Novak, Anne J.

Abstract Massively parallel sequencing analyses have revealed a common mutation within the MYD88 gene (MYD88L265P) occurring at high frequencies in many non-Hodgkin lymphomas (NHL) including the rare lymphoplasmacytic lymphoma, Waldenström’s macroglobulinemia (WM). Using whole exome sequencing, Sanger sequencing and allele-specific PCR, we validate the initial studies and detect the MYD88L265P mutation in the tumor genome of 97% of WM patients analyzed (n=39). MYD88L265P was detected at lower frequencies in other indolent lymphomas including LPL (0%), MALT (4%), nodal MZL (5%) and splenic MZL (8%); all but one MYD88L265P was heterozygous. Due to the high frequency of MYD88 mutation in WM and other NHL, and its known effects on malignant B cell survival, therapeutic targeting of MYD88 signaling pathways may be useful clinically. However, while the effects of MYD88L265P on the activity of IRAK1/4 and NF-κB are have been studied previously, we are lacking a thorough characterization of the role of intermediary signaling proteins such as TRAF6 and TAK1 on the biology of MYD88L265P-expressing B cells. A better understanding of the proteins involved in MYD88L265P signaling may lead to the development of more targeted and effective therapeutic approaches. In an attempt to identify MYD88L265P –specific therapeutic targets we first wanted to characterize the role of intermediary signaling proteins that facilitate the downstream activation of NF-κB. Upon activation of TLRs or IL-1b receptors, MYD88 forms a homodimer and recruits IRAK1/4 and TRAF6 into a complex resulting in association and phosphorylation of TAK1 followed by activation of NF-κB. We monitored the formation of a complex comprised of MYD88, IRAK1, IRAK4 and TRAF6 and immunoprecipitation of either endogenous IRAK4 or IRAK1 revealed constitutive association of IRAK with TRAF6 and MYD88L265P. To assess if the formation of a MYD88L265P/IRAK/TRAF6 complex results in downstream activation of TAK1, constitutive TAK1 phosphorylation was measured and detected in all three cell lines that express MYD88L265P. An association between TAK1 and TRAF6, another measure of TAK1 activation, was also detectable. When a similar analysis of TAK1 was performed in DLBCL cells expressing wild-type MYD88, no phosphorylation of TAK1 was detected, nor was TAK1 associated with TRAF6. IRAK1, IRAK4, TAK1, TRAF6, and MYD88 were expressed at similar levels in all cell lines studied and therefore did not contribute the differences in MYD88 complex formation observed between cell lines. These studies were further confirmed using HEK 293T cells that were transduced with either a vector control plasmid or HA-tagged MYD88WT or MYD88L265P expression plasmids. Together, these studies suggest that MYD88L265P forms a complex with IRAK and TRAF6 resulting in constitutive activation of TAK1 and NF-κB. To confirm the significance of TAK1-mediated MYD88L265P signaling on lymphoma cell growth, the effect of the selective TAK1 inhibitor, (5Z)-7-Oxozeaenol, on cell proliferation was tested. All MYD88L265P-expressing cell lines were sensitive to TAK1 inhibition in a dose-dependent manner (0-10 μM). In contrast, NHL cells expressing MYD88WT were found to be insensitive to TAK1 inhibition. We next tested the impact of the TAK1 inhibitor on a MYD88L265P positive WM patient sample. Similar to what was seen in the WM cell lines, the TAK1 inhibitor inhibited WM cell growth and survival in a dose dependent manner. Additionally, the TAK1 inhibitor significantly reduced the level of IL-10 secreted by each of the cell lines. Together, these data suggest that MYD88L265P drives cell proliferation and cytokine secretion through a TAK1-dependent mechanism. In conclusion, we are the first to validate by NGS in a large patient cohort the high prevalence and specificity of MYD88L265P in WM. Cells harboring the L265P mutation but not wild-type MYD88 exhibit constitutive signaling leading to the hyperactivation of NF-κB. We have established the role of TAK1 as an integral component of MYD88L265P signaling in both WM and DLBCL cell. Our data suggest that targeting TAK1 clinically may be an effective strategy for the treatment of WM and other lymphomas driven by MYD88L265P signaling. Disclosures: Fonseca: millennium: Consultancy; amgen: Consultancy; Binding site: Consultancy; onyx: Consultancy; medtronic: Consultancy; Genzyme: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; lilly: Consultancy; Onyx: Research Funding; cylene: Research Funding.

