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To design a simple and reproducible classifier predicting the overall survival (OS) of patients with acute myeloid leukemia (AML) ≥60 years of age treated with 7 + 3, we sequenced 37 genes in 471 patients from the ALFA1200 (Acute Leukemia French Association) study (median age, 68 years). Mutation patterns and OS differed between the 84 patients with poor-risk cytogenetics and the 387 patients with good (n = 13), intermediate (n = 339), or unmeasured (n = 35) cytogenetic risk. TP53 (hazards ratio [HR], 2.49; P = .0003) and KRAS (HR, 3.60; P = .001) mutations independently worsened the OS of patients with poor-risk cytogenetics. In those without poor-risk cytogenetics, NPM1 (HR, 0.57; P = .0004), FLT3 internal tandem duplications with low (HR, 1.85; P = .0005) or high (HR, 3.51; P < 10−4) allelic ratio, DNMT3A (HR, 1.86; P < 10−4), NRAS (HR, 1.54; P = .019), and ASXL1 (HR, 1.89; P = .0003) mutations independently predicted OS. Combining cytogenetic risk and mutations in these 7 genes, 39.1% of patients could be assigned to a “go-go” tier with a 2-year OS of 66.1%, 7.6% to the “no-go” group (2-year OS 2.8%), and 3.3% of to the “slow-go” group (2-year OS of 39.1%; P < 10−5). Across 3 independent validation cohorts, 31.2% to 37.7% and 11.2% to 13.5% of patients were assigned to the go-go and the no-go tiers, respectively, with significant differences in OS between tiers in all 3 trial cohorts (HDF [Hauts-de-France], n = 141, P = .003; and SAL [Study Alliance Leukemia], n = 46; AMLSG [AML Study Group], n = 223, both P < 10−5). The ALFA decision tool is a simple, robust, and discriminant prognostic model for AML patients ≥60 years of age treated with intensive chemotherapy. This model can instruct the design of trials comparing the 7 + 3 standard of care with less intensive regimens.

Acute myeloid leukemia (AML) with t(8;21) (q22;q22) is considered to have favorable risk; however, nearly half of t(8;21) patients are not cured, and recent studies have highlighted remarkable genetic heterogeneity in this subset of AML. Here we identify somatic mutations in additional sex combs-like 2 (ASXL2) in 22.7% (25/110) of patients with t(8;21), but not in patients with inv(16)/t(16;16) (0/60) or RUNX1-mutated AML (0/26). ASXL2 mutations were similarly frequent in adults and children t(8;21) and were mutually exclusive with ASXL1 mutations. Although overall survival was similar between ASXL1 and ASXL2 mutant t(8;21) AML patients and their wild-type counterparts, patients with ASXL1 or ASXL2 mutations had a cumulative incidence of relapse of 54.6% and 36.0%, respectively, compared with 25% in ASXL1/2 wild-type counterparts (P = .226). These results identify a high-frequency mutation in t(8;21) AML and identify the need for future studies to investigate the clinical and biological relevance of ASXL2 mutations in this unique subset of AML.

Mutation of the MYD88 gene has recently been identified in activated B-cell-like diffuse cell lymphoma and enhanced Janus kinase/signal transducer and activator of transcription (JAK-STAT) and nuclear factor κB (NF-κB) signaling pathways. A whole exome-sequencing study of Waldenstrom macroglobulinemia (WM) suggested a high frequency of MYD88 L265P mutation in WM. The genetic background is not fully deciphered in WM, although the role of NF-κB and JAK-STAT has been demonstrated. We analyzed MYD88 mutation in exon 5 and characterized the clinical significance of this genetic alteration in 67 WM patients. Clinical features; immunophenotypic markers; and conventional cytogenetic, fluorescence in situ hybridization, and single nucleotide polymorphism array data were analyzed. MYD88 L265P mutation was acquired in 79% of patients. Overall, we have identified alteration of the MYD88 locus in 91% of WM patients, including 12% with gain on chromosome 3 at the 3p22 locus that included the MYD88 gene. Patients with absence of MYD88 mutation were WM characterized with a female predominance, a splenomegaly, gain of chromosome 3, and CD27 expression. Importantly, inhibition of MYD88 signaling induced cytotoxicity and inhibited cell growth of cell lines issued from patients with WM. In conclusion, these results confirm a high frequency of MYD88 L265P mutation in WM. The discovery of MYD88 L265P mutation may contribute to a better understanding of the physiopathogeny of WM.

Mutations of the CCAAT/enhancer binding protein alpha (CEBPA) gene have been associated with a favorable outcome in patients with acute myeloid leukemia (AML), but mainly in those with a normal karyotype. Here, we analyzed the impact of associated cytogenetic abnormalities or bad-prognosis fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) in 53 patients with CEBPA+ de novo AML treated in the Acute Leukemia French Association trials. We found that only those with a normal karyotype and no FLT3-ITD displayed the expected favorable outcome. In this context, relapse-free, disease-free, and overall survival were significantly longer than in corresponding patients without the CEBPA mutation (P = .035, .016, and .047, respectively). This was not observed in the context of an abnormal karyotype or associated FLT3-ITD. Furthermore, after adjustment on age, trial, and mutation type, these features were independently predictive of shorter overall survival in the subset of patients with CEBPA+ AML (multivariate hazard ratio = 2.7; 95% confidence interval, 1.08-6.7; and 2.9; 95% confidence interval, 1.01-8.2; with P = .034 and .05, for abnormal karyotype and FLT3-ITD, respectively).

