Fonseca, Rafaël

Abstract Primary central nervous system lymphoma (PCNSL) is an aggressive and incurable variant of non-Hodgkin lymphoma (NHL) that is confined to the central nervous system. Most PCNSL (90%) are part of the immune-privileged site-associated diffuse large B-cell lymphomas (DLBCL). Unlike nodal DLBCL, only a limited number of genetic studies have been performed in PCNSL, partly due to lack of available tissue specimens. Because of the fragmented knowledge of the genomic basis, it is still a matter of debate whether they differ from systemic DLBCL with respect to their molecular features and pathogenesis and also if there is a CNS specific signature. We performed a comprehensive genomic study in a cohort of 22 immunocompetent (HIV- and EBV-) PCNSL. DNA was extracted from FFPE tissues (N=15) and frozen tissue (N=7). Samples were analyzed using a combination of aCGH (N=18), exome sequencing (N=10), mate-pair genome sequencing (N=2) and targeted sequencing (N=8). We found a complex karyotype with a median of 21 copy-number abnormalities (range 10–47), 6 structural abnormalities, 6 frameshift indels and 67 nonsynonymous mutations. By integrating mutation and copy number data we found a group of genes recurrently mutated and/or deleted in PNCSL. Remarkable findings were the high prevalence of MYD88 activating mutations (L265P/M232T/V217F), found in 69% of cases and the biallelic loss of CDKN2A (60%). A subset of recurrent abnormalities was exclusively found in PCNSL, and not being previously identified in systemic DLBCL. Thus, 11% of PCNSL have biallelic inactivation of TOX (a regulator of T-cell development) and PRKCD (protein kinase C delta), while another 17% of cases show focal monoallelic deletions/mutations in these genes. Finally, recurrent mutations have been identified in ATM, which have not been found in nodal DLBCL. Several other genes affected have been previously identified in nodal DLBCL, such as biallelic loss of TNFAIP3 (16%), PRDM1 (16%), GNA13, TMEM30A, B2M and CD58 (11% each), activating mutations of CD79B (28%) and CARD11 (19%) and translocations of BCL6 (22%). Components of the NF-kB pathways were altered in >90% of PNCSL. Pathway analysis also showed an enrichment of networks associated with immune response, proliferation, regulation of apoptosis and lymphocyte differentiation and activation. Finally, we searched for associations between genetic alterations and clinical outcome. We showed that deletions of 6q21 (PRDM1) and 6q23 (TNFAIP3) were both associated with shorter overall survival (p=0.007 and p=0.03, respectively). In summary, we report a genomic background in PCNSL similar to post-GC DLBCL but reinforcing the existence of a subset of abnormalities specific to PCNSL, suggesting their potential relevance in the disease pathogenesis. Additionally, the results obtained from FFPE samples are encouraging and larger archival tissue collections can now be analyzed in order to complement the still fragmented knowledge we have of the genetic basis of the disease. Disclosures: Stewart: Onyx: Consultancy, Research Funding; Millenium: Honoraria, Research Funding; Celgene: Honoraria; BMS: Honoraria. Fonseca:Medtronic: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; Genzyme: Consultancy; BMS: Consultancy; Lilly: Consultancy; Onyx: Consultancy, Research Funding; Binding Site: Consultancy; Millennium: Consultancy; AMGEN: Consultancy; Cylene: Research Funding; Prognostication of MM based on genetic categorization of the disease: Patents & Royalties.

