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Chromosome 11q22.3 deletion (del(11q)) is one of the most common cytogenetic alterations in CLL and usually involves both ATM and BIRC3 genes. Concomitant mutations in ATM and/or BIRC3 in the remaining allele have been associated with poor survival. Despite the encouraging efficacy of novel agents targeting BCR and BCL2 pathways, del(11q) patients still have an inferior outcome and the development of resistance to these drugs has been increasingly reported. We therefore investigated the functional impact of del(11q) together with loss-of-function mutations in ATM and/or BIRC3 and whether CLLs harboring these alterations could benefit from novel combinatorial therapies. To address these questions, we used the CRISPR/Cas9 system to generate an isogenic CLL cell line to model del(11q) derived from HG3 cells by introducing two guide RNAs targeting the 11q22.1 and 11q23.3 regions. The presence of a monoallelic deletion (size ~17 Mb) was confirmed in 100% of the cells by FISH. Truncating mutations in ATM and/or BIRC3 were introduced in the remaining allele, generating HG3 del(11q) ATMKO, del(11q) BIRC3KO and del(11q) ATMKOBIRC3KO (three clones per condition). In addition, single ATMKO and BIRC3KO mutations, or the combination of both, were introduced into both HG3 and MEC1 CLL-derived cells (three clones per condition). Functional in vitro studies revealed that del(11q) BIRC3KO cells had increased growth rates compared to del(11q) BIRC3WT clones (P We next assessed the response of these CRISPR/Cas9-edited CLL cell lines to therapy. Of note, only TP53KO clones (also generated by CRISPR/Cas9), and not del(11q) BIRC3KO cells, showed resistance to fludarabine (mean IC50 16.9 uM vs. 4.1 uM; mean apoptotic cells (5 uM) 5.5% vs. 22.5%; P Moreover, IBRU potentiated the effects of OLA in cell viability (72h) in all the del(11q) clones (combination indexes 0.69-0.85), leading to an increased necrotic cell death, as shown by annexin V/PI staining (P In conclusion, we demonstrate that del(11q) CLL cells with biallelic inactivation of BIRC3 and ATM show enhanced proliferation through activation of the non-canonical NF-kB pathway, and accumulation of DNA damage contributing to genomic instability. We show that these defects on the DDR can be therapeutically targeted by synthetic lethal approaches using PARP inhibitors either alone or in combination with BCR inhibitors, providing a rationale for the study of this combination in relapsed del(11q) CLL patients. PI15/01471 SA085U16 JCyL-MQ FEHH-MH Disclosures García-Tuñón: Novartis: Research Funding. Wu:Neon Therapeutics: Equity Ownership.

Cytogenetic abnormalities, evaluated either by karyotype or by fluorescence in situ hybridization (FISH), are considered the most important prognostic factor in multiple myeloma (MM). However, there is no information about the prognostic impact of genomic changes detected by comparative genomic hybridization (CGH). We have analyzed the frequency and prognostic impact of genetic changes as detected by CGH and evaluated the relationship between these chromosomal imbalances and IGH translocation, analyzed by FISH, in 74 patients with newly diagnosed MM. Genomic changes were identified in 51 (69%) of the 74 MM patients. The most recurrent abnormalities among the cases with genomic changes were gains on chromosome regions 1q (45%), 5q (24%), 9q (24%), 11q (22%), 15q (22%), 3q (16%), and 7q (14%), while losses mainly involved chromosomes 13 (39%), 16q (18%), 6q (10%), and 8p (10%). Remarkably, the 6 patients with gains on 11q had IGH translocations. Multivariate analysis selected chromosomal losses, 11q gains, age, and type of treatment (conventional chemotherapy vs autologous transplantation) as independent parameters for predicting survival. Genomic losses retained the prognostic value irrespective of treatment approach. According to these results, losses of chromosomal material evaluated by CGH represent a powerful prognostic factor in MM patients. (Blood. 2004;104:2661-2666)

Cytokine genes are targets of multiple epigenetic mechanisms in T lymphocytes. 5-azacytidine (5-azaC) is a nucleoside-based DNA methyltransferase inhibitor that induces demethylation and gene reactivation. In the current study, we analyzed the effect of 5-azaC in T-cell function and observed that 5-azaC inhibits T-cell proliferation and activation, blocking cell cycle in the G0 to G1 phase and decreasing the production of proinflammatory cytokines such as tumor necrosis factor-α and interferon-γ. This effect was not attributable to a proapoptotic effect of the drug but to the down-regulation of genes involved in T-cell cycle progression and activation such as CCNG2, MTCP1, CD58, and ADK and up-regulation of genes that induce cell-growth arrest, such as DCUN1D2, U2AF2, GADD45B, or p53. A longer exposure to the drug leads to demethylation of FOXP3 promoter, overexpression of FOXP3, and expansion of regulatory T cells. Finally, the administration of 5-azaC after transplantation prevented the development of graft-versus-host disease, leading to a significant increase in survival in a fully mismatched bone marrow transplantation mouse model. In conclusion, the current study shows the effect of 5-azaC in T lymphocytes and illustrates its role in the allogeneic transplantation setting as an immunomodulatory drug, describing new pathways that must be explored to prevent graft-versus-host disease.