To the editor: The CCAAT/enhancer binding protein α (C/EBPA) is a stage-specific transcription factor that controls proliferation and differentiation toward a myeloid and against a T-lymphoid fate.[1][1] Nonconditional targeted disruption of C/EBPA leads to a selective early block to granulocyte

Fanconi anemia (FA) is a genetic condition associated with bone marrow (BM) failure, myelodysplasia (MDS), and acute myeloid leukemia (AML). We studied 57 FA patients with hypoplastic or aplastic anemia (n = 20), MDS (n = 18), AML (n = 11), or no BM abnormality (n = 8). BM samples were analyzed by karyotype, high-density DNA arrays with respect to paired fibroblasts, and by selected oncogene sequencing. A specific pattern of chromosomal abnormalities was found in MDS/AML, which included 1q+ (44.8%), 3q+ (41.4%), −7/7q (17.2%), and 11q− (13.8%). Moreover, cryptic RUNX1/AML1 lesions (translocations, deletions, or mutations) were observed for the first time in FA (20.7%). Rare mutations of NRAS, FLT3-ITD, MLL-PTD, ERG amplification, and ZFP36L2-PRDM16 translocation, but no TP53, TET2, CBL, NPM1, and CEBPα mutations were found. Frequent homozygosity regions were related not to somatic copy-neutral loss of heterozygosity but to consanguinity, suggesting that homologous recombination is not a common progression mechanism in FA. Importantly, the RUNX1 and other chromosomal/genomic lesions were found at the MDS/AML stages, except for 1q+, which was found at all stages. These data have implications for staging and therapeutic managing in FA patients, and also to analyze the mechanisms of clonal evolution and oncogenesis in a background of genomic instability and BM failure.

Mutations of the ten eleven translocation 2 gene (TET2) have recently been reported in myelodysplastic syndrome and myeloproliferative neoplasms. We analyzed the incidence and prognostic value of TET2 point mutations and other genomic alterations by direct sequencing and single nucleotide polymorphism microarray analysis in 111 de novo acute myeloid leukemia, who had all achieved complete remission (CR). Mutations were observed in 19 (17%) of the 111 patients compared with 10 (27%) of 36 patients who had failed to achieve CR (P = .2). In the 111 patients who had achieved CR, TET2 alterations were only significantly associated with NPM1 mutations but not with other pretreatment characteristics. TET2 gene status was not significantly correlated with disease-free survival and overall survival, both in the entire cohort and in patients with normal karyotype.

Background. Core binding factor (CBF) acute myeloid leukemia (AML) includes AML with t(8;21) and inv(16) leading to RUNX1-RUNX1T1 or CBFB-MYH11 fusion genes. These recurrent genetic abnormalities are both associated with disruption of genes encoding subunits of the CBF, a heterodimeric transcription factor involved in hematopoiesis. Although the fusion proteins appear to be crucial for the leukemogenic process, considerable experimental evidence indicates that they are not sufficient to induce AML on their own. Due to their high sensitivity to chemotherapy with high complete remission rates and their relatively favorable outcome, CBF-AML is considered to have a good prognosis. Nonetheless, about 30-40% of these patients relapse after standard intensive chemotherapy. In this context, identification of additional genetic or molecular abnormalities could allow better understanding of CBF-AML leukemogenesis, prediction of clinical outcome and identification of novel therapeutic targets. Methods. This study focuses on 73 patients with CBF-AML [43 t(8;21) and 30 inv(16)-AML] enrolled in the pediatric trial ELAM02. Single nucleotide polymorphism array (SNP-A) was performed for all patients using Cytoscan® HD arrays according to the manufacturer instructions. In order to distinguish somatic from constitutional SNP-A lesions, we excluded known copy number abnormalities (CNA) if there was >50% overlap with variants from public database, except for breakpoints-related alterations. Interstitial uniparental disomies (UPD) Results. Among the 73 cases, 145 SNP-A lesions were found in 58 patients (81%) with a median of 2 lesions per case (range, 0-8). CNA was more frequent (84 losses, 47 gains) than UPD (n=14). No significant difference was noted between the number of CNA and UPD in inv(16) and t(8;21)-AML. Small lesions were common at breakpoints involved in the t(8;21) and inv(16) (respectively 4/43 and 6/30). Additional recurrent CNA mostly involved entire chromosomes, chromosomal arms or large chromosomal regions. Del(9q) and loss of sex chromosome were restricted to t(8;21)-AML (respectively 6/43 and 20/43). Trisomy 22 was restricted to inv(16)-AML (2/30). Other recurrent CNA included trisomy 8 (3/43 vs 1/30) and gains of 13q (2/43 vs 1/30) in both subtypes, gains of 1q and del(2q) in t(8;21)-AML (each 2/43). Del(7q) was among the most common aberrations regardless of subtype (7/43 and 7/30). The minimally deleted region of 7q contained 57 genes including MLL3 and EZH2. Additionally, we found focal deletions of IKZF1 in one patient, NF1 in another and 3 deletions of CCDC26. Except for known mutations (KIT, RAS, FLT3), NGS did not reveal any other alterations in inv(16)-AML. By contrast, t(8;21)-AML was marked by the frequency of mutations in ASXL1/2 (8%/24%) and cohesin genes SMC1A, SMC3, RAD21, STAG2, NIPBL (27% combined). Mutations were also detected in epigenetic-related genes EZH2 (5%), TET2 (8%), IDH1/2 (5%) and WT1(11%). Conclusions. SNP-A karyotyping of 73 pediatric CBF-AML revealed several recurrent alterations, with differing distribution between the 2 subgroups. Moreover, t(8;21) and inv(16)-AML appeared to have distinct mutational profiles, leading us to consider them separately for future studies. We recently reported high frequency of ASXL mutations in t(8;21)-AML and their absence in inv(16)-AML (Micol, Duployez and Boissel et al, Blood 2014). We now report high frequency of mutations in cohesin genes with the same distribution. Recent description of functional relations between cohesin and polycomb proteins, together with our results, suggest an important pathway in t(8;21) leukemogenesis. Concurrent ASXL and cohesin mutations were found in several patients, suggesting they could cooperate in some cases. Interestingly, ASXL mutations were exclusive of del(7q), suggesting that disruption of the ASXL-associated proteins MLL3 and EZH2 could be of great interest in the physiopathology of t(8;21)-AML. Finally, correlations with clinical outcome are in progress. Disclosures No relevant conflicts of interest to declare.

