Chng, Wee Joo

Abstract Abstract 88 Introduction: The biology and genetics of anaplastic large T-cell lymphomas (ALCLs) lacking ALK translocations remain poorly defined. We recently identified recurrent translocations involving the DUSP22-IRF4 locus on 6p25.3 in ALK-negative ALCL (Blood 2011;117:915–9). This translocation is present in about 20% of ALK-negative ALCLs and is absent in other T-cell lymphomas; however, the biologic consequences are unknown. In this study, we utilized gene expression profiling (GEP) to compare T-cell lymphomas with and without this translocation. Methods: cDNA was prepared from total RNA extracted from frozen tissue from 25 T-cell lymphoma patients under a Mayo Clinic IRB-approved protocol as follows: 5 ALK-negative ALCLs with 6p25.3 translocations (2 systemic, 3 cutaneous), 12 ALK-negative ALCLs without 6p25.3 translocations (11 systemic, 3 cutaneous), 5 ALK-positive ALCLs, and 3 CD30-positive peripheral T-cell lymphomas (1 transformed mycosis fungoides and 2 not otherwise specified). Partner loci in cases with 6p25.3 translocations were 7p32.3 in 2, 9p24.3 in 1, and unknown in 2. GEP data (Affymetrix U133 plus 2.0) were analyzed by unsupervised clustering; in addition, a GEP signature was developed using genes with raw values >200 in at least 2 cases with at least 2-fold, statistically significantly different expression between 6p25.3-translocated and non-translocated cases (p<0.05 after Benjamini-Hochberg multiple testing correction). Representative overexpressed genes were confirmed by quantitative real-time (qRT-) PCR. Supervised pathway analysis was performed using signatures derived from other studies or curated from public pathway databases. Results: By unsupervised clustering, the 5 cases with 6p25.3 translocations clustered together (281 probe sets). A GEP signature segregating translocated from non-translocated cases included 283 genes (195 up- and 88 down-regulated). Key features of this signature included altered expression of histone-modifying genes (e.g. EZH2, SUZ12, MLLT3, MLL; HDAC4) and up-regulation of cancer-testis antigen (CTA) genes (e.g. SSX4, TEX14, MAGEA12, SSX1, CTAG2). The signature included down-regulation of DUSP22 but not of other genes on 6p25 in the signature. Additional overexpressed genes were validated by qRT-PCR (relative expression: TEX14, 2.9, p=0.01, t test; MAL, 6.5, p=0.003; CCR8, 7.6, p=0.0004). Supervised pathway analysis demonstrated a high proliferation signature in cases with 6p25.3 translocations (p=0.006 vs. untranslocated cases). Conclusions: ALK-negative ALCLs with translocations involving the DUSP22-IRF4 locus on 6p25.3 have a distinct gene expression signature characterized by alterations of histone-modifying genes and CTA genes (which typically are regulated epigenetically, either through histone modifications or changes in methylation). This signature appears independent of anatomic site (systemic vs. cutaneous) and partner locus (7p32.3 vs. other). DUSP22, encoding a dual-specificity phosphatase involved in modulating mitogen-activated protein kinase (MAPK) signaling, is significantly down-regulated in translocated cases; however, pathway analysis did not show significant alterations in MAPK-associated genes. Other known genes in the region of the breakpoint on 6p25 were not affected substantially. These findings suggest the possibility of an unrecognized function of DUSP22. Contributions of the varying partner loci remain unclear. The distinct GEP signature associated with 6p25.3 translocations provides strong support for their biologic relevance. Finally, the genes involved in the histone-modifying signature show overlap with histone-modifying genes mutated in B-cell lymphomas, suggesting potential commonalities in the biology and possibly targeted treatment of B- and T-cell lymphomas. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 5060 Introduction In multiple myeloma, cytogenetics is informative in only about a third of patients. Despite this limitation, it remains a commonly available laboratory investigation that has prognostic utility and allows detection of global changes including ploidy. While cytogenetics abnormalities in myeloma have been well documented, a direct comparison between Western and Asian patients has not been performed. Methods Retrospective analysis of 190 newly diagnosed multiple myeloma patients presenting to the National University Hospital Singapore (NUH) between 2000–2009, and 494 newly diagnosed multiple myeloma patients from the Mayo Clinic myeloma database from 2000 to 2005 were analyzed. All abnormalities were logged onto an excel spreadsheet where all cytobands were represented. Gains, losses and structural abnormalities including specific translocations were documented separately. Abnormalities present in 5% or more samples with cytogenetic abnormalities were further analysed and compared between the 2 cohorts using chi-square test. Bonferroni correction is used when multiple comparisons were made. Results 80/190 (42%) of NUH patients had an abnormal karyotype compared with 180/494 (36%) in the Mayo clinic cohort (not statistically significant p=0.17). Distribution of ploidy status was very similar between the NUH and Mayo cohort with 35% and 27% being hyperdiploid in the NUH and Mayo cohort respectively (p=0.57). In both cohorts, the most common gains are whole chromosome gains involving chromosomes that are commonly trisomic in hyperdiploid myeloma. However, the pattern of trisomies is different between the NUH and Mayo cohort, with trisomies of chromosome 5, 7, and 19 more common in NUH (p-values <0.0001 for all three comparisons). Loss of 1p21was more common in NUH compared with Mayo (9% vs 2%, p=0.01) although loss of chromosome 7 was more common in Mayo compared to NUH (15% vs 0%, p=0.0002). t(11;14) is the most common translocations for both cohorts. Translocations involving chromosome 1 with different partners are also common in both cohorts. However, there are also interesting differences. Rearrangements involving the IgH (14q32) [10% NUH vs 4% in Mayo] and MYC (8q24) [6% in NUH vs 1% in mayo] loci are more common in the NUH cohort. In fact, when MYC translocations with IgH or IgL are included, 9 (11%) cases in the NUH cohort were affected versus 4 (in the Mayo Clinic cohort (p=0.0002). Recurrent rearrangement of 3q27 was observed in 5% of the NUH cohort while no cases were noted in the Mayo cohort. A search of the Mitelman Database of Chromosome Aberrations and Gene Fusion in Cancer (http://cgap.nci.nih.gov/Chromosomes/Mitelman) showed that only 4 out of 1643 Caucasian myeloma karyotype recorded had this abnormality (p<0.0001). 1q21 deletion and MYC rearrangements are associated with poorer prognosis. Conclusion From this comparison, the general patterns of cytogenetic abnormalities are similar between Asian and Western patients. The most important difference between Asian patients and western patients was the higher incidence of poor prognostic cytogenetic abnormalities in Asian patients including 1p21 deletion and gene rearrangements involving the MYC gene locus. 3q27 was predominantly seen in the Asian patient cohort and rarely in western patients. This could be a unique feature of Asian myeloma patients and more studies will have to be done to confirm this. These differences may have important implication on outcome of patients in Asian and Western Countries, and also unique biology that could potentially be exploited therapeutically. Disclosures: Fonseca: Consulting:Genzyme, Medtronic, BMS, Amgen, Otsuka, Celgene, Intellikine, Lilly Research Support: Cylene, Onyz, Celgene: Consultancy, Research Funding.