CCND2 is highly expressed in most of multiple myeloma (MM) samples without CCND1 or CCND3 overexpression. D-type cyclins are highly homologous proteins and there is a growing body of evidence that the functions of the D cyclins are mostly exchangeable. During mouse development, the three D-type cyclins are expressed following an often mutually exclusive pattern and their function may be tissue-specific. Likewise in MM CCND1 or CCND2 expression is commonly mutually exclusive. The mechanisms by which CCND2 is upregulated in a set of MMs are not completely deciphered. In this study, we investigated the role of post-transcriptional regulation through the interaction between miRNAs and their binding sites at3'UTR in CCND2 overexpression in MM. First, we observed that ectopic transfection of MM cell lines with several miRNAs, directly targeting CCND2 3'UTR according to luciferase reporter assay, decreased the level of cyclin D2, although not in all the cell lines. This fact suggested the possible disruption of miRNA target sites. Indeed, we detected the presence of short CCND2 mRNA, both in MM cell lines and primary cells, using four different methodological approaches: qRT-PCR, Northern blot, mRNA FISH and 3’RACE PCR with product sequencing. The short CCND2 isoform was observed by qRT-PCR in the majority of patients and in all MM cell lines expressing CCND2. This finding was confirmed by Northern blot results. The abundance of each CCND2 mRNA isoform was also assessed by two-color mRNA FISH designed to discriminate the two mRNA different in length. This approach also enabled us to notice that no subpopulation of cells distinguishable by the load of one isoform with respect to another was present. The results obtained by RACE experiments in MM cell lines support the idea that changes in CCND2 3’UTR length are explained by alternative polyadenylation (APA). The functional consequences of 3’UTR shortening is the mRNA stabilization due to the loss of miRNA sites and regulatory elements located in the 3’UTR. Accordingly, the luciferase assays using plasmids harboring the truncated CCND2 mRNA strongly confirmed the loss of miRNA sites in the shorter CCND2 mRNA isoform. The short 3’UTRs lacking miRNA-binding sites have been associated with increased expression of different genes at both the mRNA and the protein level. Here, we observed significant higher level of overall CCND2 mRNA expression in those MMs with greater abundance of the shorter 3'UTR isoform. The previous intriguing observation showing that the level of cyclin D2 increased in U266 when cyclin D1 was silenced, was confirmed by our experiments. Moreover, functional analysis showed significant mRNA shortening after CCND1 silencing, suggesting that cyclin D1 could downregulate CCND2 level by modification of polyadenylation/clavage reaction. Since CCND2 expression is undetectable in myeloma cells with t(11;14), we extended our investigation to explore if DNA methylation might play a role in abolishing CCND2 expression. We observed that the CpG island more proximal to CCND2 TSS was highly methylated in MM cell lines with t(11;14). In summary, our results reveal that CCND2 expression in MM is mainly regulated by post-transcriptional mechanisms. Downregulation of specific miRNAs directly targeting CCND2 contributes to overexpression of CCND2 in a set of MM. Moreover, the shortening of CCND2 3'UTR by alternative polyadenylation with the consequent loss of miRNA binding sites is also participating in CCND2 upregulation. In fact, this mechanism seems to play a decisive role in the regulatory network between CCND1 and CCND2 in MM. Disclosures No relevant conflicts of interest to declare.