The nucleoside analogue cytarabine (AraC) has served as the backbone of acute myeloid leukemia (AML) treatment for nearly forty years. About one-third of expressed genes are abnormally spliced in AML yet alternative exon usage (AEU) plays a role in the plasticity of tumor cells and may influence the response to treatment. Here the exon expression profiles of the erythroleukemia K562 cell line were compared to that of its AraC-resistant variant K562/AraC through Affymetrix HTA2 exon arrays. 5140 exon events harbored by 2583 genes distinguished the 2 cell lines. Among these, the skipping of TET2 exon 2 was identified in K562 cells sensitive to AraC whereas TET2 gene expression remained unchanged at the whole transcript level. The results were confirmed by exon-specific RTPCR (ESPCR). Microarray analysis did not evidenced any significant change in mRNA splicing for the 10 remaining exons of the TET2 gene. TET2 is a dioxygenase that catalyzes the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) and promote DNA demethylation. TET2 somatic mutations occur in about 25% AML, distributing across the whole coding sequence without obvious hot spots. These mutations decrease TET2 enzymatic activity by truncating the protein or affecting its catalytic activity. TET2 exon 2 is spliced in a mutually exclusive manner with exon 1 yet it is used as an alternative promoter (https://fasterdb.lyon.unicancer.fr/). However, TET2 exon 2 is not translated into protein and its role in TET2 regulation is still unknown. Having found that AraC sensitive cells harbor the spliced TET2 isoform, we investigated whether or not skipping of TET2 exon 2 correlate with disease outcome in AML patients treated with AraC-based intensive chemotherapy (AraC-IC). The discovery cohort included 106 consecutive AML patients treated with AraC-IC (median age 57.91, 64 males). RNA was extracted from bone marrow MNCs and assayed for TET2 exon 2 skipping through real-time quantitative ESRTPCR (qESRTPCR) amplification of E1E3 (spliced) and E2E3 (unspliced) TET2 isoforms. TET2 exon 2 skipping was quantified by calculating the ratio E1E3/E2E3. For statistical analysis, the ratio E1E3/E2E3 was dichotomized using median value as the cutoff value. Skipping of TET2 exon 2 was associated with a significantly lower response rate: 65% vs 92%, p = 0.001 but with a significantly lower relapse rate: 39% vs 85%: p In conclusion TET2 exon 2 skipping is associated with a low relapse rate and possesses a strong favorable prognostic impact in AML treated with AraC-CTI. The mechanism linking this alternative exon usage with resistances to AraC are currently investigated while our results suggest that the determination of TET2 exon 2 splicing status might assist risk stratification. Disclosures Nicolini: Novartis: Consultancy.

Background. Whole-genome sequencing has revealed MYD88 L265P and CXCR4 mutations (CXCR4mut) as the most prevalent somatic mutations in Waldenstrom’s macroglobulinemia (WM). Interestingly, while MYD88 L265P mutation may be considered as a founder event because of it high frequency in WM. CXCR4 is a G-protein-coupled receptor that promotes migration and activation of several pathways including RAS, Akt and NFKB. CXCR4 S338X may have a prognostic impact with induction of mechanisms of resistance. Our aim was to screen CXCR4 mutation in a large cohort of WM at diagnosis and in the relapse setting, and to analyze the genomic landscape of WM using targeted next generation sequencing (NGS) and genome wide single nucleotide polymorphism array (SNPa). Method. BM samples of 96 WM (mean age: 68 years) were analyzed. Tumoral DNA was extracted following CD19 B cell selection. Paired samples (tumor/T lymphocytes) were used as an intra-individual reference. In 22 pts, sequential tumor samples collected >6 months apart were analyzed. CXCR4 mutation was analyzed by sanger sequencing (SaS) and/or targeted NGS, along with MYD88L265P, CD79A, CD79B, N RAS, K RAS, PTEN, NOTCH1(exon34). Genome-Wide Human SNP Array 6.0 (Affymetrix chips) was performed in 52 pts, and analyzed with UCSC Genome Browser HG18 assembly. CN-LOH (copy neutral- loss of heterozygosity) and CNA (copy number aberration) were mapped using console 3.02 software (Affymetrix). Flow cytometry was performed to assess CXCR4 and CD49d (VLA4) expression. Results. We have first identified all CXCR4 mutations to be located in the C-terminal domain using NGS at a mean depth of 2000X to scan the coding exons of CXCR4 in a first group of 34 WM. We have next screened the C-terminal domain of CXCR4 in 96 WM using SaS; and confirmed CXCR4 mutations in 24% of WM (CXCR4mut). Among the CXCR4 mutations observed, 10 and 13 were non-sense (CXCR4NS) and frameshift mutations (CXCR4FS), respectively. All mutations were heterozygous and led to a truncated receptor protein. The most frequent mutation was the C1013G (S338X) mutation (5/96) followed by C1013A (S338X) (4/96). No CN-LOH was observed at CXCR4 locus nor variation of copy gene number (gain or deletion) in our cohort using SNP array. Interestingly, CXCR4mutwas associated to higher expression of CXCR4 protein by flow cytometry, independently of the type of CXCR4 mutation (n=45) (p=0.03); with no impact on the expression profile of the integrin VLA4 (CD49d) which directly interacts with CXCR4. Finally we attempted to delineate the genomic signature related to presence of CXCR4mut using targeted NGS for genes involved in RAS pathway (PTEN, K Ras, NRAS), TLR pathway (MYD88) and BCR pathway (CD79A, CD79b, CARD11). All CXCR4mut had MYD88L265P mutation except one case. We have identified no mutational signature in RAS pathway related to CXCR4mut. We then sought to understand whether the mutational profile might change overtime in WM using longitudinal samples. Variation of variant allele frequency of CXCR4 was observed in 3/22 cases. Interestingly, we have found that loss of CXCR4 mutation was concomitant to progression of BCR mutant clone, suggesting occurrence of CXCR4 mutation in a different clone from BCR pathway mutation and may be mutually exclusive to mutations in the BCR pathway in WM. Finally, the CXCR4mutWM are characterized by a higher frequency of gain of chromosome 4 (p Conclusion. Our data has confirmed the high frequency of CXCR4 mutation in WM. We identified a genomic signature associated to their presence. Interestingly we found presence of CXCR4 and BCR pathway mutations to be mutually exclusive and to participate to clonal evolution over time. Although this remains to be validated in a larger cohort, it seems that WM is characterized with multiple alterations of molecules pertaining to several pathway leading to NFkB deregulation in WM rather than to a unique deregulation of the signaling pathway. Disclosures No relevant conflicts of interest to declare.

