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The t(4;14)(p16.3;q32) leads to the ectopic expression of two oncogenes, the Multiple Myeloma Set Gene (MMSET) and the Fibroblast Growth Factor 3 (FGFR3). It is found in 15% of patients with multiple myeloma (MM) and indicates a poor prognosis. To identify the clinico-biological features associated with this adverse prognosis, we prospectively studied a series of 79 patients with t(4;14) MM.Between March 2002 and July 2007, a t(4;14) was detected by real time quantitative PCR of the IGH/MMSET and FGFR3 transcripts in 79 patients. The clinico-biological data were analysed at diagnosis and at relapse. Response rate and time to progression (TTP) after successive lines of chemotherapy, and overall survival (OS), according to beta2m and haemoglobin level, and expression of FGFR3, were evaluated. Among the 79 patients (median age:55 years), 18 (22.8%) had a MGUS or smouldering myeloma and 61 (77.2%) had a symptomatic MM. At diagnosis, no clinical or radiological feature was significantly associated. As expected, 15 patients (18%) had a t(4;14) without expression of FGFR3. IgA isotype was found in 41% and del(13) in 90% of patients. Contrasting with previous studies, only 52% of symptomatic MM had elevated beta2m (>3mg/L). Fourty-two patients (53.1%) received autologous stem cell transplantation (ASCT). In those, the overall response (OR) was 90%. Among those responders, 16% had a complete response (CR) and 53% had a very good partial response (VGPR). Median TTP after ASCT was only 12 months. At relapse, aggressive features (plasmocytoma, cytopenias, acute renal failure, circulating plasma cells) were observed in 23% of cases. After 2nd line chemotherapy, response rate was only 49% and TTP was 6.4 months. Noteworthy, there was a trend toward a better TTP in patients treated with bortezomib as compared to thalidomide in 2nd line therapy (p=0.06). Median OS after ASCT was 31 months, independently of FGFR3 expression and isotype. OS was not significantly different in patients with and without high beta2m (>3mg/L) or low hemoglobin (

The Bone Marrow Failure (BMF) syndromes comprise a variety of distinct acquired or inherited clinical entities. Early distinction between syndromes has clear implications in the disease management and outcome. Fanconi anemia (FA), the most frequent cause of inherited BMF, is usually associated with congenital abnormalities, progressive cytopenia, chromosome fragility, and cancer susceptibility. However, due to a high clinical variability and/or the potential emergence of revertant hematopoietic cells (somatic mosaicism), identifying patients with FA or ruling out this diagnosis can be challenging. If undiagnosed, FA patients who initially present with bone marrow failure will die of toxicity after standard-dose conditioning regimens for HSCT. In this study, we evaluated FA diagnosis in patients with BMF but no clear initial evidence of FA, using of a combination of classical and innovative tests in blood and fibroblasts. A cohort of 82 patients with BMF and no strong clinical evidence of FA was analysed (patients with a clear FA diagnosis were not included). Based on the likelihood of an underlying inherited condition associated with the BMF, we classified patients in 3 groups: those likely to have idiopathic aplastic anemia (IAA) [n=38, group 1], those likely to have a constitutional condition other than FA [n=26, group 2], and those likely to have IAA but who had isolated clinical findings which could also be present in FA [n=18, group 3]. Chromosome breakage test and analysis of the FA/BRCA pathway by FANCD2 immunoblot were performed in PBL in all patients [n= 82]. To overcome potential somatic mosaicism, skin primary fibroblasts were analysed [n= 52]. Also, to rule out FA/BRCA downstream groups, we developed a new flow cytometry test based on MMC-sensitivity in fibroblasts. In total, 6 patients with FA were identified: 1/38 in group 1 (aplastic anemia at 10 yo, no positive clinical findings), 2/26 in group 2 (one with MDS at 48 yo with precocious menopause and vocal cord neoplasia at 38 yo; one with hypoplastic MDS at 50 yo), and 3/18 in group 3 (one with short stature and aplastic anemia at 37 yo; one with MDS and borderline physical abnormalities at 26 yo; and one with a single cafe-au-lait spot and aplastic anemia at 10 yo). Chromosomal breakage tests in PBL were sufficient to diagnose 4 of these FA patients (further classified as FA core using FANCD2 immunoblot). Additional fibroblast analyses were necessary to identify 2 more FA patients (both with complete somatic mosaicism) and, importantly, to definitely exclude FA diagnosis in other patients. In conclusion, underdiagnosing FA is rare if careful history and physical exam are done together with chromosome breakage test in PBL. However, in clinical situations where the suspicion of FA persists despite negative breakage tests, then fibroblasts should be tested. Because no cases of FA were found among patients with IAA and negative breakage tests in PBL, we suggest that FA screening can be limited to this technique in this population (group 1). The strategy here presented allowed us to identify a few unexpected FA cases in a cohort of BMF patients, and importantly, to definitely rule out FA in others, with clear clinical impact for patients who undergo HSCT.