Abstract Abstract 987 Background: IMiD® compounds are effective in immunoglobulin light-chain amyloidosis (AL), a systemic plasma cell disorder related to multiple myeloma (MM). Pomalidomide is a new IMiD® compound. Methods: Persons with symptomatic, biopsy-proved, previously-treated AL were eligible for treatment with pomalidomide if they had measurable hematologic disease, ECOG PS≥2, adequate hematologic reserve and creatinine ≤2.5 mg/dL. Pomalidomide, 2 mg/d, and dexamethasone, 40 mg/w, were given orally along with low-dose aspirin. Dose-escalation to 4 mg/d was allowed for non-responders. Exclusion-criteria were uncontrolled infection, cancer, NYHA classification >II, serum troponin-T >0.1 ng/mL, ≥grade-3 neuropathy, thrombosis or other AL therapy within 2 weeks of enrollment. 31 subjects enrolled, but only 29 are evaluble for toxicity and response due to recent enrollment. Results: Median age was 66 y (range, 52 82 y); 19 were male. Median time from diagnosis to study-entry was 36 mo (range, 0.9–104.1 mo). 24 had cardiac involvement, 10, renal involvement and 8, nerve involvement. Cardiac biomarker staging was: I, N=3, II, 21, and III, N=7. Subjects were extensively pretreated: 28 received an alkylator (including an autotransplant in 13), 15, prior lenalidomide or thalidomide and 12, prior bortezomib. 193 cycles of pomalidomide were given to 29 subjects. 11 subjects had protocol-specified dose-increases (no response) and 16, dose-reductions (AEs). 20 subjects had dexamethasone dose-reductions. ≥Grade-3 AE's ≥possibly attributed to therapy occurred in 21 subjects. 7 of these subjects had only hematologic AE's. Neutropenia was the most common severe AE (N=10); severe thrombocytopenia occurred in 2 subjects. Other grade-3/-4 AEs included infection/fever (N=3); fatigue (N=4), and pleural effusion, ascites/edema, and edema each in one patient. 1 subject each had dyspnea, increased creatinine, thrombosis, muscle weakness, atrial tachycardia, and diarrhea. There were 2 episodes of syncope attributed to AL rather than therapy. A subject died 5 d after beginning therapy presumably from cardiac AL. As of 7/27/010, median time on-study is 8.4 mo (range, 0.2–16.2 mo). 18 subjects discontinued therapy for: progression/death (N=9); consent withdrawal (N=4), AE (N=3) and physician choice (N=2). Overall hematologic response rate (N=29) is 38% (95% CI, 20–58%) There were 8 PRs and 3 VGPRs including 4 PRs and 2 VGPRs among 14 subjects failing prior lenalidomide and/or thalidomide. 3 subjects had confirmed organ responses (2 heart and 1 kidney) and 4 unconfirmed organ responses. 1 y survival and PFS are 77% (95% CI, 60–98) and 56% (95% CI, 36–85). Conclusion: These data suggest that pomalidomide could provide a safe and effective therapy in previously-treated persons with AL including those failing lenalidomide and/or thalidomide. Disclosures: Dispenzieri: Celgene: Honoraria, Research Funding; Binding Site: Honoraria. Off Label Use: Non-FDA approved indication. Gertz:Celgene: Honoraria. Kumar:Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding. Fonseca:Amgen: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy, Research Funding; Genzyme: Consultancy; Onyx: Research Funding; Otsuka: Consultancy; Medtronic: Consultancy. Bergsagel:Celgene: Consultancy; Centocor: Consultancy; Genentech: Consultancy; Amgen: Consultancy; Novartis: Consultancy. Stewart:Millennium: Consultancy; Celgene: Honoraria. Lacy:Celgene: Research Funding.

Abstract Abstract 1570 B-cell non-Hodgkin lymphoma encompasses a heterogeneous group of B-lymphocyte-derived malignancies that are characterized by chromosomal translocations involving the immunoglobulin (IG) gene loci and specific histological subtypes of disease are associated with a different spectrum of IG translocations. Marginal zone derived B cell lymphomas encompass three distinct entities: extranodal marginal zone B lymphoma (MZL) of mucosa associated lymphoid tissue (MALT), nodal MZL (NMZL), and splenic MZL (SMZL). MALT lymphoma is genetically unique and four mutually exclusive chromosomal translocations have been identified in this disease thus far. However, the known translocations are only present in a subset of cases suggesting that additional uncharacterized translocations or other genetic events may exist that contribute to disease development. In previous studies we characterized the novel t(X;14)(p11.4;q32) translocation in a patient with MALT lymphoma and found that GPR34, an orphan G-protein coupled receptor (GPCR), was highly expressed due to its juxtaposition to the IGHSA2 switch region. We then measured GPR34 mRNA expression in tissue biopsies from a panel of MALT, NMZL, SMZL, lymphoplasmacytic (LPL), diffuse large B cell (DLBCL), follicular (FL), and mantle cell (MCL) lymphomas. Expression of GPR34 was detected in all tissues examined, but was significantly increased in MALT (37 fold), LPL (23 fold), NMZL (18 fold), and SMZL (21 fold) compared to normal CD19+ B cells. GPR34 mRNA expression was also analyzed by gene expression profiling (GEP) of a panel of lymphomas or control lymphoid tissue and we confirmed elevated expression of GPR34 in the t(X;14) case and detected elevated