Witzig, Thomas E.

Abstract Abstract 1586 Transformation growth factor (TGF-β) is a highly pleiotropic cytokine critical to a variety of cellular events such as cell differentiation and apoptosis. TGF-β is synthesized as a prepro-TGF-β precursor and secreted after being processed in Golgi apparatus as a latent form that non-covalently combines both TGF-β and latency-associated protein (LAP). Our previous work in B-cell NHL has shown that the intratumoral T cell composition results in the establishment of a profoundly inhibitory tumor microenvironment. However, the underlying mechanism is only partially understood. In this study, using patient specimens and lymphoma cell lines, we evaluated the role of TGF-β in the tumor microenvironment and determined the effect of TGF-β on the generation of intratumoral TH1, TH17 and Treg cells in B-cell NHL. First, we determined expression of TGF-β and found that a latent form of TGF-β was specifically expressed on the surface of CD19+ B cells, but not on other types of cells from B-cell lymphoma biopsy specimens. By screening cell lines, we found that latent TGF-β was also expressed on the surface of lymphoma cell lines, confirming the finding. Second, we tested whether surface expression by lymphoma cells led to the secretion of TGF-β in culture medium. Using an ELISA assay, we detected variable levels of latent TGF-β in the culture medium of primary malignant B cells (median 100 pg/ml per million cells, range: undetectable −229 pg/ml, n=7). Similarly, lymphoma cell lines secreted variable amounts of TGF-β from undetectable to 200 pg/ml per million cells. Next, we determined the effect of TGF-b on intratumoral T cell proliferation and differentiation. As expected, exogenous addition of TGF-β inhibited the proliferation of T cells. Notably, the proliferation of intratumoral T cells was significantly reduced when co-cultured with lymphoma cells bearing an active form of TGF-β compared to that with lymphoma cells without TGF-β. Using flow cytometry, we showed that the addition of exogenous TGF-β enhanced Foxp3 expression in activated CD4+, CD4+CD45RA+ or CD4+CD45RO+ intratumoral T cells, suggesting that TGF-β promotes the generation of Treg cells in tumor microenvironment. In contrast, TGF-β suppressed the expression of IFN-γ in activated CD4+ T cells and inhibited the up-regulation of IL-12 and IL-23-induced IFN-γ expression in CD4+ cells, indicating that TGF-β suppresses the generation of TH1 cells. TGF-β alone slightly inhibited IL-17 expression in CD4+ T cells; however, TGF-β, in the presence of IL-6 and IL-23, up-regulated IL-17 expression in CD4+ T cells, suggesting proinflammatory cytokines are able to reverse the suppression induced by TGF-β. These results suggest that TGF-β controls the generation of TH1, TH17 and Treg cells contributing to the imbalance of effector TH cells and inhibitory Treg cells in the tumor microenvironment of B-cell NHL. Since malignant B-cells produce TGF-β, these results further support the important role of malignant B cells in the regulation of intratumoral T cell differentiation and the host immune response. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: Adult T-cell acute lymphoblastic leukemia / lymphoma (T-ALL) is an rare and aggressive hematological malignancy with overall survival (OS) according to the UKALL XII/E2993 of 48% at 5 years (Marks et al, Blood 2009). Over the last decade, the incorporation of L-asparaginase, dose intensification (similar to pediatric protocols), availability of newer agents such as clofarabine (Dec 2004) and nelarabine (Oct 2005) have been shown to improve outcomes. We carried out this study to see if the above mentioned changes in treatment have translated to an OS benefit. Methods: After due IRB approval, adults diagnosed with T-ALL from 1990-2014 at Mayo Clinic were identified. All clinical and pathologic data was retrospectively reviewed Comparative analysis was performed based on year of diagnosis before and after 2005 (Group 1, diagnosis prior to 2005, and Group 2, diagnosis post 2005). Survival was estimated using the Kaplan-Meier Method and log-rank test. Chi-square test was used to compare variables. Results: A. Patient Characteristics: Between 1990 and 2014, a total of 92 consecutive patients (pts) with T-ALL were identified. Median age at diagnosis was 33 years (range; 18-88 years) with 72% males. Distribution of pts by year of diagnosis was as follows: Group 1(n= 47) (51%), and Group 2 (n=45) (49%). Median overall survival was 97.2 months. Median follow up for Group 1 was 50 months, during which time 23 (39%) deaths were documented, and 22.8 months (0.9 - 115.4) for Group 2 at which time19 deaths (42%) were documented (p= 0.04). Pts in Group 2 were older than Group1 (median age 41 vs 27 years (p= 0.004). Apart from age, the two groups were similar in other characteristics (Table 1). B. Therapy received by patient groups: We observed a high use of L-asparaginase containing regimens in Group 1 vs Group 2 [35(74%) pts vs 19 (42%), p=0.0013]. In contrast there was an increase in use of Hyper-CVAD in Group 2; 23(51%) vs. 3 (6%) (p<0.0001). There was no difference in the use of intensive pediatric protocols (p=0.11). There was a trend of increased use of nelarabine in Group 2, however clofarabine usage was not different (p=0.09, and p=0.6 respectively) (Table1). Allogenic stem cell transplant was offered more in Group 2 compared to Group 1, 16 (36%) patients vs. 10 (21%) patients (p=0.0009). In contrast, 7 (19%) patients underwent autologous transplantation in group 1 vs. none in group 2 (p=0.0009). C. Overall outcome by patient groups: We did not observe any difference in CR, or relapse rates among the two groups. The median OS and time to relapse were also not statistically different among Group 1 and 2 [102.6 vs 61.8 months Figure A, and 7.1 vs 14.1 months respectively]. Furthermore, age adjusted survival analysis was also not statistically significant. Conclusion: In this large cohort of adult T-ALL patients, in spite of significant advances in treatment strategies over the last two decades, we observed no difference in overall and relapse free survival prior to and after 2005. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Al-Kali: Celgene: Research Funding. Thompson:Kite Pharma: Research Funding. Witzig:Spectrum: Research Funding; Novartis: Research Funding; Celgene: Research Funding; Amgen: Honoraria; Valeant Pharma: Equity Ownership.

Abstract While extensive, the genetic studies of DLBCL tumors to date have primarily focused on somatic mutations that are associated with development of lymphoma, i.e., driver mutations. Identification of key genetic events that impact and/or predict response to therapy and clinical outcome of lymphoma patients has not been accomplished. In an exploratory study, we used whole-exome sequencing to identify novel biomarkers that can predict which patients have an aggressive form of DLBCL, and therefore would likely benefit from a different and more aggressive treatment plan. Using whole-exome sequencing data from 54 newly diagnosed DLBCL patients, we evaluated the association of somatic coding single nucleotide (cSNV) and copy number (CNV) variants with aggressive DLBCL. All patients were treated with R-CHOP or immunochemotherapy, and disease aggressiveness was based on relapse, with patients classified as having aggressive disease (AD) (n=13, defined as treatment failure, relapse, or progression within 24 months of diagnosis) versus non-aggressive disease (n=41, defined as achieving at least 24 months of relapse-free survival). An in-house algorithm, patternCNV was used to detect somatic CNVs. Logistic regression was used to assess statistical significance. In the cSNV analysis, 24 genes were found to be significantly (p ≤ 0.05) associated with AD. Of these genes, CIITA (p=0.01) was previously identified as a mutational target in DLBCL and is a recurrent gene fusion partner in lymphoid cancers. Metacore analyses showed that seven of these genes, including CIITA, can be placed in the same regulatory network centered around CREB1. Interestingly, the CREB binding protein (CREBBP) is a known target of pathogenic mutations in DLBCL. In the CNV analyses, we identified 245 gene amplifications and 209 gene deletions associated with AD (p ≤ 0.05). Deleted genes were localized in three major chromosomal regions, 6p22.3-6p22.1 (26 genes), 6q13-6q16.3 (31 genes), and 6q21-6q24.2 (86 genes). In the CNV amplification analysis, the genes were localized in two major chromosomal regions, 3q12.3-3q29 (90 genes) and 19q13.12-19q13.43 (122 genes). We also quantified the association of each regional deletion/amplification with