Background: The invention of Next Generation Sequencing (NGS) has spurred research into human diseases, especially in the field of malignancy. In acute myeloid leukemia (AML), a plethora of novel alterations have been identified, including mutations in epigenetic regulator genes (e.g. IDH1, IDH2, DNMT3A), genes coding for proteins of the cohesin complex (e.g. SMC1A, SMC3, STAG2) and spliceosome genes (e.g. SF3B1, U2AF1). Although the diagnostic and prognostic implications of many of these alterations are not yet clear, there is increasing evidence that several of them might have major implications for understanding the disease biology or for patient-treatment. Thus, there is increasing need to reliably detect these mutations in large patient groups in clinically relevant time-frames and at an affordable cost. Due to the large number of genes to be screened, amplicon-based NGS represents an attractive detection method. Although, several assays have been reported, integrating different numbers of genes, it is currently unclear whether they really allow reliable detection of alterations in a reproducible way. Here we report our results from a round robin comparison of the detection of known AML-variants using a highly multiplexed, single tube assay coamplifying a total of 568 amplicons covering 54 entire genes or hot spot gene regions involved in leukemia (TruSight Myeloid sequencing panel; Illumina), with respect to the sensitivity, reproducibility and quantitative accuracy. Material and Methods: Ten European laboratories routinely involved in molecular AML diagnostics participated in this study. All groups performed two sequencing runs, each containing 8 samples. These samples were centrally aliquoted and distributed, the analyses were done in a blinded fashion. Six out of the 8 samples on each run were derived from a set of 9 samples composed of DNA isolated from the blasts of 18 different newly diagnosed AML patients mixed at a 1:1 ratio, with 50 ng of DNA being used for the library preparation. Three of these 9 samples were analyzed in replicate in separate runs by each group. The remaining two samples were a commercial test DNA containing 10 known single nucleotide variants (SNV) or insertion/deletion (InDel) alterations with defined variant allele frequencies (VAF) between 4 and 25% and DNA derived from the OCI-AML3 AML cell line (mutant for DNMT3A and NPM1). Sequencing was performed on MiSeq NGS systems (Illumina) using 2x151 bp-runs. Sequencing data were analyzed by all laboratories using the VariantStudio software (Illumina), with the threshold for mutation calling set at 3%. Results: Analysis of data quality indicated that 85% of the samples met the predefined acceptance criteria (>=95% amplicons with at least 500 reads/amplicon), the median coverage was 7379 reads/amplicon (range 0-47403 reads). Of the 9 mutations present in the positive control, 7 were called at least once in the two replicates by all labs, two mutations with a VAF of 5% were missed by 1 and 4 participants, respectively. Overall, the VAF calls for this sample showed a high level of accuracy across the participants (median coefficient of variation 5%, range 0-22.5%) as well as excellent intra- and inter-laboratory reproducibility (Fig.1). In total, the 9 primary leukemic samples contained 43 known variants in 19 genes, including all commonly mutated genes in AML, i.e. CEBPA, DNMT3A,RUNX1, NPM1, FLT3, WT1. For these samples, the sensitivity was 95.7%. Based on the entire data set (positive control and leukemic samples), the calculated sensitivity of the assay for known variants with an expected VAF>=5% was 93.3%. The rate of non-calls was slightly higher for InDels (14/179; 7.8%) than for SNVs (25/407; 6.1%; P=.47). Two 57-bp long insertions in FLT3 exons 14/15 were not called, which is expected due to the specifications of the assay (max. detectable InDel length Conclusions: This inter-laboratory comparison shows a high sensitivity and impressive quantitative accuracy of NGS-based characterization for known variants down to a minor VAF of 5%. Although additional optimization in individual parameters might be necessary, these initial results clearly indicate that rapid comprehensive molecular characterization of patient samples appears feasible, even in the clinical setting. Figure 1 Figure 1. Disclosures Thiede: AgenDix GmbH: Equity Ownership, Research Funding; Illumina: Research Support, Research Support Other. Bullinger:Illumina: Research Support Other. Hernández-Rivas:Illumina: Research Support Other. Heuser:Illumina: Research Support Other. Preudhomme:Illumina: Research Support Other. Lo Coco:Illumina: Research Support Other. Martinelli:Illumina: Research Support Other. Schuh:Illumina: Research Support Other. Enjuanes:Illumina: Research Support Other. Lea:Illumina: Research Support Other. Schlesinger:Illumina: Employment.