GPR34 mRNA expression in MALT, LPL, NMZL, SMZL, and ABC DLBCL. When we grouped specimens by high and low expression of GPR34, a clear molecular subtype of SMZL could be identified and suggests that GPR34 high expressing tumors have a unique GEP. To confirm the qPCR and GEP analysis of GPR34 expression, we analyzed surface expression of GPR34 by flow cytometry of normal and malignant tissues and found that expression of GPR34 was detected on all MALT and SMZL B cells with an average DMFI of 2.08 (DMFI range 1.26–3.18, n=12) and on the JeKo-1 lymphoma B cell line (DMFI= 4.75). Analysis of CD19+ cells from normal controls (n=3) revealed a low level of GPR34 on (average DMFI =1.12). Little is known about the function of GPR34 and to further characterize the impact of GPR34 overexpression on cell signaling, OCI-LY19 lymphoma B cells and HeLa cells were transduced with GPR34 or a vector control. To determine if MAPK activity, a pathway regulated by GPCRs, was upregulated, the phosphorylation status of ERK1/2 in GPR34 or control cells was analyzed and we found that ERK1/2 was constitutively phosphorylated at significantly higher levels in GRP34 expressing cells. To further explore which signaling pathways were affected by GPR34 overexpression, GPR34 or vector control cells were transfected with an AP-1, CRE, NF-kB, E2F, SRE, NFAT, or ISRE- luciferase reporter plasmid and GPR34 expressing cells had significantly increased luciferase activity driven by AP-1 (5.35-fold), CRE (4.7-fold), NF-kB (2.6-fold), and E2F (2.1) when compared to vector control cells. To determine the biologic impact of GRP34 overexpression, the proliferation rates of vector control and GPR34 cells were compared and we found that proliferation of GPR34 WT cells was significantly higher than that seen in the control. We next tested the effect of the MEK inhibitor on proliferation and saw a dose dependent decrease in proliferation of GPR34 expressing cells. We then tested the ability of GPR34 cells to generate colony formation in a soft-agar colony formation assay. After 3 weeks of culture, the GPR34 cells had formed significantly more colonies than the vector control suggesting that overexpression of GPR34 can result in increased anchorage-independent cell growth. Taken together, our results identify a novel translocation in MALT lymphoma, emphasize a novel role for GPR34 in regulation of gene expression and tumor cell growth, and suggest that MEK inhibitors may be useful in a subset of translocation–harboring or GRP34 high-expressing tumors. Disclosures: No relevant conflicts of interest to declare.

Abstract Introduction: Single-agent carfilzomib has shown encouraging activity in patients with relapsed and refractory multiple myeloma who harbor high-risk cytogenetic abnormalities (Jakubowiak et al, Leukemia 2013;27:2351-56). The phase 3 study ASPIRE (NCT01080391; N=792 patients) demonstrated that progression-free survival (PFS) was significantly improved with carfilzomib, lenalidomide, and dexamethasone (KRd), compared with lenalidomide and dexamethasone (Rd) in patients with relapsed multiple myeloma (RMM) (Stewart et al, N Engl J Med 2015;372:142-52). We present a pre-planned subgroup analysis of the efficacy and safety of KRd vs Rd in the ASPIRE study according to baseline cytogenetic risk status. Methods: Adults with RMM (1-3 prior lines of therapy) were eligible. Patients were randomized (1:1) to KRd or Rd. Treatment was administered in 28-day cycles. Patients in the KRd arm received carfilzomib as a 10-minute intravenous infusion on days 1, 2, 8, 9, 15, and 16 (starting dose, 20 mg/m2 on days 1 and 2 of cycle 1; target dose, 27 mg/m2 thereafter) during cycles 1-12; carfilzomib was omitted on days 8 and 9 during cycles 13-18 and was discontinued after 18 cycles. All patients received lenalidomide 25 mg on days 1-21 and dexamethasone 40 mg on days 1, 8, 15, and 22. The primary end point was PFS. Secondary end points included overall survival, overall response rate (ORR), duration of response (DOR), health-related quality of life, and safety. Cytogenetic risk status was assessed using fluorescence in situ hybridization. The high-risk group consisted of patients with the genetic subtype t(4;14) or t(14;16) or with deletion 17p in ≥60% of plasma cells, according to central review of bone marrow samples obtained at study entry. The standard-risk group consisted of all other patients with known baseline cytogenetics. The cutoff value of 60% for the proportion of plasma cells with deletion 17p was used on the basis of recommendations from the International Myeloma Workshop Consensus Panel 2 (Munshi et al. Blood 2011;117:4696-700). Results: A total of 792 patients were randomized to KRd (n=396) or Rd (n=396). In patients with known baseline cytogenetics, baseline cytogenetic risk status was similar between the treatment arms (high-risk: KRd, 24.6%; Rd, 23.4%; standard-risk: KRd, 75.4%; Rd, 76.6%). Efficacy outcomes by cytogenetic risk status are presented in the Table. Median PFS in the high-risk group (n=100) was 23.1 months (95% confidence interval [CI]: 12.5-24.2) for KRd vs 13.9 months (95% CI: 9.5-16.7) for Rd (hazard ratio [HR]: 0.639; 95% CI: 