patient outcome. As expected, each deleted region was univariately associated with time to relapse: 6p22.3-6p22.1 (HR = 7.19, 95% CI: 2.66-19.46, p=1.33x10-5), 6q13-6q16.3 (HR = 4.44, 95% CI: 1.74-11.32, p=6.84x10-4), and 6q21-6q24.2 (HR = 5.42, 95% CI: 2.12-13.87, p=9.23x10-5) as well as the two amplification regions: 3q12.3-3q29 (HR = 4.60, 95% CI: 1.49-14.24, p=4.27x10-3) and 19q13.12 -19q13 (HR = 13.79, 95% CI: 4.39-43.34, p=2.81x10-8). Deletions at 6q21-6q24.2 have been previously reported in DLBCL and two tumor suppressor genes associated with lymphoma pathogenesis are localized in the region, TNFAIP3 (p=0.14) and PRDM1 (p=0.02), the latter of which was significantly associated with AD. However, the genes in the 6p21 locus that were most significant (p=0.002) were SLC22A16, GPR6, SOBP, MATTL24, DDO, and REV3L. SLC22A16, which also had cSNVs in two tumors, is an organic cation transporter that has been shown to transport chemotherapeutic drugs including doxorubicin. Successful drug response has been correlated with the level of activity and expression of this transporter in tumor cells. To determine if expression of SLC22A16 would have an impact on doxorubicin transport, and may therefore impact response to R-CHOP we overexpressed either an empty vector control or SLC22A16 in OCI-Ly7 DLBCL cells, which do not express endogenous SLC22A16 mRNA. We found that OCI-Ly7 cells expressing SLC22A16 have increased 14C-doxorubicin uptake and are more sensitive to doxorubicin-induced cell death when compared to the empty vector control cells. These data suggest that loss of SLC22A16 expression through gene deletion could impact the ability of DLBCL cells to respond to therapy and indicates an aggressive form of the disease. In summary, our data are the first to use whole exome sequencing to identify genetic variants associated with aggressive DLBCL, which will require replication in additional samples. Nevertheless, our data highlight the role for somatic CNVs in patient outcome and we biologically validate the potential impact of deletions in SLC22A16 at 6p21. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1790 CLL is incurable with conventional therapies and prognosis is poor in patients with relapsed or treatment refractory disease. Patients with purine analogue refractory disease and defective TP53 function have limited therapeutic options. Based on: 1) The ability of purine analogues to decrease tumor burden; 2) Higher response rates in patients treated with alemtuzumab and rituximab vs. alemtuzumab alone; and 3) Data showing that more frequent lower doses of rituximab could be more effective than standard therapy, we hypothesized that combination therapy with pentostatin, alemtuzumab and low dose rituximab (PAR) would be effective and tolerable therapy for patients with relapsed/refractory CLL and those with previously untreated progressive CLL who had 17p13-. This two stage phase II University of Iowa/Mayo Clinic Lymphoma SPORE clinical trial (NCT00669318) was approved by Mayo Clinic and the University of Iowa IRBs according to the principles of the Declaration of Helsinki. The aims are to assess: 1) Complete (CR) and overall responses; and 2) Progression-free survival (PFS), duration of response, and time to next treatment (TTT). Patients with progressive CLL (including the SLL variant) defined by standard criteria are eligible for this study if they had either previously treated CLL or previously untreated CLL with 17p13-. Cycle 1 of therapy is 5 weeks with rituximab 20 mg/m2/d IV Mon-Wed-Fri starting day 1, subQ alemtuzumab beginning days 3–5 with a dose escalation (3 mg–10 mg– 30 mg/d) and then 30 mg/d Mon-Wed-Fri, and pentostatin 2mg/m2/dose/IV days 8 & 22. Cycles 2–3 are each of 4 weeks duration and are the same as weeks 2–5 of cycle 1. Patients who achieve CR after cycle 2 with a negative CT scan and bone marrow study with no evidence of residual CLL cells after immunohistochemical staining, do not receive cycle 3 of therapy. All patients receive granulocyte growth factor support after pentostatin therapy as well as PCP and herpes virus prophylaxis. Patients are tested for CMV reactivation by PCR weekly during therapy and treated with valganciclovir if reactivation is detected. We report the results of the planned interim analysis performed after completion of therapy for the