D-type cyclins are key regulators of progression through G1 phase of the cell cycle. Strong expression of at least one of the three D cyclins is common in human cancers. However, while the cyclin D1 and D3 genes (CCND1 and CCND3) are recurrently involved in genomic rearrangements, especially in mantle-cell lymphoma and multiple myeloma, no clear involvement of the cyclin D2 gene (CCND2) has been reported to date in human malignancies. In T-cell acute lymphoblastic leukemia (T-ALL), the T-cell receptor genes TCRA/D and TCRB are frequently involved in chromosomal rearrangements. In order to identify new genomic rearrangements and oncogenes in human T-ALL, we performed an interphasic FISH screening of T-ALL cases using TCR flanking probes. Using this approach, we identified two new chromosomal translocations: t(7;12)(q34;p13) and t(12;14)(p13;q11), involving the TCRB and TCRA/D loci, respectively. Molecular analysis of the breakpoint sequences demonstrated the involvement of the CCND2 locus at 12p13. Expression analysis using RQ-PCR and immunoblotting demonstrated dramatic cyclin D2 overexpression in the translocated cases (n=3) compared to other T-ALLs (total, n=86), whereas other genes located near the translocation breakpoints were not deregulated on microarray analysis. To further evaluate expression in T-ALL with respect to normal T-cell differentiation, we analyzed CCND2 expression in purified subpopulations from normal human thymus. CCND2 levels were downregulated through progression from the early stages of normal human T-cell differentiation and transition to beta-selection. In the most immature T-ALLs, a moderate CCND2 expression was observed, consistent with their differentiation stage, while low expression was found in other T-ALL. By contrast, the massive and sustained expression in the CCND2-rearranged T-ALL cases strongly suggested an oncogenic role due to the TCR translocation. T-ALL oncogenesis is a multi-step process; we here found that the TCR-CCND2 translocations were associated with other oncogene expression (TAL1, HOXA, or TLX3/HOX11L2), NOTCH1 activating mutations, and/or CDKN2A/p16/ARF deletion, showing that cyclin D2 dysregulation could contribute to multi-event oncogenesis in various T-ALL groups. In conclusion, this report is the first clear evidence of a direct involvement of cyclin D2 in human cancer due to recurrent somatic genetic alterations. This reinforces the view that the strong expression of cyclin D2 which is detected in various types of cancer can contribute to oncogenesis, and points to cyclin D2 as a potential target for therapy in these tumors.

Fanconi Anemia patients have an increased toxicity of drugs used in conditioning regimen (CT) for hematopoietic stem cell transplantation (HSCT), due to defective DNA repair processes. This observation has led to diminish the doses of drugs (such as cyclophosphamide and busulphan) and irradiation in the preparative regimen. We and others have reported that genetic polymorphisms, which interfere with metabolisms of drugs used in CT, are associated with toxicities after HSCT. In order to test the hypothesis that probably Fanconi patients have a difference in prevalence of pharmacogene polymorphisms, we have compared the samples of 49 Fanconi patients (median age of 6,2 years) with 107 samples of normal population of the same ethnic group. Polymerase chain reaction (PCR) and restriction-digestion protocols were used to identify most of the polymorphisms. We have selected pharmacogenes knonwn to interfere with the metabolisms of drugs used in the CT or GVHD prophylaxis. The following gene polymorphisms were analyzed: P450 cytochrome family [CYP2B6*2(C64T), *4(A785G), *5(C1459T), *6(G516T)]; glutathion-S-Transferases [GSTM1 (null), GSTT1(null), GSTP1(G313A)], Thiopurine-S-methyl transferase [TPMT *3B (G460A) and *3C (G238C)], Multiresistance-drug [MDR1 (C3435T)], Methylenetetrahydrofolate reductase [MTHFR (C677T, A1298C)], Vitamin D receptor [VDR (ApaI, TaqI and BsmI)]. All gene polymorphisms studied were found in the same frequency in FA as in normal controls, except polymorphisms of GSTP and GSTT. In fact, FA patients had a higher frequency of mutant alleles (AG or GG) of GSTP (86%; n=42) compared to 51% (n=55) in normal controls (p