0.369-1.106). Median PFS in the standard-risk group (n=317) was 29.6 months (95% CI: 24.1-not estimable) for KRd vs 19.5 months (95% CI: 14.8-26.0) for Rd (HR: 0.657; 95% CI: 0.480-0.901). ORRs were 79.2% (KRd) vs 59.6% (Rd) in the high-risk group, and 91.2% (KRd) vs 73.5% (Rd) in the standard-risk group. In the high-risk group, 29.2% (KRd) and 5.8% (Rd) of patients achieved a complete response (CR) or better, including 16.7% (KRd) and 3.8% (Rd) with a stringent complete response (sCR). In the standard-risk group, 38.1% (KRd) and 6.5% (Rd) of patients achieved ≥CR, including 15.0% (KRd) and 3.5% (Rd) with an sCR. Median DOR in the high-risk group was 22.2 months for KRd vs 14.9 months for Rd. Median DOR in the standard-risk group was 30.4 months for KRd vs 20.4 months for Rd. The rate of grade ≥3 adverse events was 89.1% (KRd) vs 78.4% (Rd) in the high-risk group, and 85.6% (KRd) vs 84.5% (Rd) in the standard-risk group. Rates of grade ≥3 adverse events of interest (dyspnea, hypertension, acute renal failure, cardiac failure, ischemic heart disease, and peripheral neuropathy) by cytogenetic risk status are presented in the Table. Conclusion: In patients with high-risk cytogenetics, treatment with KRd resulted in a median PFS of nearly 2 years, which was a 9-month improvement relative to that in patients treated with Rd. Treatment with KRd also led to a 10-month improvement in median PFS vs Rd in patients with standard-risk cytogenetics; similar reductions in the risk of progression or death with Kd vs Vd were observed in both cytogenetics risk groups. Treatment with KRd also led to higher response rates, greater response depth, and a longer DOR compared with Rd in patients with high- or standard-risk cytogenetics. KRd had a favorable benefit-risk profile in patients with RMM, irrespective of baseline cytogenetic risk status, and improved outcomes in patients with high-risk disease. Disclosures Fonseca: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Applied Biosciences: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Onyx/Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Binding Site: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Siegel:Celgene Corporation: Consultancy, Speakers Bureau; Amgen: Speakers Bureau; Takeda: Speakers Bureau; Novartis: Speakers Bureau; Merck: Speakers Bureau. Dimopoulos:Celgene: Honoraria; Onyx: Honoraria; Genesis: Honoraria; Janssen-Cilag: Honoraria; Novartis: Honoraria; Janssen: Honoraria; Amgen: Honoraria. Spicka:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Masszi:Novartis: Consultancy; Janssen-Cilag: Consultancy; BMS: Consultancy; Takeda: Consultancy. Hájek:Janssen-Cilag: Honoraria; Celgene, Merck Sharp & Dohme: Consultancy, Honoraria. Rosiñol:Celgene: Honoraria; Janssen: Honoraria. Mateos:Janssen-Cilag: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Takeda: Consultancy; Onyx: Consultancy. Wang:Celgene: Research Funding. Niesvizky:Celgene: Consultancy, Speakers Bureau. Oriol:Celgene: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau. Jakubowiak:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SkylineDx: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: institutional funding for support of clinical trial conduct, Speakers Bureau; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SkylineDx: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Palumbo:Celgene, Millennium Pharmaceuticals, Amgen, Bristol-Myers Squibb, Genmab, Janssen-Cilag, Onyx Pharmaceuticals: Consultancy, Honoraria; Novartis, Sanofi Aventis: Honoraria. Bensinger:Onyx: Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Acetylon: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millenium: Research Funding. Kukreti:Lundbeck: Honoraria; Ortho: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Amgen: Honoraria. Tonda:Onyx: Employment. Obreja:Amgen Inc: Employment. Moreau:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract Abstract 3605 CLL is a malignant B-cell disorder characterized by the accumulation of small B lymphocytes with a mature appearance in blood, marrow and lymph nodes. Despite effective treatment options, all patients with CLL will eventually relapse after therapy. This could be due in part to the presence of subclones of the CLL cell population that harbor genetic abnormalities, which confer resistance to treatment. The aims of this study were to investigate the clonal evolution in longitudinal samples of CLL patients and to identify genetic alterations associated with disease progression and resistance to therapy. Sequential analyses were performed in 51 samples from 23 patients who were included in a previously reported clinical trial of pentostatin, cyclophosphamide and rituximab (PCR) given every 3 weeks for 6 cycles in previously untreated CLL (Blood 109:2007). In all cases the first sample analyzed was prior to therapy. In 5 of 23 patients, three time points were analyzed: >6 months prior to entry onto PCR trial (time point A), just before starting with the PCR regimen (time point B), and the time of relapse after PCR trial (time point C). Seven patients were analyzed at time points A and B; 9 at time points B and C and 2 at time points A and C. The