Abstract To further understand the molecular biology of mucosa-associated lymphoid tissue (MALT) lymphoma, we conducted a comprehensive gene expression study on the Affymetrix platform using total RNA extracted from biopsy specimen of 35 well-characterized pulmonary MALT lymphoma samples and compared them to gene expression profiles of a range of B and T cell lymphoma, such as Burkitt’s lymphoma, diffuse large B-cell lymphoma, Waldenstrom macroglobulinemia, chronic lymphocytic leukemia, multiple myeloma, and peripheral T-cell lymphoma, and normal cellular counterparts, such as different B-cell and T-cell populations, normal plasma cells and lung tussue. Analytical strategies were adopted to negate tissue specific effect such that our results can be generalized to MALT lymphoma from other anatomical sites. We further analyzed the data using functional tools such as gene-set enrichment analysis and gene ontology. Network/pathway analysis was performed using Metacore. Unsupervised clustering of whole dataset (including other lymphoid malignancies and normal cellular counterpart) using genes with variable expression, showed that compared to other B-cell lymphoid malignancies, MALT lymphoma have a prominent T-cell signature. Using established gene expression of different B-cell compartment, we confirmed that MALT has a marginal zone B-cell and memory B-cell cell-of-origin signature. Using ANOVA with post-hoc pair-wise comparison, we identified 4 novel transcripts over-expressed in MALT but not other B-cell tumors, and validated one of these using immunohistochemistry. MALT lymphoma with t(11;18) or t(14;18) over-expressed genes enriched for pathways other than the NFKB pathway such as JAK-STAT and SRC signaling pathways, which may represent novel therapeutic targets. We identified a number of genes with ‘outlier’ expression including RARA and RGS13. However, translocation involving RARA and the immunoglobulin heavy chain locus was not detected by FISH, suggesting other mechanisms of deregulating gene expression were involved. Using unsupervised clustering of only the MALT samples, we found that the molecular heterogeneity within MALT is composed of a group characterized by MALT1 translocations and another group with plasmacytic differentiation. Interestingly, further refinement of this grouping can be achieved by clustering the samples based on ‘outlier’ gene expression. Samples with MALT1 translocations have high expression of RARA, samples with plasmacytic differentiation have high FKBP11 expression and samples with high RGS13 expression tend to have trisomy 3 and reactive follicles. Our study therefore identified novel molecular markers, deregulated pathways and genetic defects in MALT. Furthermore, subgroups with distinct pathological features and anchored by unique pattern of ‘outlier’ gene expression were identified.

Abstract Abstract 4954 Background Multiple Myeloma (MM) remains incurable with current approaches and newer therapies targeting the abnormal disease biology need to be developed. The malignant plasma cell is highly dependent on the marrow microenvironment, which promotes tumor cell proliferation as well as survival. There is increased bone marrow vascular density in patients with MM and the degree of neo-angiogenesis appear to be prognostic in this disease. Studies have also shown that vascular endothelial growth factor (VEGF) is intricately involved in the interaction between the marrow microenvironment and the myeloma cell. These findings suggested that the VEGF pathway may be a valuable target for therapeutic approach in this disease. Patients and Methods We designed a phase 2 trial of Sunitinib in patients with relapsed multiple myeloma who had received less than three prior therapies. Sunitinib malate (SU11248; Sutent) is a novel, multi-targeted, small molecule inhibitor of the receptor tyrosine kinases (RTKs), including vascular endothelial growth factor receptor-1 (VEGFR-1), -2, and -3, and platelet-derived growth factor receptor (PDGFR) -α and -β. The treatment consisted of Sunitinib 37.5 mg given daily in a continuous fashion in six-week cycles. Toxicities were graded according to CTCAE response version 3.0 and responses were assessed according to the International Myeloma Working Group consensus criteria. Response rate of single agent Sunitinib was the primary endpoint of the study. Results 13 patients were accrued between July of 2007 and April 2009 before the study was closed. The median age of the study population was 61 years (range, 53 - 84), and included nine females (69%). The median time from diagnosis to registration was 39.2 months (range, 4.5 - 88) and 10 patients had received previous stem cell transplant. Previous therapy included lenalidomide in 7 patients, thalidomide in 5 patients and bortezomib in 2 patients. All 13 patients were alive at last follow up with a median follow-up of 11.2 months (range, 1.3 -21.2). All patients have gone off study, disease progression being the most common cause (9 patients), followed by adverse events (3 pts) and alternate treatment in the remaining patient. A hematological grade 3 or higher adverse event was documented in 6 patients and a grade 3 or higher non-hematological adverse event was seen in 8 patients. A grade 3 or higher neutropenia was observed in 5 pts and thrombocytopenia in 2 pts. Fatigue was common and was seen in 11 pts. The median number of cycles administered was one (range, 1-5) and the total number of cycles across 13 patients was 22. Treatment delays or interruptions were seen in five and six patients respectively. The most common reason for treatment delays was hematologic adverse events. No responses (PR or better) were seen, and stable disease was seen in eight of the 13 patients as the best response. The median time to progression was 2.9 months. Conclusions Single agent Sunitinib has no clinically relevant efficacy in this group of patients with relapsed myeloma with one or two previous therapies. Hematological toxicity was higher than anticipated. Whereas these results indicate that VEGFR TK inhibitors are not active in previously treated myeloma, it does not preclude the possibility that VEGF targeting strategies may enhance the efficacy of other therapies. Disclosures Kumar: CELGENE: Research Funding; MILLENNIUM: Research Funding; BAYER: Research Funding; GENZYME: Research Funding; NOVARTIS: Research Funding. Gertz:celgene: Honoraria; millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees. Dispenzieri:Celgene: Research Funding.