first 19 patients (July 2008 - October 2010). The median patient age was 63 years (range 47–78) with 74% males. Two patients with 17p13- were previously untreated. Of the 17 previously treated patients (median 2 prior regimens, range 1–6), 8 (47%) had purine analogue refractory CLL (disease progression < 6 months of treatment). Nine (47%) patients had advanced clinical stage (Rai III-IV). Adverse molecular prognostic factors were 17p13- (n=8, 42%, 2 also had 11q22-), 11q22- (n=1), unmutated IGHV (12/17, 71%), ZAP70+ (≥20%, 12/17, 71%) and CD38+ (≥30%, 6/18, 33%). The median beta-2-microglobulin level was 4.3 (range 2.4 – 12.6). Grade 3–4 treatment related toxicities were 9 non-hematological and 20 hematological events. Grade 3–4 infections occurred in 3 patients including one patient requiring hospitalization for CMV re-activation. Fourteen (74%) patients responded to treatment with 6 (32%) CR (includes 2 CRi) and 8 (42%) partial responses (PR). Five (26%) patients had stable disease (SD). Eleven (58%) patients completed 3 cycles of therapy. One patient achieved a CR with no residual disease after 2 cycles of therapy and received no further treatment per protocol. Median duration of response is 7 months (95% CI: 4–not reached (NR)). Among the 8 patients with 17p13- there were 3 CR/CRi, 4 PR, and 1 SD. Four patients proceeded to allogeneic transplant with reduced intensity conditioning (RIC) and were censored for TTT at the initiation of transplant related therapy. With a median of 16 (range 6 – 35) months of follow up, median PFS is 7 months (95% CI: 5–NR), median TTT is 27 months (95% CI: 5-NR) and the calculated median overall survival is 23 months (95% CI: 14–NR). We report that PAR is effective and tolerable therapy in this population of relapsed refractory patients with high to very-high risk CLL. In addition, the responses in patients with 17p13- were similar to that of the patients who did not have 17p13-. The treatment regimen was useful for disease control in 3 patients who proceeded to reduced intensity conditioning allogeneic transplantation. This study shows that PAR could play an important role in the treatment of recurrent and high risk CLL. Supported by the University of Iowa/Mayo Clinic Lymphoma SPORE CA097274 Disclosures: Zent: GlaxoSmithKline: Research Funding; Genentech: Research Funding; Genzyme: Research Funding.

Abstract Abstract 1580 Background: Extranodal Marginal Zone Lymphomas of Mucosa Associated Lymphoid Tissue (MALT) account for approximately 5% of all non-Hodgkin Lymphoma cases. Specific MALT lymphomas are characterized by distinct clinical features, treatment strategies, and outcome, depending on site of the disease. While previous reports have described clinical course and treatment of gastric and pulmonary MALT lymphomas, a paucity of data exist describing primary parotid MALT lymphoma. This is a single institution experience. Methods: Patients with primary parotid MALT lymphoma were identified from the Mayo Clinic Lymphoma Database since 1986. This database includes all consenting patients with lymphoma seen at Mayo Clinic Rochester. Clinical characteristics, treatment, and outcomes were collected. Pathology was reviewed by a lymphoma pathologist; WHO criteria were used for diagnosis. PFS and OS were assessed using the Kaplan-Meier method and Cox proportional hazard model was used to asses the impact of variables on PFS and OS. Results: Seventy-six consecutive patients with primary parotid MALT lymphoma were evaluated and treated at Mayo Clinic from 1986 to 2010. The median age at diagnosis was 60 years (18–90). 58 (76%) were female. 42 (55.3%) patients had a history of Sjögren's Disease and 51 (67.1%) had a history of autoimmune disorders. 52 (68.4%), 4 (5.3%), and18 (23.7%) had stage I, II, and IV disease, respectively. 2 cases did not have complete staging information. 