Late events are observed in B-lineage ALL with no plateau seen if a sufficient observation period is provided. The prognosis of childhood ALL associated to the t(1;19)(q23;p13)/E2A-PBX1 translocation has improved over the two last decades. The purpose of this study is to define the kinetics of relapse in a large cohort of children and to raise the possibility of a plateau in that entity. We identified 110 children treated for a B-lineage ALL harbouring a t(1;19(q23;p13) and/or E2A-PBX1 fusion transcript from 1987 to 2004 in the FRALLE protocols (FRALLE 83: n= 3, FRALLE 87–89: n=18, FRALLE 92 (phase II): n=10, FRALLE 93: n=48, FRALLE 2000 (still opened: n=31). Analysed features included: male sex (50, 45%), age (median: 6.9y; Q1–Q3: 3.5–12), CNS disease (4, 3.5%), leucocytosis (median: 21.4 G/L; Q1–Q3: 10.8–53.4), D8 poor prednisone response (PPR) (5/79, 6%), M1 D21 marrow response (103, 94%), and CR (108, 98%). For an accurate comparison, the subgroup of pts with t(1;19) included in the FRALLE 93 protocol (n=48) was compared to the 1,147 children aged more than 1 y with B- lineage ALL treated in the same protocol. Significant differences in the t(1;19) cohort were: excess of girls (65% vs. 46%; p= .01), and older age (median: 7 vs. 4.7; p= .01)). No differences in early response to treatment (D8, D21, D35) were seen. All the 110 patients received chemotherapy only, except 3 who received an autologous BMT for D8 PPR as recommended by the FRALLE 93 protocol. At a median FU of 65 months, 18 events have been recorded including 17 relapses (bone marrow: 13, CNS: 4) and 1 secondary tumor (abdominal Burkitt lymphoma) giving a 5 y and 10 y EFS of 78.7%. If “modern era” only is considered (> 1992), 5 y and 10 y EFS is 85.5%. Relapses occurred at a median time of 422 days (range: 72–899) in the whole t(1;19) cohort. In those from FRALLE 93, the median time to relapse (MTTR) was 280 days, that is significantly lower than the one observed in the control group defined above (MTTR = 894 days, Wilcoxon test, p = .01). Conclusions: We confirm that a high cure rate is now associated to that subgroup of childhood ALL. The main finding is that no event except from the secondary Burkitt lymphoma (1705 d) was registered after 30 months from diagnosis in that cohort of 110 children with t(1;19)/E2A-PBX1 translocation associated ALL. It is then conceivable to announce cure at 36 months in that subgroup with an extremely low possibility of mistake, thus much earlier and with more confidence than in the other B-lineage ALL.

Early diagnosis of Fanconi Anemia (FA) in patients with bone marrow failure is critical for optimal clinical management. However, the remarkably high clinical variability and the potential emergence of revertant hematopoietic cells (somatic mosaicism) can obscure and delay the diagnosis of FA. Here we addressed FA diagnosis in a prospective series of adult and pediatric patients who presented with bone marrow failure without clear overall clinical picture of FA. Sixty-six patients were classified into three groups: (1) bone marrow failure likely to be congenital, based on dysmorphic features or a family history [n=18], (2) aplastic anemia likely to be idiopathic [n=32], (3) patients with intermediate clinical features not classified into the former groups [n=16]. Of note, FA patients with typical clinical features were not included in the present study. FA diagnosis was evaluated using chromosome breakage test and FANCD2 immunoblot in PHA-stimulated-PBL. In addition, skin primary fibroblasts were analysed in order to overcome potential hematopoietic FA reversion. For that purpose, and considering that chromosome breakage tests are barely efficient in fibroblasts, we used FANCD2 immunoblot and also developped a new flow cytometry test based on MMC-sensitivity in fibroblasts (to detect downstream FA/BRCA groups). Using these approaches, we detected FA in 4 previously undiagnosed patients: a 35-years old patient from the congenital-like group; a 10-years old patient presenting as an idiopathic aplastic anemia without any FA signs; and two patients from the intermediate group: a 10-years old patient with an isolated thrombocytopenia, and a 50-years old patient presenting with pancytopenia/MDS and complete hematopoietic reversion. Importantly, FA diagnosis was definitely excluded in all other patients. In conclusion, we could identify a few unexpected FA cases in a series of patients with bone marrow failure. Therefore, the comprehensive use of a large set of tests is useful for accurate FA diagnosis. Classical chromosomal breakage tests in PBL appeared to be sufficient to exclude FA in idiopathic aplastic anemia, whereas fibroblast analysis can be necessary to definitely diagnose or exclude FA in other patients.

We have identified a new recurrent chromosomal translocation, targeting the major homeobox gene cluster HOXA in human T-cell acute lymphoblastic leukemia (T-ALL). Four cases were characterized using a combination of FISH, Southern blot, breakpoint region sequencing, and a large scale expression analysis of a series of T-ALL. Specific RQ-PCR analysis of the HOXA1 to HOXA13 transcripts showed that the whole HOXA gene cluster expression was dramatically deregulated in the HOXA-rearranged cases, and also in the MLL and CALM-AF10-related T-ALL, strongly suggesting that HOXA genes are oncogenic in these types of leukemia. The HOXA-rearranged cases were included in a general portrait of T-ALL based on large scale expression analysis, showing that a new homogeneous T-ALL subgroup is defined by this chromosomal rearrangement. Moreover, patterns of gene expression associated to the distinct T-ALL oncogenic subgroups were compared with gene expression in normal human thymic sub-populations (11 purified sub-populations). Inappropriate use or perturbation of some specific molecular networks involved in thymic differentiation could be detected in the T-ALL cells. Also, we found that abnormal, frequently ectopic, expression of at least one developmental gene, including HOXA, TLX1/HOX11, TLX3/HOX11L2 and a few more, could be identified in most of the T-ALL cases. Our data strongly support the view that the abnormal expression of developmental genes, including the prototypical major homeobox genes HOXA in some cases, is critical in T-ALL oncogenesis.