Abstract Abstract 3931 Poster Board III-867 Extranodal nasal-type Natural Killer/T-cell lymphoma (NKTCL) is a distinct clinicopathologic entity most commonly affecting Asians and Central and South Americans, and characterized by a clonal proliferation of NK or T cells with a cytotoxic phenotype. There is a strong association with Epstein-Barr Virus. The tumor is aggressive with patient usually surviving short duration even with chemotherapy. We performed the first comprehensive genome-wide gene expression profiling (GEP) of extranodal nasal-type Natural Killer/T-cell lymphoma (NKTCL) using formalin-fixed paraffin embedded (FFPE) tissue (n=25) and NK cell lines (n=5) and compared the results to the GEP of normal NK cells using the Illumina DASL whole genome array, with the objective of understanding the oncogenic pathways involved in the pathogenesis of NKTCL and to identify potential therapeutic targets. Quantitative-PCR validation of the GEP findings revealed over-expression of candidate genes BIRC5 (survivin), EZH2 and STMN1 in NK cell lines compared to normal NK cells, consistent with the GEP data. We then extracted a list of genes that are differentially expressed between NKTCL and normal NK cells and tissue controls. We than subjected this list of genes to pathway and network analysis using Metacore. This revealed a significant enrichment for cell cycle related genes and pathways. Furthermore, the network analysis results demonstrated a pro-proliferative and anti-apoptotic phenotype in NKTCL characterized by activation of Myc and nuclear factor kappa B (NF-KB), and deregulation of p53. This was further corroborated using immunohistochemistry on tissue microarray of NKTCL (n=33, including all cases with GEP performed). We observed a significant percentage of NKTCL showing overexpression for c-Myc (45.4%), p53 (87.9%) and NF-KB p50 (67.7%) on immunohistochemistry. Notably, overexpression of survivin was observed in 97% of the cases. Based on our findings, we propose a model of NKTCL pathogenesis where deregulation of p53 together with activation of MYC and NF-KB, possibly driven by EBV LMP-1, result in the cumulative upregulation of survivin. When KHYG and NKYS cell lines were treated with a compound IDR E804 which inhibited survivin, there is significant inhibition of cell growth as assess by MTS assay and induction of apoptosis as measured using Annexin V staining by flow cytometry. This suggests that compounds inhibiting survivin may be a potentially useful novel therapeutic approach in NKTCL. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1824 Introduction. Chromosomal abnormalities are prevalent in multiple myeloma (MM) and have been useful in delineating disease subtypes and prognosis groups. Globally, a dichotomous developmental pathway based on ploidy status seems to exist. The accumulation of chromosomal abnormalities in MM often gives rise to complex copy number profiles which are difficult to be captured due to the limitations of karyotyping in a relatively non-proliferative tumor. It is therefore unclear if genomic complexity as a reflection of chromosomal instability (CIN) is of biological and clinical relevance in MM. In this study, we introduce a novel measure of CIN, chromosome instability genome event count (CINGEC), validated its prognostic relevance in myeloma, and subsequently derived a gene expression (GEP) signature, CINGECS, investigated the gene contents of it to elucidate biological mechanisms related to CIN, and assessed its association with survival in the context of other gene expression based prognostic factors. Method. The rationale for CINGEC is that the more unstable is a genome, the more genome events does it harbor irrespective of the size of altered segments. Hence, we first obtain a copy number level sequence from a segmentation result of copy number profile signals from aCGH or SNP chip. We developed an algorithm to estimate the minimum number of genome events to account for the observed complex genome profiles. CINGEC is the sum of genome event counts from all autosomal chromosomes. The GEP signature, CINGECS, is derived from a public dataset that has both aCGH and GEP (GSE26849 