6 (7.8%) and 2 (2.6 %) had bone marrow and lung involvement, respectively. No patients had GI tract, liver, or CNS involvement. Median estimated OS (overall survival) was 18.3 years and median PFS (progression free survival) was 7.9 years. ALC<0.96 (p=0.0162) were associated with shorter survival. Age, ECOG performance score, stage, LDH, and history of Sjögren's disease were not prognostic for overall survival. The treatment approach and PFS are outlined in the table. For stage I disease, outcomes were excellent with local therapy. The median estimated PFS was 7.9 years with 72% of patients undergoing surgery surviving at 8 years. Patients receiving radiotherapy had an estimated median PFS of 15.7 years and median OS of 18.3 years. In contrast, patients being observed had a median PFS of 5.5 years. The use of systemic therapy with chemotherapy, immunotherapy or in combination was associated with a median PFS of 7.4 years. Summary: Primary parotid MALT lymphoma is strongly associated with Sjögren's disease. This lymphoma is usually limited to the parotid and associated with a favorable prognosis. For limited disease, local treatment with radiation or surgery appears to be an effective treatment options. For extensive disease, both immunotherapy and chemotherapy are efficacious treatments. Further prospective studies and long-term follow up are needed to determine the optimal treatment approaches and efficacy of chemotherapy/immunotherapy regimens. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1572 Background: The incidence of histologic transformation (HT) of follicular lymphoma (FL) to an aggressive lymphoma has been reported to range from 10–75%. The British Columbia Cancer Agency reported a risk of 3% per year (Al-Tourah AJ, et al J Clin Oncol 2008;26: 5165–5169). The hallmark of HT is the acquisition of new somatic mutations and genomic instability within the malignant clone. DNA damaging agents such as radioimmunotherapy (RIT) may increase the risk of HT by inducing additional DNA damage. The incidence of HT after RIT is unknown. This single institution study analyzes the risk of histologic transformation (HT) in patients with FL after RIT treatment with yttrium-90 ibritumomab tiuxetan. METHODS: The Mayo Clinic Lymphoma Data Base were queried for all patients having received a single dose of yttrium-90 ibritumomab tiuxetan for FL. The Mayo Clinic Lymphoma Database includes all consecutive adult patients diagnosed with lymphoma evaluated at Mayo Clinic Rochester. The prospective radioimmunotherapy lymphoma database was also utilized (GAW). The diagnosis of FL was defined by current WHO Classification, and all cases were centrally reviewed. HT was defined as the development of histologically proven diffuse large B cell lymphoma in patients with history of Grade 1 or 2 FL (change to Grade 3A/B FL was not included). Patients having demonstrated evidence of HT prior to RIT were excluded from this analysis. RESULTS: 119 consecutive patients were included. 64(54%) were male. The median follow-up was 57(range 2–175) months. RIT was administered as treatment for progressive FL in 96.6% of patients. RIT was administered as a consolidation therapy in 4(3.4%) patients. The median number of prior systemic treatments was 2 (range 0–10). The median time from diagnosis to RIT was not different in patients with HT vs. those without HT 44 (10–186) vs. 40 months (range 1–394) (p=0.30), respectively. The median time to HT from RIT was 13 months (range 1–97). Four patients had HT within 3 months of RIT. The median survival after HT was 15 months (range 1–147). The estimated five year risk of transformation from RIT by Kaplan-Meier in patients with fludarabine exposure and those without exposure was 55% and 29% (p=0.0498). 10 year risk from the time of RIT was 64% with fludarabine exposure and 31% without (p=0.0498). The relative risk of HT after RIT in patients previously treated with fludarabine was HR=4.1 (95% CI: 2.0–7.9). The median number of prior therapies in patients with HT vs. patients without HT was 2 (0–6) and 1 (0–10) (P=0.33). CONCLUSIONS: To our knowledge, our data is the first to estimate the risk of HT after the use of RIT. The estimated overall risk of HT of 31% at 10 years from diagnosis in patients without fludarabine exposure is similar to that of patients in whom RIT has not been administered. The estimated overall risk of HT of 64% in those patients having received purine analogues appears to be increased. We conclude that RIT appears to not increase the risk of HT in patients who did not receive prior purine analogue therapy. The differences in extent and mechanisms involved in DNA damage induced by radiation vs. purine analogues may play a role in clinical differences in observed rates of HT. Disclosures: No relevant conflicts of interest to declare.