Abstract Background: Elevated serum BLyS levels have been found in patients with both malignant and autoimmune diseases when compared to those of healthy donors, suggesting that BLyS may play a pathogenic role. While it is clear that BLyS expression is required for normal B cell development and homeostasis, the mechanistic details underlying control of BLyS expression remain to be defined. In previous work we found that a single nucleotide polymorphisms (SNP) in the BLyS promoter (−871C→T) resulted in increased BLyS transcription suggesting that genetic variation in the BLyS gene may influence its expression. Because elevated BLyS levels have been detected in patients with NHL we wanted to determine if additional SNPs in the BLyS gene were associated with high serum BLyS levels and risk of developing of NHL. Methods: We genotyped 9 tagSNPs within the BLyS gene in a clinic-based study of 441 incident Caucasian NHL cases and 475 frequency matched Caucasian controls seen at the Mayo Clinic from 2002–2005. We evaluated the association of individual SNPs as well as haplotypes from the BLyS gene with risk of NHL. We also jointly tested the main effects for all independent (r2<0.25) SNPs using a multivariate logistic regression (MLR) model. As a secondary analysis, for those SNPs showing significant results (< 5% significance), we evaluated associations between those who had all high risk alleles at the SNPs of interest compared to those who had all low risk alleles at the SNPs of interest. Additionally, we evaluated serum BLyS levels by ELISA in these same subjects. Serum BLyS levels were detemined by ELISA. Results: In the individual single SNP logistic regression analysis, 3 of the 9 tagSNPs were significant at p<0.05. Haplotype and MLR results were nonsignificant. However, when we categorized participants into low and high risk groups based on risk alleles at the three statistically significant SNPs, we found the high risk variant had an odds ratio (OR) of 2.086 (p=0.0001) for risk of B-cell NHL. When the analysis was restricted by histologic subtype we found that diffuse large B-cell lymphoma or follilcular lymphoma grade 3 had an OR of 3.163 (p=0.01), follicular lymphoma grade I/II had an OR= 2.547 (p=0.046), and chronic lymphocytic leukemia (CLL) had an OR=1.238 (p=0.13). Because there was not a significant correlation of the high risk variant with CLL, we performed an additional analysis in which we included all B cell NHL cases excluding CLL and the OR was 2.799 (p=0.0001). We next wanted to determine if serum BLyS levels correlated with either the high or low risk variant. The mean serum BLyS level in those individuals (untreated cases and controls) who carried the low risk variant at all three SNPs was significantly lower (p=0.0061) at 1.3 ng/ml (n=25, range: undetectable-4.4 ng/ml) compared to 4.3 ng/ml in those with the high risk variant (n=74, range: undetectable- 66.8 ng/ml). Conclusions: In summary, we have found that genetic variation in the BLyS gene is significantly associated with an increased risk of developing B-cell NHL, particularly follicular and large B-cell lymphoma. Further, we found that genetic variation is also associated with an increase in serum BLyS levels.Taken together, our data further highlight the role of the BLyS gene in B cell malignancies and characterization of the precise genetic and environmental mechanisms that regulate BLyS may have significant clinical impact.