and GSE26760, n=246) by performing differential gene expression analysis (SAM, p<0.001, q<0.001, fold-change 2) using GEP data between samples grouped into the top and bottom quartiles based on their CINGEC obtained from the analysis of aCGH data. The biological mechanisms distinctive between high and low CINGEC samples are assessed with biological pathways such as Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). Results. We first show that CINGEC is associated with MM patient survival by analyzing a previously published Mayo Clinic aCGH dataset (n=64, Chng WJ et al. Leukemia 2010; 24: 833–842). Furthermore, CINGEC is a stronger prognostic factor than another aCGH-based measure of CIN derived from breast cancer, the Genome Instability Index (Chin et al. Genome Biol 2007;8; R215). The CINGECS comprise of 160 differentially expressed genes with 144 up-regulated and 16 down-regulated. KEGG pathway and GO analysis of this set of genes showed enrichment by those involved in (1) cell cycle checkpoints and progression such as cell cycle, cell division, spindle organization, mitosis, (2) DNA damage responses such as response to DNA damage stimulus, DNA repair, nucleotide-excision repair, DNA gap filling, and (3) generic cancer related processes such as DNA replication, cell proliferation. Finally, CINGECS is shown to be significantly associated with poorer overall as well as progression free survival in both newly diagnosed (UAMS TT2 dataset, n=351, GSE 2658) and relapsed patients (Millenium Apex Dataset, n=188, GSE9782). Furthermore, it is found to be a significant prognostic signature independent of diverse GEP signatures known to be associated with survival for MM patients, including the Proliferation Index, the Centrosome Index, the UAMS 70-gene High-Risk Signature, the IFM High-Risk Signature, the IL-6 signature derived from cell lines, as well as GEP-based signatures of genomic instability derived from other cancers such as the CIN70 signature (Carter et al. Nat Genet 2006; 38:1043–1048) and CINSARC67 (Chibon et al. Nat Med 2010; 16: 781–787). Conclusions. We conclude that CINGEC can account for CIN with high resolution copy number profile data. Also, its corresponding GEP signature, CINGECS, potentially encompasses changes reflective of both cause and consequences of the CIN phenotype and is an independent prognostic signature in MM. Disclosures: Fonseca: Consulting:Genzyme, Medtronic, BMS, Amgen, Otsuka, Celgene, Intellikine, Lilly Research Support: Cylene, Onyz, Celgene: Consultancy, Research Funding.

Abstract Background: The initial therapies for multiple myeloma have changed significantly in the recent years with routine incorporation of combinations containing IMiDs and proteasome inhibitors. However, patients with MM invariably relapse after their initial treatment and undergo successive therapies for continued control of their disease. The increasing access to new therapies for treatment of relapsed disease has led to improved survival of patients with MM. Many recent phase 3 trials have studied the impact of new agents on outcome of patients with MM who are relapsing after the initial lines of therapy for relapsed disease. However, the natural history of relapsed myeloma earlier in the course of relapsed disease has not been studied in detail. Patients and methods: We designed a global, multicenter, retrospective study to examine the current treatment approaches for initial relapses in MM and patient outcomes following application of successive treatment regimens. Patients with multiple myeloma who experience a first relapse between January 1, 2007 and December 31, 2011 were identified at participating sites. Clinical and laboratory data pertaining to the time of diagnosis and from the time of individual relapses up to and including the third relapse will be obtained from the clinical records. The first date of treatment with an anti-myeloma regimen for first disease relapse was termed time zero (T0). Results: Three hundred and fifteen patients were enrolled; median age at diagnosis was 59 years (range: 35 - 86), 59% were male. Patients were enrolled from North America (33%), Europe (35%) or Asia (32%). The