Abstract Background WM remains an incurable cancer despite recent advances. While a phase II trial has demonstrated efficacy of dexamethasone, rituximab and cyclophosphamide (DRC) in the treatment-naïve (TN) patients, this regimen has not been studied systematically in the relapsed/refractory (R/R) setting. Recent data has suggested the MYD88mutation status in WM patients can serve as a predictive marker with therapies such as ibrutinib. We examined the efficacy and tolerability of up to 6 cycles of DRC, with focus on R/R population and activity of this regimen with respect to the patients' MYD88L265Pstatus. Methods Records of WM patients seen consecutively at Mayo Clinic campuses in Rochester, Arizona and Florida from 01/2007 to 12/2014 were reviewed. MYD88L265P status, as assessed by AS-PCR, was recorded when available. Data obtained from symptomatic patients treated with DRC (dexamethasone 20 mg intravenously followed by rituximab 375 mg/m2 intravenously on day 1 and cyclophosphamide 100 mg/m2 orally bid on days 1 to 5) were analyzed. Time-to-event analyses were performed from DRC therapy using the Kaplan-Meier method. We used the Consensus response criteria (6th International Workshop). Results Of 100 symptomatic patients who received DRC, 50 had R/R WM (40% refractory) and 50 were TN. Table 1 shows the patients' baseline characteristics. In the R/R population, DRC was 2nd line (range 2-8) therapy in 58% of patients. The median IgM levels declined from 3,870 mg/dL to 1,846 mg/dL (p=0.0001) at best response. Median follow-up from DRC was 51 months (95% CI: 38-55). The median time-to-best response was 6.8 (0.5-28) months. Overall response rate (ORR) was 87% [VGPR 4%, PR 64%, MR 19%]. Four patients (9%) achieved SD and 2 patients (4%) had PD. Of the sixteen deaths (32%) noted at time of analysis, 11 were related to progressive disease. The median disease-specific survival (DSS) from DRC was not reached (NR) (95% CI: NR-NR). The median progression-free survival (PFS) and time-to-next-therapy (TTNT) were 32 months (95% CI: 15-51) and 50 months (95% CI: 35-60), respectively. In contrast, in the TN patients the median IgM levels declined from 4,130 mg/dL to 1,250 mg/dL (p=0.0001) at best response; median time to best response was 11 (0.6-47) months; median follow-up from DRC was 30 months (95% CI: 21-36). ORR was 96% [VGPR 17% (n=8), PR 70% (n=32), MR 9% (n=4)]. Of the 7 deaths (14%) at time of analysis, 4 were related to WM progression. Median DSS from DRC was NR (95% CI: NR-NR); median PFS and TTNT were 34 months (95% CI: 23-NR) and NR (95% CI: 37-NR), respectively. Among 29 genotyped patients in whom MYD88 mutation status was available, MYD88L265P was present in 25 patients (8 of 10 TN and 17 of 19 R/R patients). The response rates and the time-to-event outcomes were similar in the MYD88L265P and wild-type patients. Grade ≥3 adverse effects included neutropenia (20%), thrombocytopenia (7%) and infections (3%). Conclusions Similar to the frontline setting, DRC is an active and well-tolerated salvage regimen. In contrast to ibrutinib, DRC offers a less expensive, limited-duration treatment option, with preliminary data suggesting activity independent of patients' MYD88 mutation status. Disclosures Ailawadhi: Pharmacyclics: Consultancy; Novartis: Consultancy; Amgen Inc: Consultancy; Takeda Oncology: Consultancy. Kumar:Glycomimetics: Consultancy; Sanofi: Consultancy, Research Funding; Noxxon Pharma: Consultancy, Research Funding; Onyx: Consultancy, Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Kesios: Consultancy; Array BioPharma: Consultancy, Research Funding; Skyline: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy. Dispenzieri:Jannsen: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; pfizer: Research Funding; Celgene: Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees; Alnylam: Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees. Ansell:BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding. Kapoor:Amgen: Research Funding; Celgene: Research Funding; Takeda: Research Funding.