Abstract Abstract 770 Background: Waldenstrom's macroglobulinemia (WM) is a B-cell lymphoma characterized by high serum monoclonal IgM and infiltration of lymphoplasmacytic cells into the bone marrow. As with many hematologic malignancies, cytokines within the tumor microenvironment play an important role in supporting the growth and survival of malignant WM cells. IL-21 is a pleiotropic cytokine involved in the differentiation of B cells into plasma cells. During malignancy, IL-21 has demonstrated diverse effects promoting the growth of myeloma and Hodgkin lymphoma cells while inducing apoptosis in chronic lymphocytic leukemia. However, the biologic significance of IL-21 has not been examined in WM. Our objective here was to assess the expression of IL-21 and its receptor in WM cells and to examine whether IL-21 contributes to the biology of WM. Results: When compared to normal bone marrows, immunohistochemistry revealed significant IL-21 staining in the bone marrow of patients with WM (n=5). To determine whether WM cells are susceptible to IL-21 in the microenvironment, expression of the IL-21 receptor (IL-21R) was assessed via PCR in CD19+CD138+ cells isolated by positive selection from patients with WM (n=8) and in the newly characterized WM cell line, MWCL-1. Nearly all (7/8) CD19+CD138+ WM cells expressed IL-21R, as did MWCL-1 cells. Using flow cytometry we detected expression of IL-21R protein on the surface of WM cells as well. The contribution of microenvironmental IL-21 to the biology of WM tumors was then examined. In MWCL-1 cells, IL-21 (100 ng/mL) increased proliferation by 37% (p=0.005) over untreated controls as determined by thymidine incorporation at 72 hr, and in primary WM cells, proliferation increased by nearly 50% (p=0.003). Interestingly, the immortalized B cell line, IM-9, responded to IL-21 with a significant decrease in proliferation, consistent with previous data indicating differential effects of IL-21 depending on the pathological status of the B-cell in question. IL-21 also significantly induced (p<0.0005) IgM secretion in WM as measured by ELISA (MWCL-1 5,956 +/− 393 ng/mL vs. 10,013 +/− 730 ng/mL; CD19+138+ WM 504 +/− 33 ng/mL vs. 811 +/− 32.5 ng/mL). Annexin/PI staining was used to assess viability, but no apoptotic effects were associated with IL-21 in WM. To better understand the mechanisms through which IL-21 increases cellular proliferation and IgM secretion in WM, we characterized STAT activation in response to this cytokine. In MWCL-1 cells, IL-21 significantly increased the phosphorylation of both STAT1 and STAT3, and to a lesser extent, STAT5. Treatment with a STAT3 inhibitor completely abolished the effects of IL-21 on cellular proliferation and IgM secretion suggesting IL-21 mediates its biologic activity through a STAT3-dependent mechanism. The expression of transcription factors involved in B-cell differentiation was also measured in MWCL-1 cells treated with IL-21. Both BLIMP-1 and Bcl-6 levels significantly increased upon addition of IL-21, whereas PAX5 was significantly decreased. IL-21 had no effect on the expression of XBP-1, which is involved in regulating Ig secretion, suggesting that the increase in IgM secretion in MWCL-1 cells may occur secondary to the increase in proliferation, as opposed to an actual increase in the production of IgM. Lastly, IL-21 significantly enhanced IL-10 secretion from MWCL-1 cells (669 +/− 152 pg/mL vs. 1,948 +/− 279 pg/mL, p=0.0002). While the interplay between IL-10 and IL-21 in WM remains to be examined, IL-10 is known to be involved in normal B-cell development and may have synergistic effects with IL-21 in malignant WM cells. Overall our data indicate that IL-21 in the bone marrow microenvironment significantly affects the biology of WM tumor cells through a STAT3-dependent mechanism. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: Cutaneous T-cell lymphoma (CTCL) is an incurable lymphoproliferative disorder characterized by the clonal expansion of malignant CD4+ T cells. Currently there is a poor understanding of the underlying mechanisms in the development and progression of this disease. Members of the TNF superfamily have been shown to be important mediators in the formation and regulation of normal T cell responses. A proliferation-inducing ligand (APRIL), a newly identified member of the TNF superfamily, has been shown to stimulate tumor cell growth as well as proliferation of primary B and T lymphocytes. APRIL transcript levels are reported to be low in normal tissues with the highest levels found in peripheral blood leucocytes. In contrast, higher mRNA levels have been detected in tumor cell lines and primary tumor tissues and various tumor cell lines have been shown to respond to APRIL with increased tumor cell proliferation and decreased apoptosis. The striking effects of APRIL on tumor cell growth and survival raise the possibility that the APRIL and its receptors-TACI and BCMA may be involved in the pathogenesis and maintenance of T cell malignancies. Goal: To explore the involvement of APRIL on normal and malignant T cell growth and survival. Results: Under resting conditions, there was minimal APRIL binding to normal T-cells and no TACI or BCMA expression. However, stimulation of resting T cells with OKT3 (anti-CD3) or a combination of PMA and ionomycin resulted in an increase in APRIL binding and TACI expression on a subset of activated T-cells. When CD3+ resting human T cells were cultured in the presence of APRIL, IL-2, or OKT3 alone or a combination, normal T cells had greater viability compared to the nil control and APRIL mediated-T cell survival was similar level to that seen with IL-2 or OKT3 alone. CSFE labeled T cells were activated with suboptimal dose of OKT3 and incubated in the presence of APRIL and /or IL-2. Both APRIL and IL-2 alone induced cell proliferation, while treatment with the combination of APRIL and IL-2 had a synergistic effect on T cell proliferation. Malignant T cells isolated from the blood of patients with Sezary Syndrome exhibited a similar pattern of APRIL binding and APRIL also enhanced their survival. Gene array experiments were performed to identify genes involved in APRIL-mediated survival. In normal and primary malignant T cells the anti-apoptotic genes BCL2A1, BIRC6, CHK1, MCL-1, and MDM2 were upregulated and the pro-apoptotic genes CASP5, DR5, and TNFR2 were downregulated in response to APRIL. Conclusion: APRIL promotes the survival of normal and malignant T cells and co-stimulates CD3-mediated T cell proliferation. The gene array experiments suggest that APRIL regulates the expression of anti-apoptotic and pro-apoptotic genes in both normal and malignant T cells. Taken together, these data indicate an important role for APRIL in the biology of normal T cells and in the pathophysiology of T cell lymphoma.

Abstract Abstract 429 BACKGROUND: While standard clinical prognostic factors such as those included in the International Prognostic Score (IPS) predict outcome in cHL patients, predicting the outcome of patients might be further refined using biological factors. We therefore tested whether pretreatment serum cytokines, that measure the biological activity of the tumor as well as the host response, could provide additional prognostic information in cHL patients. METHODS: Newly diagnosed cHL patients were prospectively enrolled in the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER). Serum cytokines were measured from pretreatment blood draws using a multiplex ELISA (Invitrogen, Camarillo, CA). Thirty cytokines, including pro-inflammatory, Th1 and Th2 associated cytokines, were analyzed using the Luminex-100 system Version 1.7 (Luminex, Austin, TX). Data analysis was performed using the MasterPlex QT 1.0 system (MiraiBio, Alameda, CA). The assay was also performed on research serum draws from 50 non-lymphoma epidemiology controls enrolled on a corresponding lymphoma etiology case-control study. The upper limit of normal for each cytokine was defined as the 95th percentile from the control distribution. Clinical data were abstracted using a standard protocol and all 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 association between cytokines and outcome. RESULTS: 140 HL patients with cHL diagnosed between September 2002 and February 2008 with available pretreatment serum were included. The median age was 40 years (range 18–89) and 53% were male. Seventy-seven patients (55%) had limited stage disease and 63 (45%) had advanced disease. One hundred and twenty one patients (86%) were treated with ABVD chemotherapy, while the remainder received the Stanford V regimen (9%), BEACOPP (4%) or MOPP chemotherapy (2%). At a median follow-up of 47 months (range 0–87), 25 patients (18%) had an event and 16 patients had died (11%). The serum levels of 12 cytokines (EGF, FGFb, GCSF, HGF, IL-6, IL-8, IL-12, IL-2R, IP-10, MIG, TNFa and VEGF) were significantly higher in cHL patients than controls. When correlated with clinical factors, IL-2R, IP-10, MIG, IL-6 and HGF were associated with an increased sedimentation rate, B-symptoms, advanced stage and splenic involvement. Elevated IL-8 levels were associated with bulky disease. Elevated levels of HGF, IL-6, IL-8, IL-2R, IP-10 and MIG were all associated with a poorer EFS, however only IL-2R (p=0.002) and IL-6 (p<0.001) were independently prognostic for both EFS and OS. Furthermore, using IL-6 and IL-2R in a 2-cytokine model (see Figure), patients with elevation of both cytokines had a dramatically greater likelihood of early relapse and death than those with normal levels or elevations of only one of the cytokines (1-year EFS 40% compared to 97% and 93% respectively, p<0.0001; 2-year OS 49% compared to 98% and 95%, p<0.0001). While elevated IL-6 and IL-2R levels correlated with the IPS (p=0.002), this 2-cytokine model remained independently predictive of prognosis. In fact, when only good risk patients (IPS 0–3) were considered, the 2-cytokine model remained predictive of patients at risk for early relapse (1-year EFS 54% versus 95% and 97%, p<0.0001). CONCLUSION: Elevated pretreatment serum cytokines are associated with an increased likelihood of disease relapse and an inferior survival in patients with cHL. The pretreatment cytokine profile, particularly serum levels of IL-6 and IL-2R, may identify cHL patients at high risk for early disease relapse who may benefit from novel therapeutic approaches. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3927 Poster Board III-863 In previous studies we characterized the 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. As part of a larger MALT gene expression-profiling project, we have now acquired gene expression analysis on the patient carrying the t(X;14)(p11;q32) translocation and have confirmed overexpression of GPR34. We then measured GPR34 mRNA expression in a panel of MALT lymphomas (n=17) and found that GPR34 was expressed at levels higher than that seen in normal B cells (mean, 11.3 fold; median, 5.5; range, 1.4-64 fold). When analyzed separately, 70% (12/17) had an expression level greater than 3-fold over normal B cells. Of note, in a gastric MALT lymphoma specimen, we found a 64 fold increase in GPR34 mRNA expression. FISH studies performed on this specimen showed an extra intact GPR34 signal but no translocation involving IGH or GPR34, suggesting that other mechanisms, including gene dosage effect, can upregulate GPR34. Elevated expression of GPR34 mRNA was also detected in other histologic types of NHL, but not to the extent seen in MALT lymphoma. Taken together, these data suggest that GPR34 is commonly overexpressed in MALT lymphoma and that deregulation of GPR34 expression can occur independent of a t(X;14)(p11.4;q32) translocation. The receptor encoded by GPR34 is most similar to the PY2 receptor subfamily of GPCR and GPR34 mRNA transcripts are abundant in mast cells while lower levels were detected in other immune cells including B cells. Signals from GPR34 have been briefly described and the results to date suggest that overexpression of GPR34 results in an accumulation of inositol phosphates. To further characterize the impact of GPR34 overexpression on cell signaling, HeLa cells were transduced with a retroviral expression plasmid (pBMN-GFP) that expresses GPR34 and GFP. GFP expressing cells were isolated and overexpression of GPR34 mRNA was confirmed by PCR and GPR34 protein expression was detected by flow cytometry. When normalized to the isotype control, pBMN-GPR34 cells expressed 17-fold more GPR34 on their cell surface compared to the pBMN-vector control cells. To determine which signaling pathways were affected by GPR34 