patients were diagnosed between 1997 and 2011 and the median time to T0 from diagnosis was 23.9 months (range: 0.8 - 131.9 months). An IMiD (thalidomide or lenalidomide), bortezomib or alkylator (melphalan or cyclophosphamide) was part of the initial therapy in 63%, 36% and 51% of patients, respectively; 93 (33%) patients had stem cell transplantation as part of initial treatment. The median follow up from the study entry was 56 months; 117 were alive at the time of data extraction. A second line, third line and fourth line of treatment were recorded in 307, 163 and 84 patients respectively. An IMiD (thalidomide, lenalidomide, or pomalidomide), proteasome inhibitor (bortezomib or carfilzomib) or alkylator (melphalan or cyclophosphamide) was part of the therapy for first relapse in 50%, 59% and 33% of patients, respectively. The responses to the regimens after T0, the TTNT, PFS and OS to each line of therapy are provided in the Table. Conclusion: The current study provides an assessment of the current treatment approaches used for initial treatment of MM and the salvage treatment options utilized. The study again demonstrates the progressively shorter duration of disease control with each successive treatment regimen, reflecting ongoing development of drug resistance. The overall survival of over 3 years from the time of therapy for first relapse highlights the impact of the newer therapies that have been introduced for this disease. Table. Treatment outcomes after initial relapse Endpoint 2nd line therapyN=307 3rd line therapyN=163 4th line therapyN=84 Partial response or better Median in Months (95% Confidence Interval) Overall Survival 36.1 (30.2, 42.4) 18.4 (14.5, 26.8) 12.6 (10.4, 19.0) Progression-Free Survival 13.4 (11.8, 15.8) 8.3 (6.5, 11.2) 6.4 (5.0, 8.1) Time to Next Treatment 15 (12,17) 9.8 (7.4, 12.0) 6.6 (5.8, 8.7) Figure 1. Figure 1. Disclosures Kumar: Onyx: Consultancy, Research Funding; Skyline: Consultancy, Honoraria; BMS: Consultancy; Sanofi: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Novartis: Research Funding; Takeda: Consultancy, Research Funding; Celgene: Consultancy, Research Funding. Dimopoulos:Novartis: Honoraria; Amgen: Honoraria; Janssen-Cilag: Honoraria; Genesis: Honoraria; Janssen: Honoraria; Onyx: Honoraria; Celgene: Honoraria. Leleu:Janssen: Honoraria; Celgene: Honoraria; Takeda: Honoraria; Amgen: Honoraria; TEVA: Honoraria; Novartis: Honoraria; BMS: Honoraria; Pierre Fabre: Honoraria; LeoPharma: Honoraria; Chugai: Honoraria. Moreau:Celgene, Janssen, Takeda, Novartis, Amgen: Membership on an entity's Board of Directors or advisory committees. Lahuerta:Janssen Cilag, Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Vij:Takeda, Onyx: Research Funding; Celgene, Onyx, Takeda, Novartis, BMS, Sanofi, Janssen, Merck: Consultancy.

Abstract Abstract 4160 We propose that the pathophysiology of marginal zone B-cell lymphomas (MZL) could allow for their classification into lymphoid organs located in MZL and extra-nodal MZL of MALT type; the former category encompassing splenic MZL (SMZL) and nodal MZL (NMZL). The rarity of these lymphomas and difficulty in distinguishing them from other low-grade B-cell lymphoma such as lymphoplasmacytic lymphomas hampers further understanding of disease biology. In an attempt to better characterize the genetic basis of these entities and compare acquired genetic changes based on this categorization we performed array-based comparative genomic hybridization (using the 244A array, Agilent) in a cohort of MZL cases. In total we analyzed 101 MZL, including 46 MALT (including 8 cases with t(11;18)(q21;q21)), 35 SMZL and 20 NMZL. Overall, 90.1% cases have copy-number abnormalities (CNA). Genomic complexity was greatest in MALT without t(11;18)(q21;q21)(4 CNA), followed NMZL (3.5 CNA), SMZL (3 CNA) and MALT with t(11;18)(q21;21)(1 CNA). Seventeen minimal deleted regions (MDRs) and 9 minimal amplified regions (MARs) were defined based on the presence in at least one entity with a frequency of 10% or higher. Gains of chromosomes 3, 12 and 18 were found in all entities. Deletion of 6q was the most common abnormality in MALT (21.1%) and, to less extent, SMZL and NMZL. Biallelic