Background: In B-progenitor acute lymphoblastic leukemia (B-ALL) the identification of additional genetic alterations associated with treatment failure is still a challenge. Aims: 1.To identify genomic gains and losses in B-ALL at the time of diagnosis and to correlate these abnormalities with the genetic characteristics and the patients outcome. 2.To assess the prevalence and prognostic impact of genetic lesions in IKZF1, TP53, CRLF2, IL7R, PAX5, JAK2 and LEF1 genes in B-ALL. Methods: A total of 215 B-ALL patients were eligible for this study. 115 (53.5%) had less than Results: DNA copy number aberrations (CNAs) were observed in 96.5% of cases. Gains on chromosomes 4 (10%), 6 (10%), 10 (12%), 14 (13%), 21 (31%) and X (18%) were more common in children, whereas losses in 7p (13%) and 9p21 (24%) were frequent in adults. In children, gains on chromosomes 6, 10, 14q, 17, 18, 21 and X were associated with longer overall survival (OS) while loss on 7p and 17p were associated with shorter OS in adults (Table 1). The integrative analysis of aCGH and NGS showed that 60% of patients carried at least one alteration (deletion and/or mutation) in the seven genes analyzed. Focal deletions were common in IKZF1, CRLF2, PAX5, and LEF1 genes while broad deletions were more frequent in TP53, JAK2 and IL7R. Forty-one percent of patients harbored IKZF1 alterations (IKZF1+), 22.9% in PAX5, 18.6% in JAK2 and 11% had TP53 or CRLF2 alterations. LEF1 and IL7R genomic lesions were only present in 4.2% and 2.5% of the B-ALL, respectively. JAK2 and CRLF2 mutations were associated (p=0.01). Moreover abnormalities in IKZF1+ were associated with alterations in JAK2 (p=0.004), TP53 (p=0.04) and PAX5 (p=0.03). The presence of alterations was most frequent in high-risk (HR) B-ALL (38% vs 18%, p=0.02), although 8% low-risk (LR) childhood patients showed genetic alterations. Fourteen B-ALL patients carried mutations in TP53, CRLF2 and/or JAK2 genes: all of them were Ph-, and 11 were classified as HR. Of note, 3 cases were children. In the HR group the presence of mutation in TP53, CRLF2 and/or JAK2 was related to a shorter OS (3-year OS: 56% vs. 36%; P=0.034) and event-free survival (3-year EFS: 56% vs. 11%; P The presence of IKZF1 alterations stratified HR-Ph- B-ALL cases. Thus HR-Ph- patients with normal IKZF1 were associated with longer EFS compared to HR-Ph- B-ALL IKZF1+ (3-year EFS: 60% vs. 33%; P=0.005) (Figure 2). The presence of IKZF1 deletion was associated with shorter EFS (89% vs. 67%, P= 0.04) and increased CIR (3-year CIR: 12% vs. 33%; P=0.05) in children. Summary/Conclusions: CNAs are frequent in B-ALL and are associated with genetic subtype, age and overall survival. The integrative analysis by aCGH and NGS techniques demonstrated that alterations in IKZF1 gene and mutations in TP53, CRLF2 and/or JAK2 genes could stratify high-risk Ph- B-ALL patients. Subvention:FP7/2007-13, Nº306242-NGS-PTL; HUS272U13, JCyL,Consejería de Educación; BIO/SA31/13 Gerencia Regional de Salud, SACYL, Spain Table 1. DNA copy number aberrations (CNAs) associated with overall survival in 215 B-ALL patients Association CNA n Median (months) P value Short overall survival 7p-ChildrenAdults 19613 9615 0.05 17p-ChildrenAdults 1257 694 0.029 +19ChildrenAdults 351421 Not reached10 0.001 22q+ChildrenAdults 261214 Not reached7 Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Atypical chronic myeloid leukemia (aCML) is a chronic myeloproliferative disorder with a clinical and hematologic picture similar to chronic myelocytic leukemia (CML) but lacking Philadelphia chromosome and BCR-ABL rearrangement. Cytogenetic studies have shown either a normal karyotype or numeric

Abstract 1704 Gene expression profiling studies have been performed in MDS to better characterize these diseases. However, the molecular pathogenesis of low-risk MDS is not yet fully understood. Furthermore, the transcriptional activity is dependent on many factors including epigenetic modifications. Therefore the integration of genome-wide epigenetic regulatory marks along with gene expression levels would provide additional information regarding the biological characteristics of low-risk MDS. A total of 83 low-risk MDS patients and 36 age-matched controls were included in the study. A cohort of 18 patients with low-risk MDS and seven controls were included in a simultaneous integrative study of methylation and expression, while the whole series was used as a control group of expression data. Both the RNA and the DNA were isolated from BM mononucleate cells and hybridised with the Human Genome Expression Array (U133 Plus) from Affymetrix and MCAM Array from University Health Network (Canada), respectively. For analysis and interpretation of the hybridisation results, the R/Bioconductor program, DAVID bioinformatic resource, the web-delivered bioinformatics tool set Ingenuity Pathway Analysis and Metacore Analytical Suite were used. The results generated by expression and methylation microarrays