FA is an autosomal recessive disorder with diverse clinical symptoms, including developmental anomalies, progressive bone marrow failure, and a predisposition to the development of malignancies. FA cells are hypersensitive to DNA cross-linking agents, and display chromosomal instability and defective DNA repair. FANCG-deficient pts accounts for 10% of all FA pts, and are a high risk FA population for acute myeloblastic leukemia. FANCG protein is part of the FA nuclear core complex, interacting with FANCA and FANCF. It can also interact with FANCD1, RAD51, and CYP2E1, suggesting roles in homologous recombination DNA repair and oxidative DNA-damage protection. However, FANCG lack apparent homology to other proteins involved in such processes. To address whether FANCG mRNA expression level was associated with some microarray-based functional signatures using Affymetrix HG-U133A Genechips, we first used a dataset including 111 T-acute lymphoblastic leukemia (T-ALL) samples. This large series was selected because FA pts do not usually develop T-ALL (risk of FANC gene mutation/inactivation low), FANCG is highly expressed in thymus and lymphoblasts, FANCG gene expression was significantly varying across these samples. We first selected samples having the lowest (FANCGlow) and highest (FANCGhigh) FANCG expression levels (n=15 per group, median expression values: 36 vs. 329), and identified genes differentially expressed between these 2 groups using SAM (fold change>3; qvalue.75) when analyzing all 111 T-ALL samples. These genes were mainly involved in cell proliferation, mitotic cell cycle and its regulation (DNA replication, traversing Start control point, G2/M transition, chromosome segregation and cytokinesis), and were also representing cellular components involved in such processes (kinesin complex, spindle, and kinetochore). FANCA, RAD51, RAD51C, and PIR51 were also among the genes having expression patterns close to FANCG one. In addition, 11 of these 55 genes, all involved in DNA replication, were also correlated with FANCA (>.75). A robust regression analysis identified CKS1B, CCNB2, RFC5, MELK, STK18, and CENPF as the genes most significantly associated with FANCG expression profile (p2; qvalue Conclusion: these results suggest that the FA core complex, and especially FANCG and FANCA, is involved in DNA replication and mitosis.

Recently, Croop et al[1][1] reported the feasibility of mobilizing and collecting peripheral blood CD34+ progenitor cells from patients with Fanconi anemia. The authors performed successful apheresis collection in 6 of 8 patients. They showed that prolonged granulocyte colony-stimulating factor (G

The Saccharomyces cerevisiae checkpoint protein Rad9 is required for transient cell-cycle arrest and transcriptional induction of DNA-repair genes in response to DNA damage [1]. It contains a carboxy-terminal tandem repeat of the BRCT (BRCA1 carboxyl terminus) motif, a motif that is also found in many proteins involved in various aspects of DNA repair, recombination and checkpoint control [2,3]. We produced yeast strains expressing Rad9 in which the BRCT domain had been deleted or which harboured point mutations in the highly conserved aromatic residue of each BRCT motif. Rates of survival and checkpoint delay of the mutants after ultraviolet (UV) irradiation were essentially equivalent to those of rad9 Δ (null) cells, demonstrating that the BRCT domain is required for Rad9 function. Rad9 hyperphosphorylation, which occurs after DNA damage [4–6], was absent in the BRCT mutants, as was Rad9-dependent phosphorylation of the Rad53 protein. A two-hybrid approach identified a specific interaction between the Rad9 BRCT domain and itself. Biochemical analysis in vitro and in vivo confirmed this interaction and, furthermore, demonstrated that the Rad9 BRCT domain preferentially interacted with the hyperphosphorylated forms of Rad9. This interaction was suppressed by mutations of the BRCT motifs that caused null phenotypes in vivo , suggesting that Rad9 oligomerization is required for Rad9 function after DNA damage.

SummaryD-type cyclins form complexes with cyclin-dependent kinases (CDK4/6) and promote cell cycle progression. Although cyclin D functions appear largely tissue specific, we demonstrate that cyclin D3 has unique functions in lymphocyte development and cannot be replaced by cyclin D2, which is also expressed during blood differentiation. We show that only combined deletion of p27Kip1 and retinoblastoma tumor suppressor (Rb) is sufficient to rescue the development of Ccnd3−/− thymocytes. Furthermore, we show that a small molecule targeting the kinase function of cyclin D3:CDK4/6 inhibits both cell cycle entry in human T cell acute lymphoblastic leukemia (T-ALL) and disease progression in animal models of T-ALL. These studies identify unique functions for cyclin D3:CDK4/6 complexes and suggest potential therapeutic protocols for this devastating blood tumor.