Abstract Abstract 2655 Diffuse large B cell lymphoma (DLBCL) has been classified into two distinct molecular subtypes: germinal center B cell–like (GCB), non-germinal centre-like (non-GCB). To improve outcomes for DLBCL patients, it is necessary to identify additional novel targets within GCB and non-GCB classifications to further stratify patients for prognosis and assist in choosing therapy for the individual patient. We have recently demonstrated that STAT3 activation is frequent in the DLBCL tumors, however the exact molecular mechanism(s) of STAT3 activation in DLBCL tumors are not known. Molecular mechanisms such as epigenetic silencing of negative regulators of the STAT3 pathway such as protein tyrosine phosphatase 6 (PTPN6) may contribute to STAT3 activation. We aimed to learn whether PTPN6 was expressed in GCB and non-GCB DLBCL, and if so, how that expression correlated with STAT3 activation and prognosis. We first performed epigenetic studies of PTPN6 in 38 DLBCL tumors and 6 DLBCL cell lines by methylation specific (MSP) PCR and high resolution melting array (HRM) methods. Surprisingly, PTPN6 promoter hypermethylation (0/38) was not detected in patient sample and cell lines by both the methods. Since the MSP PCR technique yields qualitative rather than quantitative data, we next performed pyrosequencing on the 38 DLBCL samples, and found results consistent with the MSP PCR analysis. Our data conclusively demonstrate that PTPN6 hypermethylation is absent in DLBCL tumors. We next sequenced the 600 bp upstream of the transcription initiation site of PTPN6 promoter 2 and 1 in 10 DLBCL tumors. We did not detect any point mutations in the promoter 2 and 1 core regions. Since PTPN6 promoter hypermethylation and mutations were absent in DLBCL tumors, we determined the expression level of the PTPN6 protein in 40 DLBCL tumors by molecular subtype. Formalin fixed paraffin-embedded DLBCL tumor samples were stained by immunohistochemistry (IHC) for the determination of molecular subtype using the Hans algorithm and the detection of PTPN6 expression. Using a threshold of ≥30%, 75% (30/40) of cases were PTPN6 positive with various levels of expression: 11 cases had 30–80% of tumor cells staining positive and 19 had >80% of cells PTPN6 positive. PTPN6 expression by IHC was only correlated with higher IPI scores and a trend towards a shorter event free survival (EFS) (p=0.07). Within the 29 GCB tumors 69% (20/29) were PTPN6 positive; 100% (10/10) of non-GCB cases were PTPN6 positive. These data clearly suggest that PTPN6 expression is found in both GCB and non-GCB with the latter being uniformly positive (p=0.03) and PTPN6 negative cases being uniformly GCB. PTPN6 mRNA and protein was detected in all three ABC lines (LY3, Ly10, DHL2). Within the GCB lines (DHL6, Ly1 and Ly19) DHL6 was weakly positive and Ly1 and Ly19 were negative for PTPN6 mRNA and protein. Furthermore, within the GCB group PTPN6 positive cases had inferior EFS as compared to PTPN6 negative cases. In the non-GCB group all cases were PTPN6 positive with an EFS similar to PTPN6 positive GCB cases. The role of PTPN6 in the persistent activation of the STAT3 pathway in DLBCL patients has not been investigated. We hypothesized that tumors with activated STAT3 would have loss of PTPN6. Interestingly, this hypothesis was disproven. Within the 15-pSTAT3 positive cases 12 (80%) were PTPN6 positive. Conversely, 26% (6/23) of the pSTAT3 negative cases were PTPN6 negative. The distribution of pSTAT3 and PTPN6 by IHC in samples was evaluated in both GCB and non-GCB groups. Within the PTPN6+/pSTAT3+ group out of the 12 cases most were non-GCB (8/12; 66%). However, within the PTPN6-/pSTAT3- group all the 5 cases were GCB (5/5; 100%). Survival analysis revealed that the groups with the best EFS were those with PTPN6-/pSTAT3- tumors (n=5); those with PTPN6+/pSTAT3+ group (n=12) had the shortest EFS; and those with PTPN6+/pSTAT3- tumors (n=17) being intermediate between the other groups. In summary, we have demonstrated for the first time that PTPN6 is highly expressed in DLBCL tumors, and a common abnormality in non-GCB subtypes, which is positively correlated with activated STAT3. PTPN6 expression in the DLBCLs is not regulated through SHP1 promoter hypermethylation or point mutations. The finding that SHP1 loss was found only in GCB cases and was especially favorable should be explored further and may provide an important new stratification factor for future DLBCL studies. Disclosures: No relevant conflicts of interest to declare.