and focal monoallelic deletions on 6q23.3-q24.1 identified TNFAIP3 as the sole gene inside the common MDR between entities. Deletion of chromosome 13 was observed in all entities, but NMZL and MALT were characterized by whole chromosome deletions, whereas focal deletions on 13q14.3 (MIR16-1/MIR15A) were identified in SMZL. A subset of abnormalities was shared for more than one entity: deletions 9p21.3 (CDKN2A) and 11q21-q22 (ATM) were found in NMZL and SMZL but not in MALT; deletion 7q32.1-q33 was the most common CNA in of SMZL but was also found in MALT; gain of 6p was found in MALT and NMZL but not in SMZL. Other CNA were only found in one entity such as deletion of 17p13.3-p12 (TP53) in SMZL, deletion of 15q25.3-q26.2 in NMZL. In addition to 17% of cases with the t(11;18)(q21;q21), extra copies of MALT1, focal amplifications of REL and/or monoallelic/biallelic deletions of TNFAIP3 were identified in an additional 46% of MALT lymphomas. These CNA were identified alone or in combination, but all of them were mutually exclusive with the t(11;18)(q21;21), thus totalizing 63% of MALT lymphomas with abnormalities affecting regulator genes from the NF-kB signaling pathway. Moreover, depending on the anatomic site of the MALT lymphoma, the prevalence of CNA affecting the NF-kB pathways varies from 50% in lacrimal glands to 75% in salivary glands. Abnormalities affecting regulator genes of the NF-kB signaling pathways were identified in a significantly lower frequency in NMZL (30%) and SMZL (31%) entities, suggesting its differential role in the pathogenesis of different entities. Using this high-resolution approach we show that while many abnormalities are shared unique patterns of genomic gains and losses characterize subgroups of MZL. Extranodal MZL seems to have a higher propensity of constitutive genetic upregulation of NF-kB pathways. Furthermore, this study remarks the importance of this pathway also in the subgroup of MALT lymphomas without translocations affecting the pathway. Disclosures: Fonseca: Genzyme: Consultancy; Medtronic: Consultancy; BMS: Consultancy; AMGEN: Consultancy; Otsuka: Consultancy; Celgene: Consultancy, Research Funding; Intellikine: Consultancy; Cylene: Research Funding; Onyx: Research Funding; FISH probes prognostication in myeloma: Patents & Royalties.

Previously, we have shown that centrosome amplification is common in multiple myeloma and is already present in monoclonal gammopathy of undetermined significance (MGUS), suggesting an early role in myelomagenesis. In addition, we derived a gene expression based centrosome index (CI) that correlated closely with centrosome amplification detected by immunofluorescence. We also showed in a heterogeneously treated and relatively small cohort of patients that the CI is an independent prognostic factor. In this study, we validated the prognostic significance of a gene expression based centrosome index (CI) in 2 large cohorts of homogenously treated myeloma patients entered into clinical trials. Cohort 1 consisted of newly diagnosed patients from the University of Arkansas for Medical Science (UAMS) entered treated with total therapy II. These patients have gene expression profiling performed on purified CD138+ plasma cells from pre-treatment bone marrow cells using the Affymetrix U133plus chip (data available from Gene Expression Omnibus). Cohort 2 consisted of relapsed or refractory patients entered into bortezomib trials. These patients have GEP performed on purified CD138+ PCs using the Affymetrix U133A and B chip as part of a pharmacogenomics study (data kindly provided by Millenium Pharmaceuticals). Expression values extracted using the MAS 5.0 software are log-transformed, median centered and analyzed using Genespring 7.0 (Agilent Technologies). Survival is calculated from date of entry into trial till death in both cohorts. In the UAMS dataset, a high CI is significantly associated with shorter survival and emerged as an independent prognostic factor with t(4;14) (detected by spike FGFR3/MMSET expression) when entered into a multivariate Cox proportional hazard analysis (other variables entered included CKS1B expression above median and