Abstract 163 In acute myeloid leukemia (AML), both cytogenetic and molecular abnormalities are strongly associated with prognosis. In particular, in cytogenetically normal AML (CN-AML), FLT3-ITD (internal tandem duplication) carries adverse prognostic factor whereas NPM1 or CEBPA mutations are associated with favorable outcome. Recently, mutations of the ten eleven translocation 2 gene (TET2) have been reported myeloid neoplasms. We evaluated the frequency and prognostic value of TET2 alterations, in a cohort of 111 de novo AML patients. We studied 111 patients aged between 15 years and 69 years with previously untreated de novo AML who had reached complete remission (CR) using intensive chemotherapy. 28 of them also received an allogenic bone marrow transplantation in first CR. Analysis of TET2 sequence variation was performed by direct sequencing of PCR products from 111 genomic DNA samples obtained at diagnosis. Frameshift and nonsense variations were all scored as mutation whereas missense mutations were retained when observed at diagnostic but absent in the CR paired sample obtained. Previously identified single nucleotide polymorphisms (SNP) were not considered. TET2 anomalies were numbered according to Genebank reference FM992369. Paired diagnosis and CR genomic DNAs were analyzed using Affymetrix Genome-Wide Human SNP Array 6.0 (Affymetrix, Santa Clara, CA). Data were analyzed using Gene Chip Genotyping Console 3.0.2 and Partek Genomics Suite (www.partek.com/). Comparisons were made by Fisher's exact test for binary variables and the Mann-Whitney‘s test for continuous variables. Disease Free Survival (DFS) and overall survival (OS) were calculated according to the Kaplan-Meier method. Comparisons regarding DFS and OS were performed with the log-rank test. 24 acquired TET2 mutations were observed in 19 of the 111 (17%) de novo AML patients, suggesting the alteration of the two TET2 alleles in 5 patients. They included 21 different events: 6 frameshift, 7 non-sense and 11 missense mutations. Four of the missense mutations were located in conserved regions and 7 outside. All of them were detected in the diagnostic sample but were absent in the paired remission sample. Except for two missense mutations (S282F, T492S) both detected in two patients, no recurrent TET2 mutation was observed. Acquired mutations were spread over all exons. No case of uniparental disomy (UPD) was observed and only one patient presented a small deletion of 60Kb in the TET2 gene locus without TET2 mutation. No significant difference was observed between patients with or without TET2 alterations for gender, age, hemoglobin level, platelet count, FAB subtypes distribution and cytogenetics according to MRC classification, but there was a trend for higher WBC count in patients with TET2 alteration. No significant association was observed between TET2 mutations and FLT3 or CEBPA alterations. However, TET2 alterations were significantly associated with NPM1 mutations (p=0.032). In the entire patient cohort, no difference in DFS or OS was seen between patients with and without TET2 alteration. However, a significantly worse DFS was observed for patients presenting TET2 mutations within the subgroup of patients with NPM1 mutations (3y-DFS: 0% vs 66.4%, 95% CI [45.6–87.2], p=0.008) Considering both the favorable prognosis of NPM1 mutations without FLT3-ITD in CN-AML and the absence of clear association between FLT3-ITD and TET2 alterations in this study, prognostic value of the genotype characterized by NPM1 mutation without FLT3-ITD or TET2 alteration (NPM1+FLT3-ITD-TET2-) was compared to other patients within CN-AML group (N=54). NPM1+FLT3-ITD-TET2- patients showed a significantly better DFS and OS compared to other patients in CN-AML group (3y-DFS: 82.1%, 95% CI [59.1–100] vs 37.3%, 95% CI [20.2–54.3], p=0.01; 3y-OS: 80.8%, 95% CI [56.1–100] vs 42.3%, 95% CI [23.3–61.3], p=0.04). In conclusion, we observed point mutations of TET2 in 17% of patients, whereas TET2 deletion or UPD are very rare. In our study, TET2 mutations were clearly associated with NPM1 mutations and carried a negative prognostic impact in this subgroup. Screening for TET2 mutations may improve the characterization of CN-AML and help to identify within the low-risk subgroup with NPM1 mutation and without FLT3-ITD, patients at high risk of relapse. Disclosures: Fenaux: Celgene: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Ortho Biotech: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Cephalon: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Abstract 975 Fanconi anemia (FA) is a rare genetic condition characterized by congenital abnormalities, chromosome fragility, progressive bone marrow failure during childhood, and cancer susceptibility. FA patients experience a high risk to develop myelodysplasia (MDS) and secondary-type acute myeloid leukemia (AML) during their teens or in young adulthood. Severity of the cytopenia, excess of blast cells and presence of a cytogenetic clone in the bone marrow are usual criteria to undertake hematopoietic stem cell transplantation. In order to investigate the pattern of chromosomal and genomic abnormalities during bone marrow progression in FA and their association to MDS/AML, we analyzed bone marrow samples from FA patients using a wide panel of chromosomal and molecular techniques including DNA microarrays and oncogene sequencing. This series of FA patients was enriched in patients older than 18 year-old and/or with morphological or karyotypic abnormalities on the follow up BM aspirate. 57 FA patients were included, aged 4 to 57 yo (median 18); FA groups were FA-A (n=49), FA-G (n=1), FA-D2 (n=1), FA-D1 (n=1) and undertermined (n=5). Bone marrow morphology was hypoplastic/aplastic anemia (n=20), MDS (n=18, mainly RCMD and RAEB according to the WHO 2008 classification), AML (n=11), or no abnormality except the usual mild dyserythropoiesis of FA (n=8). Bone marrow samples were analyzed by karyotype, FISH, high density array-CGH and/or SNP-arrays with respect to the paired fibroblast DNAs, and by sequencing of selected oncogenes and tumor suppressor genes. A specific pattern of genomic abnormalities due to unbalanced translocations was found in the 29 MDS/AML, which included 1q+ (44.8%), 3q+ (41.3%), -7/7q (17.2%), and 11q- (13.8%). Moreover, cryptic abnormalities (translocations, deletions or mutations) of the RUNX1/AML1 gene were evidenced for the first time in FA, in 6 out of the 29 patients with MDS or AML (20.7%). By contrast, mutations of FLT3-ITD, MLL-ITD, and N-RAS, but not TP53, CBL, TET2, CEBPa, NPM1, and FLT3-TKD, were rarely found. Frequent homozygosity regions were evidenced by SNP-array in 11 patients, but the analysis of the paired fibroblast DNA and the constitutional FANC mutations demonstrated that they were not related to somatic copy-neutral loss of heterozygosity but to consanguinity. Importantly, the RUNX1/AML1 and other chromosomal/genomic abnormalities were found at the MDS and AML stages only, except for 1q+ which could be found at any stages including normal bone marrow morphology. In our experience 1q+ does not predict systematically a transformation into MDS/AML in the following years. These data have important implications, not only for the cytogenetic staging of the bone marrow cells in FA patients with an impact for therapeutic managing, but also as a basis to investigate the multistep clonal selection and related oncogenesis in patients with hypoplastic bone marrow and genomic instability, with potential relevance for non-FA patients. Disclosures: Gluckman: Cord-use: Membership on an entity's Board of Directors or advisory committees.

