI 0.51-1.65; P=.77; CR: mutated 77% vs wildtype 76%, P=.83). We next evaluated the prognostic influence of each gene in the cohesin complex separately in all patients. STAG1, SMC3 and RAD21 mutations had no influence on OS and RFS. A trend for a reduced OS was observed for STAG2 mutated patients (OS: HR 3.02; 95%CI 0.97-9.46; P=.057), although the analysis is limited by the small number of mutated patients. In the subgroup of patients with cytogenetically normal AML we did not identify a difference in OS, RFS or CR rates for patients with (n=18) or without mutations (283) in the cohesin complex (OS: HR 1.00; 95%CI 0.52-1.92; P=.99; RFS: HR 0.64; 95%CI 0.30-1.39; P=.26; CR rates: mutated 80% vs wildtype 73%, P=.64). Conclusion Mutations in the cohesin genes represent novel, recurrent mutations in AML. We found a strong association between these mutations and mutations in NPM1. Cohesin complex mutations as a group had no prognostic impact in all and in cytogenetically normal AML patients. Disclosures: Schlenk: Amgen: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Chugai: Research Funding; Ambit: Honoraria.

Description: Abstract 1402 Over 20 different fusion partner genes of NUP98 have been reported in hematological malignancies with the majority of NUP98 fusions occurring in acute myeloid leukemia (AML). Recently, a specific fusion of NUP98 with Nuclear receptor-binding SET domain protein 1 (NSD1) has been analyzed in a large cohort of pediatric and adult AML patients. In this report, 16.1% of pediatric AML samples and 2.3% of adult cytogentically normal (CN)-AML cases were found to be NUP98/NSD1-positive. NUP98/NSD1-positive patients had an adverse outcome. The aim of our study was to investigate the frequency, clinical features and the prognostic impact of NUP98/NSD1 in another large, uniformily treated adult AML cohort, and in patients with myelodysplastic syndromes, which frequently precede overt leukemia. We studied 504 younger AML patients (

Description: Mutations in the nicotinamide adenine dinucleotide phosphate+–dependent isocitrate dehydrogenase gene 2 (IDH2) have recently been found in patients with acute myeloid leukemia (AML) as well as in patients with leukemic transformation of myeloproliferative neoplasms. We analyzed 272 adult patients with cytogenetically normal AML (CN-AML) for the presence of IDH2 mutations in codons R140 and R172. IDH2 mutations of amino acid 140 or 172 could be identified in 12.1% of CN-AML patients, with the majority of mutations (90%) occurring at position R140. The incidence of IDH2 mutations in AML patients with aberrant karyotypes (n = 130) was significantly lower (3.8%, P = .006). IDH2 mutations were mutually exclusive with mutations in IDH1. IDH2 mutation status alone or in combination with IDH1 mutations had no impact on response to therapy, overall survival, and relapse-free survival in patients with CN-AML. In conclusion, IDH2 mutations are frequently found in CN-AML, but in our analysis these mutations did not influence treatment outcome. This study was registered at www.clinicaltrials.gov as #NCT00209833.