elevated gene expression based proliferation index, both of which were significantly associated with shorter survival on univariate but not multivariate analysis). In the bortezomib cohort, a high CI is again significantly associated with shorter survival, and this effect is independent of response to bortezomib. Therefore, a high CI is a powerful independent prognostic factor in both newly diagnosed and relapsed patients even in patients treated by intensive therapy (total therapy II) and novel agents (bortezomib). We next compared the gene expression profiles of tumors with high and low CI, to define the molecular phenotype of these aggressive tumors and identify possible novel therapeutic targets. Tumors with high CI over-expressed genes coding for proteins associated with the centrosome (TUBG1, CETN2, TACC3, NEK2, PRKRA, STK6, AURKB and PLK4), cell cycle (CCNB1, CCNB2, CCND2, E2F2, CDC gene family, CDK5, CDK6, CDKN2C), proliferation (RAN, CKS1B, TOP2A, TTK, TYMS, MCM gene family, ASPM), involve in DNA repair / G2 cell cycle checkpoints (BRCA1, CHEK1, CHEK2, MAD2L1, BUB1, BUB1B, FANCD2, REV1L), kinetochore and microtubule attachment (AURKB, BIRC5, CENPA, CENPE, CENPH, ZWINT), genes coding for cancer testis antigens (GAGE and MAGE family) and implicated in other cancer (PTP4A3, EZH2). Consistent with this, the most significantly associated Gene Ontology biological process are cell cycle, mitosis, proliferation, DNA repair, microtubule related In particular, aurora kinases are significantly over-expressed in patients with high CI and may represent novel therapeutic targets in these patients with very poor prognosis.

Abstract The study of genomic aberrations in chronic lymphocytic leukemia (CLL) has yielded critical information, now routinely used for the prognostication of patients. An unbiased search for genome wide areas of copy number change is needed to identify possible primary genetic events in CLL and to elucidate their relationship to other known cytogenetic factors, such as deletions of 13q14, 11q23 and 17p13 and trisomy 12. Here we report on a study where we used a 60-mer oligo array-based Comparative Genomic Hybridization (aCGH, 44B platform from Agilent Technologies, Palo Alto, CA). We observed the most common genetic aberrations in CLL and in similar frequencies as would be predicted for CLL (e.g. 44% showing deletions of chromosome 13q14). Among those patients, 45% had no apparent other aberration. Of great interest, small bi-allelic deletions of chromosome 13, which predominantly involved the DLEU1 and DLEU2 locus, were detected in 4 patients (16%). Deletions of 11q23 were observed in 6 cases (24%), and while some involved the ATM locus one did not, casting doubt on the precise gene deregulated by these deletions. Large deletions of the whole 17p-arm were detected in three cases (12%). Trisomy 12 was present in only two cases (8%). No other trisomies were detected and also small regions of copy gain were uncommon. Monosomy of chromosomes 19, X and Y were present in three separate cases. Large deletions of chromosome 2p were seen in two cases, but an additional case had a small deletion involving the ALK gene. This case also had a breakpoint at chromosome 5 involving the NMP1 locus, suggestive of a t(2;5). A recent study (Mayr et al., Blood 2006) indicates that chromosomal translocations in CLL are present in 33% and were associated with a dismal prognosis. In our analysis we detected several breakpoints indicative of translocations. Detailed study is necessary to elucidate possible molecular events associated with these translocations. Other small regions of DNA copy loss were found at several locations throughout the genome. Regions of interest are shown in the table. In summary we show that multiple areas of recurrent genomic gain and loss can be identified in clonal cells of CLL and should lead to further insights into the understanding of disease pathogenesis. Chromosome Location Gene(s) of interest Loss 6q24.3 LATS Loss 10q23.3 LG11 Loss 10q24.2 PAX2, WNT8B, TLX1, POLL, NFKB2 Loss 11p15.4 PPFIBP2 Loss 12p13 TNFRSF1A and 7 Loss 15q22.1 TCF12 Loss 19p13.3 BAX, RUVBL2 Loss 20q11.2 TP53INP2 Loss 20q13.1 WFDC13