were confirmed using Q- PCR and pyrosequencing, respectively. A total of 817 differentially methylated genes were identified as being present in low-risk MDS (p< 0.10); hyper-methylated genes (n=457) were more frequent than hypo-methylated genes (n=360). In addition, mRNA expression profiling identified 1005 genes that significantly differed between low-risk MDS and control group. Integrative analysis of the epigenetic and expression profiles revealed that 66.7% of the hyper-methylated genes were under-expressed in low-risk MDS cases. The most represented categories were regulation of apoptosis, gene expression, immune response and RNA process. BCL2, ETS1, IL27RA and DICER1, all of them hyper-methylated and down-expressed, were the most significant genes related to these functions. 1. Regarding apoptosis and BCL2, an over-expression of BCL2L11 and MYC were found in low-risk MDS. In contrast, BAX and CUX1 were under-expressed with respect to the control group. In addition, SYK gene was also hyper-methylated and under-expressed. 2. Promoter region analysis demonstrated that ETS1 transcription factor was involved in the regulation of 83 target genes included in the down-regulation signature of the low-risk MDS patients. The most significant functions of these target genes revealed that the cell-to-cell signaling and interaction pathway were prominently affected. In addition, apoptosis was identified as the function with the most number of down-regulated target genes. Therefore, the overall apoptosis pathway could be affected in low-risk MDS patients in two ways: methylation and decreased expression of BCL2 with the deregulation of related genes, as well as methylation and decreased expression of the ETS1 transcription factor with the deregulation of the apoptosis-related targets. 3. Regarding immune response, the study showed that besides IL27RA, another nine interleukins and interleukin receptors were under-expressed in the same cohort of patients: IL16, IL32, IL1RAP, IL2RB, IL6R, IL7R, IL10RA, IL10RB and IL13RA1. Three of them (IL16, IL1RAP and IL10RB) had direct genetic interactions with IL27RA. 4. Finally, the identification of DICER1 as a gene significantly altered by methylation and expression in low-risk MDS prompted us to measure the 183 miRNAs expression. A general down-regulation of miRNAs was observed in low-risk MDS cases respect to the control group (p=0.039). Our integrative analysis revealed that aberrant epigenetic regulation is a hallmark of low-risk MDS patients and could play a central role in these diseases. Furthermore, we highlight candidate DNA methylation changes associated with low-risk MDS patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 584 The Human Germinal center Associated Lymphoma (HGAL) gene is exclusively expressed in germinal center (GC) B-lymphocytes and GC-derived lymphomas. In patients with diffuse large B-cell lymphomas (DLBCL), HGAL expression identifies a subgroup of patients with biologically distinct tumors associated with improved survival. Our previous in vitro studies demonstrated that HGAL decreases spontaneous and chemoattractant-induced cell motility by activating the RhoA signaling pathway and by directly interacting and augmenting F-actin and myosin II binding. However, the major function of HGAL in GC lymphocytes remains largely unknown. Based on our previous observation of tyrosine phosphorylation of a modified ITAM motif in the HGAL by Lyn, we hypothesized that HGAL may be involved in B-cell receptor (BCR) signaling. Indeed, following BCR stimulation of two GCB-like lymphoma cell lines (Raji and VAL), we observed marked reduction of Syk, Btk and PLCγ phosphorylation upon knockdown of endogenous HGAL by specific but not control siRNAs. Concordantly, HGAL knockdown in BCR-stimulated Raji cells reduced Ca2+ mobilization and decreased NFAT transcriptional activity as analyzed by a luciferase reporter assay. HGAL expression in the BCR-stimulated HBL1 lymphoma cell line (lacking endogenous HGAL protein) resulted in increased Syk, Btk and PLCγ phosphorylation. Syk plays a major role in coupling BCR activation to downstream effectors. Endogenous HGAL was detected in immunoprecipitates of endogenous Syk and vice versa. Nanoscope microscopy studies confirmed co-localization of HGAL and Syk proteins in cell membranes, which was enhanced following BCR stimulation. In BCR-stimulated cells, Syk kinase activity was markedly increased following addition of HGAL protein as measured by an in vitro Syk kinase activity assay. To comprehensively examine HGAL effects on immune system and BCR signaling, we generated a transgenic mouse model in which HGAL is expressed under the control of the mouse Ly-6E.1 promoter in Sca1+ hematopoietic stem cells and progenitors of C57BL/6 × CBA mice. The Sca1-HGAL transgenic mice showed normal embryonic and post natal development, and at 8 weeks of age demonstrated normal lymphoid development without any significant changes in the major