Introduction As clinical trials for relapsed Acute Myeloid Leukaemia (AML) patients do not accurately reflect the daily clinical reality, data regarding the outcome of these patients is scarce. We thus conducted a retrospective analysis to quantify the prospects of salvage treatment of primary refractory or first-relapse AML patients and to assess the contribution of allograft and intensive treatment regimens with respect to major risk groups in a real-life setting. Methods We performed a retrospective analysis of 163 patients diagnosed from 2005-2012, in 5 haematological centres in the north of France (Lille, Amiens, Roubaix, Valenciennes and Lens). We considered every patient in that time frame who was treated following an intensive pathway. Statistical analysis as performed using Kaplan-Meier survival analysis and logrank test in the SPSS software Results The mean age at diagnosis was 45 (range 16-70 years) and the median age at relapse was 48 (ranging 17-70 years). The median time from diagnosis to relapse was 8 months. 20.6% of patients were considered primary refractory (relapse within 60 days from diagnosis). The median overall survival was 28 months (95% CI was 17-38 months). There was no statistically significant survival difference between primary refractory patients and first relapsed patients. Unsurprisingly, survival was significantly (p Discussion This data suggests that for patients with favourable disease delaying transplant to first relapse and treating them with intensive salvage regimen is a valuable option. High risk patients still perform poorly. The relative low representation of these patients (23.6%) is probably due to the fact that these patients are transplanted upfront and often not treated using intensive regimens at relapse. Discussion remains for the intermediate patients. In our study, although numbers are small, they do not seem to behave similarly. The outcome of intermediate I patients resembles more unfavourable patients: this should be considered when discussing both upfront transplantation and management at first relapse. Conclusion This data suggests that delaying transplant for low risk patients is feasible and associated with a good outcome. Salvage intensive chemotherapy and transplant is an effective approach for these patients. Intermediate I patients behave like unfavourable patients and should thus be considered for upfront transplantation and experimental treatments. Disclosures: Preudhomme: CELGENE: Research Funding. Quesnel:CELGENE: Research Funding. Berthon:CELGENE: Research Funding.

Background CN-AML with NPM1 mutation accounts for 25% of AML cases and is individualized as a provisional entity in the WHO classification. In this large AML subset, additional gene mutations could influence patient outcome, like it has been shown for bad-prognosis FLT3 internal tandem duplication (FLT3-ITD). We thus reviewed the characteristics and outcome of 409 patients with de novo NPM1-mutated CN-AML treated in sequential ALFA studies. Methods Patients were treated between 1990 and 2012 (median follow-up, 31 months) according to ALFA-9000 (#16), 9801 (#91), 9802 (#89), 9803 (#19) and 0701 (#84) published protocols or in the ongoing 0702 study (#110). In addition to NPM1, the following mutations were centrally assessed using standard-PCR methods and Sanger sequencing: FLT3-ITD, mutations of FLT3 tyrosine kinase domain (FLT3-TKD, i.e. FLT3D835/I836), IDH1R132, IDH2R140, IDH2R172, TET2 (exons 3-11), and DNMT3A (exons 8-9, 11-23). Primary endpoint was cumulative incidence of relapse (CIR) after censoring the 73 patients who received allogeneic stem cell transplantation (SCT) in first CR at SCT time. Analyses were performed with multiple imputations of missing values, followed by multivariable logistic regression or Cox model. Results Median age was 52 years (17-79). Median white blood cell count (WBC) was 23.5 x 109/L (0.8-377). Gene mutations are indicated in Table 1. Multivariable analyses showed that: 1) FLT3-ITD was associated with higher median WBC (60 versus 18 x 109/L, P Conclusions This study confirms the heterogeneity of patients with NPM1-mutated CN-AML, independently of the known poor prognosis of FLT3-ITD. Higher WBC was associated with FLT3-ITD, IDH2R140 and TET2 mutations, but did not represent per se an independent risk factor for relapse. Higher incidence of unfavorable non-R882 DNMT3A mutations in older patients may explain, at least in part, why older patients do worse than younger patients in this peculiar AML subset. A potential favorable prognostic role of FLT3-TKD mutations in these patients needs to be confirmed. Disclosures: No relevant conflicts of interest to declare.