SMZL usually has an indolent clinical course. Transformation to aggressive lymphoma occurs in only 4-5% of cases. Tools to identify patients facing a more aggressive clinical course and targeted therapies are needed. In the last years, we have characterized SMZL by DNA profiling (Blood 2011) and RNA/miRNA expression profiling (Mod Path 2013), and shown that somatic mutations of NOTCH2 and other genes involved in the pathway are frequent and mutually exclusive with mutations affecting NKFB pathway (JEM 2012). Here, we report genome-wide DNA promoter methylation profiling of 137 SMZL samples with its clinical and biologic implications. Methods A test series of 101 samples, including 3 SMZL cell lines (Karpas1718, VL51, SSK41) and 98 clinical SMZL cases from 10 Centers and, a validation set of 36 SMZL cases from 2 additional Centers were analyzed with the Illumina Infinium HumanMethylation27 arrays, as described (BJH 2013). Gene GEP was available for a subset of 11/101 cases. Methylation profiles were correlated with the presence of the copy number aberrations (CNAs) and somatic mutations recurrent in SMZL, IGHV mutational status, HCV infection and clinical data. Probes mapped outside CpG islands were discarded. Supervised analyses were performed using a t-test on the continuous beta values, followed by multiple test correction. For Fisher’s test, probes were classified into unmethylated (beta-values 0.3). Results Unsupervised clustering analysis of 101 SMZL samples identified 2 clusters. Cluster one (23/101, 23%) comprised the 3 cell lines and included cases characterized by a generalized higher degree of promoter methylation (High-M), and had a significantly poorer overall survival (OS) than the remaining 79/101 (77%) cases (Low-M) (P=0.048; HR 2.4; 95% CI, 1-5.8). To better understand the biology underlying this clustering, supervised analysis comparing High-M to Low-M cases was performed. By combining t- and Fisher's tests, 3200 gene probes showed differential methylation status between High-M and Low-M lymphomas. This probes signature was significantly enriched of promoters of promoter regions of PRC2-complex targets, genes involved in chromatin remodeling, and HOX and SOX family genes, and genes down-regulated by CDH1. Genes coding for subunits of the PRC2-complex (EZH2, EED, SUZ12) appeared unmethylated in their promoter regions and over-expressed by GEP in the High-M set, while EZH2-target genes and genes harboring the H3K27me3 mark had methylated promoters and down-regulated expression. A number of reported tumor-suppressor genes were highly methylated in High-M cases, including KLF4, DAPK1, CDKN2A/B, CDKN1C, CDH1, CDH2 and TIMP3. Known pro-survival lymphoma genes, such as SYK, MCL1, CARD11, BIRC5, BTK, BCL10, FOXP1, CD40, TACI and TCL1A/B, were un-methylated and over-expressed in High-M cases. The prognostic relevance of the High-M phenotype was confirmed in an independent validation set. An unsupervised clustering using the top-100 differentially methylated probes obtained by t-test in the test series identified two clusters with one (11/36, 30%) bearing methylation profile similar to High-M and a poorer OS (P=0.038). All the samples were then pooled to improve the statistical power of further analyses regarding the impact of methylation on the clinical course and on the association with biologic variables. High-M was significantly associated with poorer OS (P=0.008; HR 2.58; 95% CI, 1.2-5.4), transformation into aggressive lymphoma (33% vs 0.02%, P In a Cox regression model adjusted for the variables significantly associated with outcome at univariate analysis [High-M, age>60, Interguppo Italiano Linfomi (IIL) SMZL score, HCV infection], High-M (HR 16; 95% CI, 1.3-201) and IIL SMZL score (HR 4.5; 95% CI, 1.3-15) retained their prognostic significance on OS (P Conclusions Genome-wide promoter-methylation profiling in splenic MZL identified a subset of patients with poorer OS, characterized by a high promoter methylation and by specific genetic and biologic features. These data may provide the rational to explore epigenetic drugs in SMZL. *, AJA and AR equally contributed. Disclosures: No relevant conflicts of interest to declare.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer that results from the malignant transformation of T-cell precursors and affects children, adolescents and adults. In T-ALL, genetic lesions in several possible oncogenes and tumor suppressors have been shown to cooperatively contribute to leukemogenesis. The TLX1 (T-cell leukemia homeobox protein-1, HOX11) oncoprotein is aberrantly expressed in in 5-10% of pediatric patients and 30% of adult T-ALL patients due to chromosomal translocations. Although many downstream protein coding targets genes of TLX1 have been identified, the non-coding network downstream of TLX1 remains elusive. In this study we expand the TLX1 regulome towards long non-coding RNAs (lncRNAs). Hereto we measured the transcriptional response of all protein coding genes and 12,000 lncRNAs following TLX1 knock down in the ALL-SIL cell line using a custom designed mRNA/lncRNA expression platform (Agilent). In addition, similar mRNA-lncRNA expression profiles of 64 primary T-ALL patient samples were generated which included five TLX1+ cases. To establish the direct transcriptional TLX1 targets, we generated TLX1 ChIP-sequencing data from ALL-SIL leukemic cells. We confirm direct regulation of previously established protein coding gene targets and de novo TLX1 motif discovery also identified RUNX1 as an important mediator of the global TLX1 transcriptional network (Della Gatta et al., Nature Medicine, 2012). Complementary to these data, our analysis for the first time establishes the TLX1 driven lncRNAome in thymocyte derived leukemic cells. Remarkably, the majority of TLX1 controlled lncRNAs were upregulated suggesting that they may be implicated in the TLX1 driven repression of protein coding gene expression. Finally, pairwise mRNA-lncRNA correlation analysis allowed functional annotation of TLX1 targeted lncRNAs. In conclusion, we present the first landscaping of the genome-wide binding pattern of TLX1 and provide evidence for a previously unestablished role of lncRNAs in the TLX1 regulatory network. Disclosures: No relevant conflicts of interest to declare.