hematopoietic compartments (bone marrow (BM), spleen, thymus and peripheral lymph nodes) and in peripheral blood. They also exhibited normal GC development in response to a T-cell dependent antigen immunization. In contrast, at 12 months of age the Sca1-HGAL mice developed a decrease in BM immature B-cells at the expense of recirculating B-cells (B220+IgDhi) compared to the age-matched normal littermates, suggesting a defect in B-cell lymphopoiesis. All the Sca1-HGAL transgenic mice became ill from approximately 12 months of age and all died between 12 to 22 months of age with statistically shorter survival as compared to the wild type controls. Analysis of these animals showed massive splenomegaly with marked white pulp hyperplasia and presence of multiple, frequently contiguous nodules predominantly composed of polyclonal follicular (B220+CD21intCD23hi) B lymphocytes. Extra-lymphatic infiltration by similar B lymphocytes was observed in the liver, lungs and kidneys of Sca1-HGAL mice with advanced disease. IgG isotype titers in these animals tended to be higher than in the wild-type controls, reaching a statistically significant difference for the IgG1 isotype. Follicular hyperplasia in the Sca1-HGAL transgenic mice is likely attributable to increased RhoA activation and enhanced BCR signalling manifested by increased Syk phosphorylation, Ca2+ mobilization and in vitro B cell proliferation following BCR stimulation, in agreement with similar data observed in human DLBCL cell lines expressing HGAL. Gene expression profiling of lymphoid tissues confirmed significantly enhanced BCR signalling and RhoA pathway activation in Sca1-HGAL transgenic mice, corresponding to similar pathway activation in human lymphoma cell lines over-expressing HGAL. Overall, our findings demonstrate that HGAL, specifically expressed in GC B cells, enhances responsiveness to antigens by stimulating Syk kinase activity that without appropriate regulation may lead to lymphoproliferation. Further studies are needed to examine the role of HGAL in the pathogenesis of GC-derived lymphomas. Disclosures: No relevant conflicts of interest to declare.

In a previous report (López et al, ASH 2007, abstract 1916) we have demonstrated the presence of cytogenetic aberrations on mesenchymal stem cells (MSCs), the bone marrow (BM) stroma progenitors, from patients with myelodysplastic syndromes (MDS). Since there is a chance that the observed changes could be the result of the expansion in the culture process the aim of the present study was to analyze whether genomic changes could be present in non-expanded BM MSC and also to assess if the genomic changes detected could be correlated with specific subtypes of MDS. To address our first issue, in four MDS cases, MSCs were enriched by sorting mononuclear cells from BM with the following phenotype: CD45−/CD73++/CD34−/CD271+++, purity being >99%. Array based comparative genomic hybridization (array-CGH) was performed with this cell population. DNA was extracted, and after it was amplificated using the GenomiPhi kit. The postamplification cleanup was achived by ethanol precipitation using the sodiumacetate/EDTA. In order to know whether the amplified products contained actual genomics a PCR, using specific primers was performed; both test and reference DNA were amplified using the same amount of starting DNA. With this approach genomic changes were observed in this sorted cell population, with gains being more frequent than loses. The more frequently affected genomic regions were: 1q31, 10q26 and 20q13. In order to be sure that these features were also present in expanded MSCs, FISH using the same bacterial artificial chromosome (BAC) probe than used in the array-CGH studies was performed on expanded MSCs from the same patients and the same genomic alteration was observed, showing that not only expanded but also freshly isolated MSCs have genomic aberrations. Once demostrated that. uncultured MSCs were genomically abnormal we wanted to know whether these aberrations could be related to any clinical parameter. For this purpose expanded MSCs from 13 MDS patients were studied using array-CGH and an unsupervised hierarchical cluster analysis performed. Two different clusters were identified: one of them included all the 5q- syndrome patients, while the other incorporated the remaining MDS subtypes. The differences between these two clusters occurred just in 26 BAC. When looking at these clones and taking into account only regions involving at least two consecutive BACS, chromosomal regions 11q13, 17q25, 19q13, 20q13, and 22q13.2 were gained in 100% or 80% of the 5q-cases; by contrast a normal copy or a deletion of these regions were observed within the rest of patients. Our results show, for the first time, that both cultured-expanded and freshly-isolated MSC from MDS display genomic aberrations, as assessed by array-CGH and FISH, some of them specially linked to a specific clinical entity, the 5q- syndrome.