Abstract 1288 Background. Approximately 30% of the patients who fulfil the criteria of Waldenström macroglobulinemia (WM) are diagnosed while asymptomatic, and will not require immediate therapy; these cases are called indolent WM (IWM). However, patients with a disease-related event will be considered for therapy, these cases are called symptomatic or aggressive WM (AWM). The physiopathology of these 2 groups remains unclear, and the mechanisms of progression have not been fully understood so far. We hypothesized that a gene signature that differentiates these two categories could be identified to better understand the underlying mechanisms of progression of WM. Methods. Seventeen patients diagnosed with WM (8 IWM and 9 AWM) were included in this study. We selected tumour cells from the bone marrow (BM) using mononuclear cell isolation, then B cell enrichment (B cell isolation kit, Myltenyi-Biotec, USA). The purity was confirmed by flow cytometry. Total RNA was extracted using the Trizol method. Gene expression profiling was performed using U133A arrays (Affymetrix, USA). Gene expression was normalized using the RMA algorithm. We ranked genes by fold-change of expression levels on a first series of 11 patients (5 IWM and 6 AWM) calculated with the ‘limma’ package in R. Next, we used a supervised classification to establish a gene expression profile to distinguish IWM from AWM. Therewith, we validated this profile on an independent set of 6 patients (3 IWM and 3 AWM). We then performed a pathway analysis using Ingenuity® analysis software. We confirmed gene expression deregulation with qRT-PCR on 3 candidate genes in the first series of patients. Genome-wide detection of copy number alteration and loss of heterozygosity were performed on 13 of the 17 WM cases, using the Genome-Wide Human SNP Array 6.0 (Affymetrix, USA). Finally, we investigated the functional consequences of the deregulation of these candidate genes in BCWM1 and MWCL1, both B cell lines originated from WM. Survival was studied using a colorimetric method with MTS (Promega, USA). Proliferation was analyzed using incorporation of a nucleoside analog (EdU) into DNA during active DNA synthesis (Invitrogen, USA). Results. The differential analysis has identified 82 probes, corresponding to 48 genes, significantly deregulated and capable of differentiating samples from IWM and AWM in an unsupervised classification. Moreover, with a supervised classification, this gene expression profile accurately classified 94% of the 17 WM samples, including the 6 WM of the independent validation set. The two molecular networks that appeared to play a major role in the physiopathology of IWM versus AWM were the plasma cell differentiation pathway and the AKT pathway. We have then identified 3 key genes in those 2 pathways, BACH2 and CIITA on the one hand and PTEN, respectively. We have then confirmed the deregulation of these gene expression levels by qRT-PCR in 3 IWM and 4 AWM; these 3 genes were over-expressed in IMW relatively to AMW. BACH2 is a B-cell-specific transcription factor known to be a tumour suppressor gene. It was shown that BACH2 reduces proliferation and induces cell death when over-expressed in B lymphoma tumour cells. We have thus pharmacologically over-expressed BACH2 in BCWM1 and MWCL1 and significantly reduced the proliferation and the survival of the two cell-lines. Further studies using BACH2 specific overexpression with lentiviral infection are underway, in vitro. The data will be presented at ASH. In order to further study the mechanisms of deregulation of BACH2 in IWM and AWM, we have conducted a genome wide SNP array study of 13 patients. Among those, 7 patients (4 IWM and 3 AWM) demonstrate a deletion of long arm of chromosome 6 (del6q), the most frequent chromosomal abnormality in WM. BACH2 gene is located on the 6q15 locus. Interestingly, we found that 3 out of the 3 AWM had a del6q that took in the 6q15 region, whereas 3 out of 4 of the IWM had a del6q preserving the 6q15 region. Therefore, haploinsufficiency could participate in the under-expression of BACH2 in aggressive WM; this hypothesis will be verified by using DNA qRT-PCR of BACH2. Conclusion. To the best of our knowledge, we have identified for the first time a specific gene expression signature that differentiates IWM and AWM. We have exposed several genes from this dataset, including BACH2, which is a candidate to better understand the underlying mechanisms of progression of WM. Disclosures: No relevant conflicts of interest to declare.

Abstract 297 Background. Current models of cancer progression are based on evolution and clonal selection i.e. evolution of tumour cells inducing expansion of cells that acquire genetic lesions over time, related to ongoing mechanisms of genomic instability. An initiating event may be followed by gene mutation, copy number alteration (CNA) or copy neutral loss of heterozygosity (CN-LOH) that drive tumor progression and emergence of mechanisms of resistance to drugs. WM is a lymphoproliferative disorder characterized by bone marrow (BM) infiltration of lymphoplasmacytic cells that secrete monoclonal IgM antibody. The clinical course is characterized either by an indolent or smoldering status or a symptomatic profile that needs chemotherapy to control the progression of tumour cells. The majority of patients (pts)evolved from indolent to symptomatic, and the mechanisms of progression of WM are not fully understood to date. We hypothesized that we could gain insights into clonal evolution underlying disease progression of WM on a paired serial analysis of WM samples using genome wide SNPa, that allow both the detection of LOH and CNA. Method. BM samples of 19 untreated pts with WM (12 males, mean age: 67 years, 11 symptomatic pts) were analysed. All patients had Genome-Wide Human SNP Array 6.0 (Affymetrix chips) on at least two sequential tumor samples > 6 months apart (42 samples from 19 patients with two to three points). Tumoral DNA was extracted following CD19 B cells selection. Paired samples (tumor/normal T lymphocytes) were used as an intra-individual reference to identify germline polymorphisms. Size, position and location of genes were identified with UCSC Genome Browser HG18 assembly, LOH and CNA using genotyping console 3.02 software (Affymetrix). FISH analysis was performed to detect deletion 6q; 13q14, 11q22, TP53, trisomy 4 and 12 chromosomal aberrations using Vysis probes. P53 and MYD88 mutation were analyzed by sanger sequencing. Results. At initial sampling, SNPa detected a total of 76 CNA genetic aberrations (range 0 – 24 per genome) including 22 gains and 54 losses; 85% of patients had MYD88 L265P mutation. During the follow-up of all indolent WM that remained indolent, we haven't observed any new genetic aberrations, gain or loss (either CNA or CN-LOH) (n=8, mean follow-up: 63 months, range: 16–107 months). Among the 11 remaining patients (mean follow-up: 44.2 months, range: 7–92 months), a clonal evolution was observed in 6 cases. Three of them were symptomatic and acquired CNA or CN-LOH during clinical evolution: one case with acquisition of several CN-LOH, including one at 3p22 involving MYD88 locus associated to a mutation, one case with an additional deletion 7q31 which was observed in a patient with a chromothripsis at diagnosis, one case with emergence of subclonal del17p. The two remaining patients evolved from smoldering to symptomatic with a major variation in CNA number. Finally, loss of CNAs displayed at diagnosis was observed in one patient after chemotherapy and during indolent follow-up. In 5 cases, no detectable CNA changes were identified between the initial and subsequent sample at relapse. Conclusion. Our results using high resolution SNP array support the hypothesis that a symptomatic WM disease will favour genetic clonal evolution of tumor cells. In our study, 3 distinct genotypic patterns were observed: (i) absence of genomic variation in stable smoldering WM disease or in symptomatic relapsed patient. (ii) unstable genotype in symptomatic or asymptomatic patients switching to symptomatic suggesting high risk tumors that are less stable and more prone to change with time. (iii) loss of abnormalities suggesting that chemotherapy eradicated the dominant clone or that a genetically distinct relapsed clone has emerged. This study refines our understanding of the dynamic genetic changes in progressive WM. Further confirmation of the role of some candidate genes is underway. Disclosures: No relevant conflicts of interest to declare.