Long noncoding RNA signatures define oncogenic subtypes in T-cell acute lymphoblastic leukemia

Cumulative evidence indicates that MYC, one of the major downstream effectors of NOTCH1, is a critical component of T-cell acute lymphoblastic leukemia (T-ALL) oncogenesis and a potential candidate for targeted therapy. However, MYC is a complex oncogene, involving both fine protein dosage and cell-context dependency, and detailed understanding of MYC-mediated oncogenesis in T-ALL is still lacking. To better understand how MYC is interspersed in the complex T-ALL oncogenic networks, we performed a thorough molecular and biochemical analysis of MYC activation in a comprehensive collection of primary adult and pediatric patient samples. We find that MYC expression is highly variable, and that high MYC expression levels can be generated in a large number of cases in absence of NOTCH1/FBXW7 mutations, suggesting the occurrence of multiple activation pathways in addition to NOTCH1. Furthermore, we show that posttranscriptional deregulation of MYC constitutes a major alternative pathway of MYC activation in T-ALL, operating partly via the PI3K/AKT axis through down-regulation of PTEN, and that NOTCH1m might play a dual transcriptional and posttranscriptional role in this process. Altogether, our data lend further support to the significance of therapeutic targeting of MYC and/or the PTEN/AKT pathways, both in GSI-resistant and identified NOTCH1-independent/MYC-mediated T-ALL patients.