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 4136 Background: The serum free light chain (FLC) assay quantitates free kappa and free lambda immunoglobulin light chains. This assay has prognostic value in MGUS, multiple myeloma (MM), solitary plasmacytoma, and amyloid light chain (AL) amyloidosis, and has been incorporated in response criteria for both MM and AL amyloidosis. We previously showed elevated FLC was associated with poor outcome in diffuse large B-cell lymphoma (DLBCL) patients from a phase II cooperative group clinical trial (Blood (ASH Annual Meeting Abstracts) 2009 November 20, 2009; 114(22): 136). Here we replicate those findings in a large clinic based epidemiology cohort, as well as examine FLC abnormalities in other lymphoma subtypes. Methods: Newly diagnosed lymphoma patients were prospectively enrolled in the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER). Serum FLC was quantitated from enrollment research serum draws within 9 months of diagnosis using the Freelite FLC assay (The Binding Site, Ltd., Birmingham, UK). Elevated FLC was defined as either kappa or lambda above the published reference range. Monoclonal elevated FLC was defined as elevated FLC with an abnormal FLC ratio; polyclonal elevated FLC was defined as elevated FLC with an FLC ratio in the normal reference range. Clinical data was abstracted using a standard protocol for the MER and patients were followed systematically for event-free (progression, retreatment, or death due to any cause) and overall survival (EFS and OS, respectively). Cox proportional hazards models were used to evaluate the unadjusted association between FLC and outcome. Results: 1118 lymphoma patients diagnosed between September 2002 and February 2008 with available serum were assayed. At a medium follow-up of 49 months (range 1–94), 484 patients (43%) had an event and 249 patients died (22%). Elevated FLC was present in 27% of lymphomas, with prevalence ranging from 13% in follicular lymphoma (FL) to 64% for mantle cell lymphoma (MCL) and 77% for lymphoplasmacytic lymphoma. Elevated FLC was present in 27% of DLBCL and associated with poor EFS (monoclonal hazard ratio (HR)=2.56, polyclonal HR=1.99, all p<0.001) and OS (monoclonal HR=3.22, polyclonal HR=2.91, all p<0.0001), replicating our previous findings. Elevated FLC was generally polyclonal (FLC ratio normal) across lymphoma subtypes, though monoclonal elevated FLC (FLC ratio abnormal) was common in MCL and the post-follicular lymphomas. Elevated FLC was associated with inferior EFS in all lymphomas combined (monoclonal HR=1.92, 95% CI:1.44-5.55, polyclonal HR=1.38, 95% CI:1.09-1.74); this association remained consistent in most subtypes (see table). In addition, elevated FLC was associated with poor EFS in MCL (monoclonal) and Hodgkin lymphoma (HL; polyclonal). Elevated FLC was also significantly associated with OS in HL (polyclonal HR=6.24, 95% CI 2.44–15.9), FL (polyclonal HR=5.85, 95% CI 2.62–13.0), post follicular lymphomas (polyclonal HR=4.16, 95% CI 1.17–14.8), as well as for all lymphomas combined (monoclonal HR=2.24, 95% CI 1.52–3.29, polyclonal HR=2.33, 95% CI 1.74–3.11). Conclusions: A significant percentage of lymphoma patients have elevated FLC. Our previous discovery of elevated FLC predicting poor outcome in DLBCL was confirmed in a large, independent dataset. Prevalence and type of elevated FLC (monoclonal vs. polyclonal) varies by lymphoma subtype. Elevated monoclonal and polyclonal FLC is associated with poor outcome broadly in lymphoma, as well as specifically for a number of lymphoma subtypes, supporting FLC as a potentially useful prognostic biomarker in many lymphoma subtypes. Disclosures: No relevant conflicts of interest to declare.