INTRODUCTION. Mantle cell lymphoma (MCL) is a well defined mature B-cell neoplasm, usually with an aggressive behaviour and a median survival of 3–5 years. It is characterized by the presence of t(11;14)(q13;q32), that leads to overexpression of CCND1. This translocation is detected by conventional cytogenetics (CC) in more than 65% of the cases. Nevertheless, cyclin D1 overexpression alone is not sufficient to give rise to a MCL. Several secondary genetic abnormalities with a potential role in the oncogenic process have been described using different molecular and cytogenetic techniques but no data from large series of MCL cases studied by CC have been correlated with survival and proliferation information. AIMS. 1. To correlate secondary chromosomal abnormalities, histological subtype and proliferative index with survival. 2. To describe the frequency of secondary chromosomal aberrations using CC and a large series of MCL patients. PATIENTS AND METHODS. We selected 145 MCL cases (94M/51F; mean age 65.4 yrs; mean survival 33 mo.), all of them with an abnormal karyotype. Histological subtype, proliferative index (Ki-67) and survival data were collected. All patients were studied by conventional GTG-banding cytogenetics, and FISH with a dual colour dual fusion CCND1/IGH probe was applied in those cases with an abnormal karyotype but without t(11;14) detected by CC. RESULTS. Translocation t(11;14) was detected as a single anomaly in 36% of patients, whereas 57% of cases displayed t(11;14) plus other additional aberrations and 6% of patients showed an abnormal karyotype without t(11;14) which was detected by FISH. We observed a total of 550 abnormalities (147 numerical and 403 structural), being the most frequent deletions of 1p (12%), 13q and 17p (10% each) and gains of 3q (9%). Recurrent breakpoints were identified at 1p31–p32, 1p21–p22, 17p13 and 1p36 (in eight, six, five and four cases, respectively). Blastoid variants of MCL displayed more karyotypic complexity with high number of structural alterations, higher number of 1p and 17p deletions, higher proliferation index and poor survival. CONCLUSIONS. 1. MCL cases associated with 17p losses (overall survival at 4 yrs: 15% vs 50%; P=0.01) and high proliferative index (15% vs 55%; P=0.005), generally corresponding to blastoid variants, display a worse outcome. 2. Our results confirm, by means of a routine technique as CC, previous observations made using more sophisticated molecular techniques in shorter series of patients.