median time between points A and B was 17.5 months (range 8–48 months) and between points B and C was 20.5 months (7–60 m). All samples were examined by array-based comparative genomic hybridization (aCGH) using the Agilent Sureprint G3 (1 million probe) array. aCGH findings were confirmed by interphase FISH using probes for D13S319 (MIR16–1/MIR15A), RB1, MDM2, CEP12, CEP6, MYB, TP53, NFKBIA, PERP and FGFR1 loci. Overall, we observed a small increase in the number of copy-number abnormalities (CNA) with disease progression. Twenty-two of the 23 patients with paired samples harbored at least one CNA that persisted in all samples, indicating clonal relationship between the sequential samples. In 15 of the 23 patients the tumor clone was stable and no CNA differences between time points were identified. Conversely, genomic evolution was found in 8 patients. In 3 cases the genetic differences were observed pre treatment (between time points A and B) and in the other 5 cases, the observed changes were found after therapy (between time points A and C or between B and C). One remarkable case with genome evolution exhibited two subclones sharing trisomies 12 and 19, but with several unique CNA confined to each subclone. The first subclone was characterized by deletions of 6q, RB1, MIR16-1/MIR15A and 3 other losses, while the second subclone showed homozygous deletion of MIR16–1/MIR15A and 5 other monoallelic deletions. The first subclone was predominant at time points A and B (60–70% of cells), but was present in only 10–20% of cells at time point C as confirmed by FISH. Conversely, the second subclone was observed in ~20% of cells at time points A and B and became predominant after therapy, found in ~80% of cells at time point C. Another case was characterized by deletion 11q32 (including ATM and others) as the sole abnormality at time point B. Significant genomic complexity was observed at time point C, including deletions of 11q32, 9p21 (CDKN2A), 9q12-q33, 14q13.2 (NFKBIA) and 17p (TP53), and gains of 2p16 (REL) and 9q34. Interestingly, the deletion 11q32 from both time points arose independently at each time point, as they exhibited different chromosomal breakpoints and copy number variants. Moreover, the other CNA found at relapse were not identified at diagnosis (confirmed by aCGH and FISH). For evolution of specific CNA, trisomy 12 was found in 5 cases at the first sample analyzed and was stable with no changes between time points. The frequency of deletions 13q14.3 (MIR16-1/MIR15A) and 17p increased at the later time points. Conversely, –6q decreased in frequency across time points (3 cases in time points A–B and 1 case in time point C). In summary, at least 35% of CLL patients exhibited clonal evolution and at least 9% showed evidence of multiple subclones. This subgroup of CLL patients provides an exceptional framework for comprehensive analysis of genome evolution during disease progression before and after therapy. Our observations also support the hypothesis of a common CLL progenitor cell can give rise to clonally related, but genetically evolving subpopulations of tumor cells. Finally, this study may bring novel information regarding the drug resistance pathways utilized by CLL B cell clones post therapy. Disclosures: Kipps: GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Genzyme: Research Funding; Memgen: Research Funding; Igenica: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Research Funding; Abbott Laboratories: Research Funding. 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.

Abstract Introduction Lenalidomide is an immunomodulatory drug (IMID) used to treat Multiple Myeloma (MM). Although a role for cereblon (CRBN)-mediated degradation of Ikaros proteins (IKZF1 and IKZF3) has been shown, the complete molecular and biochemical mechanisms responsible for lenalidomide-mediated anti-MM activity and/or resistance are undiscovered. Therefore, we aimed to analyze whether IMIDs (thalidomide, lenalidomide, and pomalidomide) are inducing oxidative stress in MM and what determines these drugs varying sensitivity and/or resistance. Methodology Amplex Red Assay has been performed to analyze IMIDs-mediated inhibition of H2O2 decomposition in both, in-vitro and in-vivo assays. Lentiviruses were prepared in 293T cells for CRBN, IgL-λ & IgL-k, and Bim knockdown experiment. Quantification of MM cellular anti-oxidative capacity for determining IMID sensitivity was standardized with H2O2-mediated oxidation of FADH2 and NAD(P)H. To measure apoptosis and gene expression analysis 106 cells were incubated with lenalidomide for 24 to 96 hours before they were examined by annexin-PI and FACS analysis. Gene and protein expression were measured by RT-PCR, western blot, and immunohistochemistry. Results We discovered that IMIDs inhibit peroxidase-mediated decomposition of H2O2 in both, in vitro horseradish peroxidase (HRP) assays and in human MM cell lines (HMCLs). Of the IMIDs analyzed, pomalidomide was the more potent inhibitor. H2O2 treatment effectively degraded IKZF1 and IKZF3 in HMCLs. To confirm the central role of CRBN in IKZF1 and IKZF3 degradation by H2O2-induced oxidative stress, we used CRBN