Abstract 659 Background: NPM1 mutations and WT1 expression are frequently identified in acute myeloid leukemia (AML). Both markers have been reported as suitable molecular target for minimal residual disease (MRD) monitoring. Recently, Gemtuzumab Ozogamicin (GO) has been shown to improve event free survival and overall survival in adult AML. Aims: To follow MRD in AML patients with NPM1 mutations (NPM1mut) and/or WT1 expression (WT1+) and treated in the randomized ALFA 0701 GO trial conducted by the Acute Leukemia French Association (Castaigne et al, Lancet 2012), in order to validate their prognostic significance and to evaluate the effect of GO on MRD response. Methods: The ALFA 0701 trial included 278 patients aged 50–70 years with newly-diagnosed de novo AML, randomized to receive or not five doses of 3 mg/m2 GO, on day 1, 4 and 7 of standard induction chemotherapy and on day 1 of two consolidation chemotherapy courses, respectively. A total of 77 NPM1mut (35 in control arm, 42 in GO arm) and 178 WT1+ (87 in control arm, 91 in GO arm) AML patients were studied. WT1 + at diagnosis was defined as a ratio WT1/ABL transcript above 25% in bone marrow (BM) or 5% in peripheral blood (PB) samples. PB and BM samples were collected at diagnosis, after induction and after each consolidation course. MRD levels were assessed using cDNA-based real-time quantitative PCR (RQ-PCR) and reported as the ratio NPM1mut or WT1 transcript/100 ABL transcript. Occurrence of a MRD level below the threshold (NPM1mut Results: 178 patients (favorable cytogenetics: 4%, intermediate: 69%, unfavorable: 27%) were WT1+. WT1 transcript levels at diagnosis had no prognostic impact on complete remission (CR) rate or CIR. In the 104 patients who achieved CR, post-induction WT1 MRD level was predictive of relapse: 18-month CIR was 70% (95%CI: [49% - 84%]) in poor responders (n=31) versus 38% [26% - 50%] in good responders (n=73) (p=0.0001). When adjusted on cytogenetics, randomization arm and FLT3-ITD status, WT1 MRD remained independently associated with relapse (HR: 2.43, [1.25 – 4.75], p=0.009). Similarly, NPM1mut transcript levels at diagnosis had no prognostic impact on CR rate or CIR. Among the 67 NPM1mut patients who achieved CR, post-induction NPM1mut MRD level was predictive of relapse: 18-month CIR was 64% [45% – 70%] in poor responders (n=50) versus 25% [10% – 42%] in good responders (n=17) (p=0.0007). When adjusted on randomization arm and FLT3-ITD status, NPM1 MRD remained independently associated with relapse (HR: 4.2, [1.2 – 14.3], p=0.02). We were able to evaluate both MRD markers in 53 patients after induction (NPM1+/WT1+ n=16, NPM1+/WT1- n=24, NPM1-/WT1- n= 13). 18-month CIR was significantly different in the 3 groups (respectively 69% [38% - 87%], 40% [19% - 60%] and 25% [5% - 51%], p=0.008). In patients with poor MRD response assessed by NPM1mut, the presence of a positive WT1 MRD level was associated with a higher CIR. NPM1 MRD good response was more frequently observed in GO arm as compared to control arm (OR: 4.38 [2.12 – 9.03], p Conclusion: Our study confirms the predictive value of MRD based on NPM1 mutations and WT1 expression in patients treated in the ALFA 0701 randomized trial. In multivariate analysis, WT1 and NPM1 MRD levels remained independent prognostic factors for relapse. We showed that treatment with GO significantly improved MRD response in adult AML. Disclosures: Castaigne: Wyeth: Consultancy.