Splenic marginal zone lymphoma (SMZL) is an indolent B cell malignancy whose diagnosis is based on lymphocyte morphology, immunophenotype and marrow and/or splenic histology. Unlike other lymphomas, there is not a common chromosomal translocation specific for SMZL, and genetic prognostic factors are poorly defined. To investigate the pattern of genomic aberrations in SMZL, we applied comparative genomic hybridization to BAC microarrays (array CGH) to a well characterized series of 75 SMZL specimens. We applied two different 1 Mb-resolution BAC arrays: UCSF HumArray 3.2 and a novel array CGH platform developed at Univ. of Salamanca. These arrays allowed us to detect DNA copy number changes across the genome with high accuracy in 67 of 75 patient samples. Data were compared with our previous array CGH studies of 170 samples from different B-cell lymphoma subgroups. FISH studies for IGH, IGK and IGL translocations and 7q deletion were performed on tissue microarrays in 24 cases. Of the 67 samples, 19 (28%) showed a normal genomic profile. The median number of genomic aberrations per tumor was 2.2 (1.3 gains and 0.9 losses), which was lower than the rates detected in other lymphoma subgroups (diffuse large cell lymphoma, 6.4; mantle cell lymphoma, 6; follicular lymphoma, 4.5) and comparable to MALT lymphomas (2 abnormalities per tumor). SMZL cells showed a genomic pattern characterized by gain of chromosomes 3q24-q29 (18%), 6p (9%), 12q (9%), and 18q (4%) and loss of 7q32 (34%), 8p21-p23 (13%), 17p13 (10%) at P53 locus and 6q21-q27 (9%). Notably, no alterations of the P16/ARF (9p21) or MYC loci (8q24) were detected. Correlation of array CGH data with conventional cytogenetics, FISH and LOH studies revealed a high concordance. Detailed mapping of 7q deletions delineated a consensus region of loss of 3 Mb in 7q32. This 7q deletion was almost exclusive to SMZL, being observed in only 5 of 170 non-SMZL B-cell lymphomas (p=0.0000001). Four cases presented IG-translocation. Mutation of IGH was observed in 62% and correlated with a complex karyotype (61 vs. 13%; p=0,0008) whereas unmutated IGH correlated with the deletion of 7q (56 vs. 23%; p=0,01). Among the various genomic abnormalities, only the deletion of 8p or the presence of a complex karyotype correlated with inferior overall survival (OS) (median OS, 58 vs. 110 months, p=0,004; and 60 vs. 105 months, p=0,01; respectively). In summary, array CGH has defined a pattern of genomic aberrations in SMZL that differs from other B-cell lymphoma subgroups and that may predict overall survival. Because the deletion of 7q32 is the most distinctive genetic marker in SMZL, the identification of a putative tumor suppressor gene inactivated within the region of deletion seems mandatory.

Background. In the World Health Organization classification system (WHO), Splenic Marginal Zone B-cell Lymphoma (SMZBCL) is described as an indolent B-cell lymphoma, which generally presents as splenomegaly with involvement of the bone marrow and peripheral blood. The immunophenotype is usually IgM+, IgD+/−, cytoplasmic-Ig−/+, pan B antigens+, CD5−, CD10−, CD23−, CD43−/+ and cyclin D1−. A minority of cases (< 20%) express CD5 antigen. The most frequent cytogenetic aberrations are deletions at 7q22–q32, gains of chromosome 3/3q and involvement of chromosomes 1 and 8. The objective of this study is the genetic characterization of SMZBCL by conventional cytogenetics and FISH. Patients and methods. We present 92 patients with SMZBCL from a multicenter study after being diagnosed morphologically and immunologically. Conventional cytogenetic studies were carried out on lymphoid cells from 72 hour-peripheral blood and/or spleen cultures stimulated with TPA. FISH was performed with centromeric probes from chromosome 3 and 12 and locus specific probes from P53 gene and 7q31 loci. Cross-species color banding FISH (RxFISH) probe was applied in 11 patients and SKY in 4 patients to redefine/confirm G-banding karyotype. Results. A clonal chromosome abnormality was found in 62/92 patients (67%) being identified in 37 (60%) as a complex karyotype. The most frequent recurrent abnormalities were deletions of 7q (30 cases) and gains of chromosome 3 (20 cases). 6/20 cases with gains #3, were gains of 3q arm, in three of them resulting from an unbalanced translocation. Thirteen cases showed rearrangements of 3q without gain of material. The chromosomes most frequently involved were: 7q, 3q, 14q, 8q and 1q (in more than 10 cases). Four cases presented gain of chromosome 3/3q and 7q deletion in the same karyotype. Four novel cytogenetic aberrations involving 14q32 were found in this series: t(6;14)(p12;q32) in two cases, t(14;19)(q32;q13) in two cases and t(10;14)(q24;q32), t(1;14)(q21;q32) and t(9;14)(pter-p22::14q32-14q11::9q22) in one case respectively. Conclusions. -In our series, the most frequent cytogenetic aberration in SMZBCL is a deletion at 7q (48%) and could be considered as a cytogenetic marker of these entity. -Gains of chromosome 3 or partial gains affecting only 3q arm are frequently observed in SMZBCL (32%) being chromosome 3 commonly rearranged. -RxFISH ans SKY techniques help us to define new aberrations not detected by conventional cytogenetics. -We describe translocations involving 14q32 that could help us to identify novel oncogenes related to SMZBCL.