knockdown OPM2 isogeneic cells and the CRBN-overexpressing OCIMY-5 cell line. We treated both sets of isogenic cell lines with lenalidomide and H2O2 for 3 hours, and we showed that H2O2 similarly mediates IKZF1 and IKZF3 degradation in a CRBN-dependent fashion. Next, we tested viability of CRBN present and absent cell lines with increasing concentrations of lenalidomide and H2O2 for 3 days. Lenalidomide-induced cytotoxicity was CRBN dependent, but H2O2 was not after 3 days, as shown by MTT assays. The capacity of MM cells to decompose H2O2 was measured via a biochemical test that quantitatively measured cellular anti-oxidative capacity. IMID sensitivity was well correlated with cellular anti-oxidative capacity, likely, cells more efficiently decompose H2O2was resistant and cells were not sensitive to IMID. This result shows that antioxidant capacity determines lenalidomide sensitivity among HMCLs with similar CRBN protein expression. We discovered that lenalidomide-mediated cytotoxicity in MM was attributable to oxidative damage of intracellular immunoglobulin proteins. By using several sets of isogenic cells lines with and without CRBN expression, we confirmed that lenalidomide treatment caused accumulation of IgL dimers only in CRBN-positive cells. Lenalidomide-induced IgL dimerization lead to decreased secretion and consequent intracellular accumulation of IgL, as evidenced by unchanged IgL mRNA expression, increased total intracellular IgL protein, and decreased secretion of IgL. After 72 hours of lenalidomide treatment we found decreased XBP-1u, increased XBP-1s, and over-expressed GRP78/BiP endoplasmic reticulum stress (ERS) marker proteins in CRBN positive cells but not in CRBN knock-down cells. We observed Bim requirement, especially BimEL, after lenalidomide treatment in CRBN-positive lenalidomide-sensitive cells. Our data reveals that lenalidomide-mediated; progressive ERS can positively enhance bortezomib-induced apoptosis in an in-vitro MM model. We pretreated MM cells with lenalidomide and then treated them with bortezomib. OPM2 cells pretreated with lenalidomide for 2 days clearly showed increased sensitivity to bortezomib-induced apoptosis compared with cells that were not pretreated. Conclusion IMIDs inhibit H2O2 decomposition. Ikaros protein degradation is a consequence of H2O2 mediated oxidative stress. Therefore, cells producing high H2O2 and with less antioxidative capacity are more sensitive to IMIDs. On the basis of this discovery, we would be able to predict which patients will benefit from IMIDs-mediated therapy and develop new drugs other than IMIDs that can inhibit intracellular H2O2 decomposition in MM. At present, CRBN may be required for IMIDs to effectively inhibit H2O2decomposition. Disclosures Chirackal: Mayo Clinic: Patents & Royalties: Filed a professional US patent for quantifying cellular anti-oxidative capacity. Fonseca:Mayo Clinic: Patents & Royalties: Filed a professional US patent for quantifying cellular anti-oxidative capacity.

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.

Abstract Abstract 4984 Background: Waldenström macroglobulinemia (WM) is a B-cell lymphoproliferative disorder characterized by lymphoplasmacytic bone marrow infiltration, immunoglobulin M (IgM) monoclonal gammopathy and increased production of interleukin-6 (IL-6). Anemia occurs in a significant number of these patients and it is postulated that this occurs independently of tumor burden. Hepcidin is a hepatic peptide hormone that plays a central role in iron homeostasis and has been implicated in anemia of inflammation. Hepcidin binds to the cell-membrane iron exporter-ferroportin, causing its internalization and degradation, leading to iron deficiency anemia. The role of hepcidin in WM patients with anemia is not clearly understood. In this study we investigated the association between anemia, inflammation and hepcidin in WM patients with an eventual goal of providing improved treatment strategies for anemia in this complex set of patients. Patients and Methods: Fifty-nine WM patients [Female N=25, Median Age 70 (49-89), Hemoglobin (Hg) 11.1gm/dl, IgM 3420mg/dl, Beta-2 Microglobulin (β2M) 3.105mcg/ml and Bone Marrow Involvement 30%] and fifteen healthy patients [Female N=6, Median Age 36 (18-51)] were analyzed. The patients were divided into anemics (Hg <12, N=38) and non-anemics (Hg >12, N=21). The serum level of IL-6 was measured using Human IL-6 ELISA Kit II (BD Biosciences) and hepcidin using Hepcidin ELISA (DRG Instruments GmbH, Germany) with protocols supplied by the manufacturers. Tumor cells from patients with WM were sorted by either CD19 or CD138 expression, RNA extracted and hybridized to Affymetrix U133A 2.0 expression arrays. Gene expression analysis was performed using Agilent Genespring GX 11.0 software. Gene expression data was summarized using GCRMA and median normalized per gene. Statistical analyses were performed by utilizing student t tests and One-way ANOVA using PRISM software. Results: WM patients with anemia had significantly higher levels of IgM (4121 vs. 2845mg/dl, p=0.021), beta-2 microglobulin (3.77 vs. 2.72mcg/ml, p=0.009) and bone marrow involvement (41.45 vs. 