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer resulting from the malignant transformation of T-cell precursors. Genetic studies in T-cell acute lymphoblastic leukemia (T-ALL) have uncovered a remarkable heterogeneity of genetic defects. Amongst these, NOTCH1activating mutations are the most frequently occurring events (>50%) in T-ALL. Since long non-coding RNAs (lncRNAs) are emerging as important players in oncogenesis, we decided to decode the NOTCH1 driven lncRNA transcriptional landscape in T-ALL and normal T-cell development. Methods and Results: RNA-sequencing was performed following pharmacological inhibition (GSI) of the NOTCH1 mutant and gamma secretase inhibitor (GSI) sensitive T-ALL cell line CUTLL1 in a time series experiment as well as for human CD34+ thymic progenitor T-cells cultured on an OP9 feeder layer with or without DL1-triggered NOTCH1 stimulation. First, we validated both model systems by confirming robust regulation of multiple canonical known protein coding NOTCH1 target genes including DTX1, NOTCH3 and NRARP. Next, we identified distinct subsets of NOTCH1 regulated lncRNAs in both experiments with an overlap of 27 commonly regulated NOTCH1 driven and previously annotated lncRNAs. An even larger number of novel, unannotated T-ALL/T-cell specific lncRNAs was found to be NOTCH1 regulated. Next, we took advantage of publically available ChIP-sequencing data for ICN1 and enhancer specific chromatin marks in CUTLL1 (Wang et al., PNAS, 2013), allowing the selection for direct regulated lncRNAs with enhancer properties. Amongst these lncRNAs, the recently described LUNAR1 lncRNA (Trimarchi et al., Cell, 2014) was present as top candidate in our dataset, thus validating this approach for further selecting bona fide NOTCH1 regulated lncRNAs. In a first step towards functional annotation of this subset of selected lncRNAs, we performed so-called guilt-by-association analysis through correlating expression levels of the individual lncRNAs with transcriptome data for all protein coding genes followed by gene set enrichment analysis in a large cohort of primary T-ALL patients. Subsequent enrichment mapping of significant gene sets yielded markedly different gene set clustering patterns for each of the individual analyzed lncRNAs, as well as distinct annotated related functionalities such as cytokine signaling, TCA-cycle, DNA replication and repair and translation. Prioritarization of lncRNAs for further functional validation was performed by measuring their expression in an extended panel of GSI-treated T-ALL cell lines (HPB-ALL, DND-41, T-ALL1 and ALL-SIL), sorted subsets of CD34+ and CD4+/CD8+ double positive thymocytes and an independent T-ALL patient cohort. Conclusion: We present the landscaping of an integrated lncRNA network acting downstream of NOTCH1 signaling in T-ALL and normal T-cells. These data pave the way towards the development of novel therapeutic strategies impacting on hyperactive NOTCH1 signaling. Disclosures No relevant conflicts of interest to declare.

Abstract 135 From December 2000 to June 2010, 1201 children with SR-BCP-ALL (age: 1–9 years, WBC Results: 1. Overall efficacy: No leukemic induction failure was observed; 4 induction deaths (0.3%) occurred leading to a 99.7% CR rate. Only 30 out 1097 evaluable pts (2.7%) had a decisional EOI MRD ≥ 1%. Eighty-two relapses have been observed (BM: 47, CNS+: 24, BM+CNS: 9, testis: 2). For the whole population 5-year EFS is 91.5% (95%CI: 89.8–93.3), 5-year OS is 97.4% (95%CI: 96.4–98.4). 5-yr EFS and OS for the A1 and A3 groups are 93.1% vs. 61.2% (p 2. Overall toxicity: 5 non leukemic deaths have been observed after CR (0.4%). Other main toxicities were attributable to ASPA (CNS thrombosis: 1.5%, grade 3–4 pancreatitis: 0.8%, allergic reaction-all grades- during DI °2: 70%). Six cases of secondary neoplasms were observed (AML: 5 pts, astrocytoma: 1 pt). 3. Randomization: 5y EFS and OS from randomization was 92.9% and 97.2% for D21 M1 pts DNR(+) versus 93.3% and 98.2% for D21 M1 DNR(-) pts (p= 0.89 and p= 0.85, respectively). Interestingly EOI MRD levels in the two arms were not different at the sensitivity cut-off of 10−2 (p=0.93) or 10−3(p= 0.74). 4. Prognostic factors: For the whole population, age < 6 years, WBC < 20.000/mm3, ETV6-RUNX1 positivity, as well as D21 marrow M1 and EOI MRD levels of 10−2 and 10−3 were all associated to EFS in univariate analysis. In multivariate analysis remain only WBC < 20.000/mm3 (p=.001), ETV6-RUNX1 positivity (p=.02), EOI MRD levels ≤ 10−3 (p=1.4×10−5). Considering only randomized pts (D21M1 pts) age Conclusions: Very good results were obtained with this VCR-DEX-ASPA oriented protocol. SR-BCP ALL pts which represents around 55 % of all children with ALL can be cured with a modest dose of anthracyclin (75 mg/m2). A further decrease in therapy based on the identified prognostic factors could be proposed. Intensification of the chemotherapy for the rare SR pts with a very high MRD has salvaged half of these pts but the addition of new drugs and/or HSCT is now to be evaluated. Disclosures: No relevant conflicts of interest to declare.