Abstract 2533 Acute myeloid leukemia (AML) is a heterogeneous disease because of different leukemogenic mechanisms and variable response to antileukemic treatment. In addition to age and leukocytosis at diagnosis, cytogenetic abnormalities are key factors to assess prognosis. Several gene alterations such as those involving CEBPa, NPM1 and FTL3 have been identified and have improved the classification of AML. However, variability remains and cannot be completely explained. In this study, we performed SNP array analysis on a cohort of 128 AML patients in order to identify new genetic alterations and potential new candidate genes involved in leukemogenesis or disease progression. Patients were classified according to MRC cytogenetics (26 favourable; 77 intermediates, 15 adverse, 10 no informative caryotype). Patients were aged from 11 to 65 years and distributed across all French-American-British (FAB) classes except M3 (8 M0, 25 M1, 38 M2, 26 M4 and 11 M4Eo, 12 M5, 2 M6), Paired DNA was extracted from bone marrow aspirates obtained at diagnosis and after achieving complete remission (CR), and analyzed using Affymetrix Genome-Wide Human SNP Array 6.0 to distinguish acquired from constitutional genetic abnormalities. Copy number variations (CNA) were validated on a custom Agilent microarray (Human Genome CGH Microarray 105k). Data were analyzed using Affymetrix' Genotyping Console 3.0.2, Agilent's GeneSpring GX. Statistical analyses were executed using R version 12.1. The Cox proportional hazard regression model was used to relate genetic abnormalities to treatment outcome, with karyotype included as the second covariate. Time was censored at transplantation date if bone marrow transplantation was performed. We found 210 genomic abnormalities in 74 patients: 197 CNA and 13 copy neutral losses of heterozygosity (uniparental disomy or UPD), resulting in 1.6 abnormalities on average per patient (range 0 to 17). Among CNA, deletions were more frequent than gains (130 vs. 66). CNA spanned from 8kb to 191MB (median of gains 24MB, median of losses 2MB). 116 of them had not been detected by conventional cytogenetics. UPD spanned from 23MB to 150MB (median, 33 MB). Abnormalities were located over all chromosomes except for chromosome14 and were particularly frequent on chromosomes 2, 7, 11, 16, 17, and 21 (54% of all abnormalities). We defined 72 minimal common regions which were altered in at least 2 patients. Among the 43 common regions shorter than 5Mb, 16 contain at least one gene reported in AML or cancer. CNA and UPD were distributed across all FAB subtypes. Of note, 7 of 8 patients with AML M0 had 1 or more abnormalities. Among the 74 patients with CNA or UPD, 36 had an intermediate caryotype. We found no significant association of number of abnormalities with known gene alterations known to influence prognosis (i.e., CEBPa, FLT3itd or NPM). As expected, there were significantly more alterations in patients with a favourable or adverse caryotype (P=0.0045, Fisher's exact test). Furthermore, the number of genetic abnormalities was significantly associated to disease-free and to overall survival (P=0.010 and 0.0016, respectively). This remained significant in a multivariate analysis including karyotype (P=0.046 and 0.01, respectively) CNA or UPD was detected in 56% of AML patients who achieved remission of these 45% have intermediate cytogenetics. We identified 43 minimal common regions shorter than 5MB, which were altered in at least 2 patients and 37% of these regions involve genes previously reported in AML or cancer. Increased genomic alterations were significantly associated with favorable and adverse cytogenetics and with disease-free and overall survival. Prognostic significance of number of abnormalities remains significant after adjustment for cytogenetics. DNA SNP array analysis may be useful to better define prognostic subgroups in addition to conventional cytogenetics and may identify candidate genes implicated in leukemogenesis or disease progression. Disclosures: No relevant conflicts of interest to declare.

A strong correlation between trisomy 13 and AML1 mutation has been recently reported in AML, particularly in M0 FAB subtype (Dicker et al, Silva et al). Whereas the exact mechanism of this coincidence has not been completely clarified, this correlation suggests a probable cooperation of these two events in leukemogenesis. FLT3, located on the 13q12 region, has been proposed to be implicated by over expression induced by gene dosage effect. However, trisomy 13 remains a rare event in AML and its association with AML1 mutations is not constant. To better document this association, we studied, a cohort of patients with trisomy 13 by array-CGH, which is a powerful tool to detect cryptic alterations as small size gene copy number variations (CNV). In addition we searched for AML1 mutations and performed the correlation with FLT3 alterations. We studied 12 AML with trisomy 13 (7 of which were M0 FAB subtype) and 6 additional patients affected by non-AML hematological malignancies (2 T-ALL, 1 NHL and 3 MPD). We performed direct sequencing of the exons 3 to 8 of AML1. Array-CGH was performed on 10/12 AML patients using Agilent CGH-Array 185k with pooled normal DNA as reference. GEP was performed using HG-U133 plus 2.0 Affymetrix Array on 5 patients characterized by trisomy 13 and AML1 mutations. AML1 mutations were observed in 8/18 patients including 6 of the 7 M0 AML, which represents a particularly high rate even in this subtype (86%). On the contrary, none of the non-AML patients harbored AML1 gene mutation. FLT3 locus analysis in the 12 AML patients showed the following results: None FLT3-ITD was observed whereas 5 FLT3-D835 mutations were detected (42%). FLT3 over expression was observed by GEP on 4/5 analyzed patients all without FLT3-D835 mutation. The remaining patient presented a FLT3-D835 mutation. CGH study confirmed trisomy 13 in 9/10 AML cases with weak amplitude for 4 patients, strongly suggesting that trisomy 13 was a sub-clonal anomaly, in accordance with caryotype results. Only one other recurrent abnormality was observed by CGH in 3 AML patients: 2 gains and one deletion affecting chromosome 21, all including AML1 locus. Moreover, CNV incidence was strong in these patients traducing a great genomic instability: there were 34 CNV gains and 23 deletions equally distributed all along the genome with a size extending from 300kb to complete chromosome. Among the interstitial CNV loci were included the following genes (CD68, CDH1, CDX2, CTNNA3, DOCK2, EIF3S9, EIF4A1, ERG, GNA12, IGF2, KLRC3, MAD1L1, MAFA, MKL2, VAV2) that could perhaps play a role in leukemogenesis. Here we confirmed the strong correlation between AML1 mutation and trisomy 13. Trisomy 13 is often sub-clonal indicating this alteration could be an additional event contributing to leukemia progression. In accordance with previous reported data, FLT3 over expression or FLT3-D835 mutation could cooperate with AML1 mutation. However, alteration of genes located on other CNV could not be excluded.