27.14%) as opposed to non-anemics. IL-6 levels were significantly higher in anemics when compared to non-anemics and controls (6.486 vs. 1.952 vs. 1.716 pg/ml, p=0.0433). Further, in WM patients with anemia serum hepcidin levels were significantly elevated when compared to non-anemics (13.03 vs. 6.08ng/ml, p=0.0123). An additional comparison was made with severe anemics (Hg <10g/dl) to non-anemics and higher mean hepcidin levels were noted in the former (16.21 vs. 6.08 gm/dl, p=0.0057). Gene expression analysis was then performed on 12 patients identified as severe anemics (Hg < 10g/dl) or non-anemics (Hg > 12g/dl). Unsupervised clustering identified 2 major groups that separated anemics versus non-anemics. Further, unsupervised clustering segregated anemic patients with the largest IgM values, suggesting that the expression and subsequent clustering of genes was driven by the level of tumor burden in this patient cohort. Also, up-regulation of genes involved in the cell cycle and proliferation such as cyclin A2, cyclin B1, PCNA and BUB1B were identified in anemic WM patients. Conclusion: A statistically significant increase in tumor burden (increased IgM, β2M and bone marrow involvement) was observed in WM patients with anemia as compared to patients without. A statistically significant increase in serum IL-6 was noted in WM patients with anemia. Interestingly, the WM patients with anemia were also noted to have higher serum hepcidin levels. Taken together, our data suggests that increased tumor burden may promote increased IL-6 and subsequently results in increased hepcidin, which correlates with anemia in WM patients. Additional studies are required to determine the specific role of hepcidin in anemia of WM. Anemia causes significant morbidity in WM patients; therefore, understanding the mechanism of anemia is important in providing superior patient care. Disclosures: Fonseca: Amgen: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy, Research Funding; Genzyme: Consultancy; Onyx: Research Funding; Otsuka: Consultancy; Medtronic: Consultancy.

Abstract Abstract 38 INTRODUCTION: Lenalidomide and bortezomib have moved into the management of newly diagnosed multiple myeloma leading to dramatically improved outcomes. As a consequence, the role of upfront autologous peripheral blood stem cell transplant (ASCT) has become more controversial. The ECOG E4A03 clinical trial randomized newly diagnosed MM patients to lenalidomide with high-dose dexamethasone (LD) vs lenalidomide with low-dose dexamethasone (Ld) (Rajkumar et al Lancet Oncol 2010; 11: 29–37). Upon completing four cycles of therapy, pts had the option of ASCT or continuing on the assigned therapy. The purpose of this abstract is to determine the outcome of patients on this trial pursuing early ASCT according to various age-groups. MATERIALS and METHODS: This is a post hoc, retrospective analysis of overall survival within age subgroups stratified by early ASCT status. This is a landmark analysis including only pts surviving the first 4 cycles of therapy. RESULTS: In all three age-groups studied, 1, 2, and 3-year survival probability estimates with ASCT were excellent (Tables 1, 2, and 3). For patients under the age of 65 who survived the first 4 cycles of therapy, overall survival at 3-years was 94% with early ASCT, 78% in pts continuing protocol therapy. Although direct comparison with patients not going to early transplant is not possible because the assignment to early ASCT versus no early ASCT was not randomized, survival with ASCT at 3-years appeared higher. While we attempt to correct for age, the differences may be influenced by other factors such as performance status, comorbidities, response to therapy, etc. The presumption that treatment related mortality (TRM) should be more problematic for older pts undergoing ASCT is addressed by looking at the >65 and >70yo cohorts. In the >65 age group, one-year mortality is similar between the early ASCT population and the no early ASCT population. In the >70 age group, no adverse impact of early ASCT was seen in the first year on overall survival but the sample size is extremely small. In all age groups early ASCT seemed to mitigate some of the survival disadvantage associated with randomization to the LD arm. CONCLUSIONS: This analysis shows that the strategy of lenalidomide plus dexamethasone induction followed by early ASCT has a remarkably good outcome in terms of overall survival in all age groups studied and supports the continued role of early consolidative ASCT in newly diagnosed patients. The risk of early mortality was notably low in the first year in all age groups. The risk of early mortality seems to increase at 2 years for the LD pts not choosing early ASCT and at 3 years for the Ld pts not choosing early ASCT. Selection bias makes it difficult to compare results for pts that chose early ASCT directly to the patients who did not receive early ASCT in this trial. As such, these results emphasize the need for randomized trials investigating the timing of ASCT in myeloma in the era of novel therapy. Disclosures: Siegel: Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck: Membership on an entity's Board of Directors or advisory committees. Off Label Use: Lenalidomide for front line therapy. Abonour:Celgene: Speakers Bureau; Millennium Pharmaceuticals: Speakers Bureau. Callander:Millennium Pharmaceuticals: Research Funding. Fonseca:Amgen: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy, Research Funding; Genzyme: Consultancy; Onyx: Research Funding; Otsuka: Consultancy; Medtronic: Consultancy. Vesole:Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Abstract Abstract 1923 Our understanding of the genetic abnormalities associated with the development of multiple myeloma has increased significantly in the last decade. However, very little is known about how, or if, myeloma tumor genomes change with time and if therapeutic interventions influence these events. To address these issues we studied a cohort of 29 patients for whom at least two serial samples (1-65 months, median 19 months) were available for analysis. Each serial pair was analyzed by both array-based comparative genomic hybridization (aCGH) and microarray gene expression profiling (GEP) to identify DNA copy number abnormalities (CNA) at a 25kb resolution and gene expression differences present in the bulk of the tumor mass. Though this does not address the intra-clonal heterogeneity that may exist at a given time point, it does answer if the bulk of the tumor mass is changing with time. This study has unearthed several surprising and clinically relevant findings. First, myeloma tumor genomes are not as unstable as previous cytogenetic analyses suggest. In 40% of patients we observed no detectable CNA changes (1-37 months, median 12 months). In 24% of patients we observed the exclusive acquisition of new CNA (1-12, median 3.5) (3-22 months, median 18 months). In 36% of patients we observed both the loss (1-20, median 3) and gain (1-33, median 21) of CNA (5-43 months, median 20 months). Because time was not a significant influence on the detection of stable or unstable genomes we compared CNA changes with TC class and found patients with the high-risk 4p16 and maf IgH translocations were over-represented in the latter subset of patients. These observations raise the question of what happens between multiple rounds of therapy and if different regimens influence these phenotypes differently. For two patients with no CNA changes between the first two time points there was an additional sample that extended the follow-up by 52 and 12 months. Again no CNA changes were seen between diagnosis and these final samples taken 63 and 50 months later. For one patient with CNA changes (5 shared, 29 lost, and 32 gained) we have a detailed time course of 5 samples from diagnosis through to end-stage plasma cell leukemia. This patient received continuous lenalidomide-dexamethasone (Rd) for 20 months and progessed with a clone containing a BIRC2/3 deletion, which activates the NFKB pathway. The patient received single agent PR-171 and a bortezomib containing regimen and unexpectedly, the tumor genome observed in the third sample was almost identical (32 shared, 2 lost, and 4 gained CNA) to the first time point, including two copies of BIRC2/3. Subsequently, the patient received melphalan-prednisone-bortezomib (MPV) and the tumor genome observed in the fourth and fifth samples, which were identical, were similar to that seen in the second sample (24 shared, 13 lost, and 39 gained CNA). To understand these observations better we performed FISH to ascertain if the observed clones were detectable earlier, albeit at a low frequency. These experiments proved that the two dominant subclones observed at time points 1 and 3 versus 2, 4, 5 were mutually exclusive at the single cell level. Moreover, both of these clones were detectable at diagnosis with 12% of the tumor mass being the second subclone that eventually evolved into plasma cell leukemia. Interestingly, we assayed 5 of the 39 unique CNA observed in the final two samples and only one, the 17p13 deletion, was detectable earlier. This suggests the MPV regimen effectively eliminated a clone that was previously sensitive to Rd and selected for a dramatically evolved subclone that was previously sensitive to two different proteasome inhibitors. Although it is clear that the high-risk patients are enriched in the subset with the most changes, it is not clear if the specific drugs used (Melphalan vs IMID vs proteasome inhibitor) or intervention strategy (Cycled vs continuous/maintenance) and perhaps the response achieved (PR vs CR) influences these events. These observations do highlight two important clinical concepts that need to be considered in the future. First, the meaning of a partial response needs further investigation as this may reflect effective elimination of one subclone but not another. Second, because some patients are not changing or can revert back to a previous subclone we need to consider re-chanllenging patients with previously effective regimens when patients progress. 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. Stewart:Millennium: Consultancy; Celgene: Honoraria. Bergsagel:Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Millennium: Speakers Bureau; Novartis: Speakers Bureau.