overexpression, pBMN-GPR34 or pBMN-vector control cells were transfected with an AP-1, CRE, NF-κB, E2F, SRE, NFAT, or ISRE- luciferase reporter plasmid. Upon normalization with renilla, pBMN-GPR34 expressing cells had increased luciferase activity (n=3) driven by AP-1 (5.35-fold), CRE (4.7), NF-κB (2.8-fold), and E2F (2.13) when compared to pBMN-vector control cells. ISRE, NFAT, and SRE mediated luciferase expression was similar in the GPR34 and control cells. AP-1 and CRE have been implicated in a large variety of cellular processes, including transformation, and both AP-1 and CRE activity is induced upon activation of MAP kinases. To determine if MAPK activity was also upregulated in GPR34 expressing cells, we analyzed the phosphorylation status of Erk1/2 in pBMN-GPR34 cells by western blot and found that Erk1/2 was constitutively phosphorylated in GRP34 expressing cells (1.8 fold increase) compared to vector control cells. Increased phosphorylation of PKC-α/β was also detected in pBMN-GPR34 cells (3.5 fold increase compared to control cells). To determine the biologic impact of GRP34 overexpression on cell growth, the proliferation rates of pBMN-control and pBMN-GPR34 cells were compared and it was found that proliferation of GPR34 expressing cells was 2.2 times higher than that seen in control cells. Because the MAPK kinase pathway was found to be active in the pBMN-GPR34 cells, we tested the effect of the MEK inhibitor PD98059 on proliferation and saw a dose dependent decrease in proliferation of GPR34 expressing cells. These results suggest that GPR34-mediated proliferation is Erk-dependent. In summary, these data suggest that deregulation of GPR34 is commonly found in MALT lymphoma and that overexpression of GPR34 results in activation of Erk1/2, phosphorylation of PKC, and results is AP-1 and CRE mediated transcription. Additionally, our data suggest that overexpression of GPR34 results in increased cell growth that is MAPK-dependent. Taken together, this studies indicate that overexpression of a GPCR, GPR34, may be a novel mechanism by which MALT, lymphoma, and potentially other subtypes of NHL, develop. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: Patients with newly diagnosed, advanced-stage, follicular B-cell non-Hodgkin’s lymphomas (NHL) commonly receive rituximab as initial therapy. While antibody dependent cytotoxicity (ADCC) is felt to be the predominant mechanism of action for rituximab, the immunologic effects of rituximab are largely unknown. We have previously shown that genetic polymorphisms in IL-8, IL-2, IL-12 and IL-1 receptor antagonist (IL-1RN) have prognostic significance in follicular lymphoma. The goal of this study was to measure changes in the abovementioned and other serum cytokine levels in patients receiving single agent rituximab as initial therapy for follicular NHL and to identify cytokines that are associated with an increased risk of disease relapse. Methods: Patients with untreated follicular grade 1 NHL, and measurable stage III/IV disease were treated with rituximab 375 mg/m2 intravenous weekly for 4 doses and were then followed for response and time to progression (TTP); no maintenance therapy was provided. Thirty-seven patients were enrolled in this clinical trial between July 1999 and May 2001. As previously reported, the overall response rate in the 36 eligible patients was 72%, with 36% complete remissions. Now, with a median follow up of 5.1 years, the median TTP is 2.2 years (95% CI, 1.2–3.3 years) and the 5-year overall survival is 77% (95% CI: 64–92%). We measured the serum levels of a variety of inflammatory cytokines by ELISA at baseline and 1 month after completing treatment. Serum specimens were available in 30 patients. Thirty two cytokines or receptors including IL-1β, IL-1RN, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 p40/p70, IL-13, IL-15, IL-17, TNF-α, IFN-α, IFN-γ, GM-CSF, MIP-1α, MIP-1β, IP-10, MIG, Eotaxin, RANTES, MCP-1, VEGF, GCSF, EGF, FGF-basic, HGF, BLyS and APRIL were analyzed. Results: A significant increase was seen in the serum levels of BLyS (p=0.0001) after rituximab therapy, while IL-12 (p=0.0002), IL-1RN (p=0.0001), IL-2R (p=0.0001), IL-1β (p=0.02), IL-7 (p=0.02), IL-10 (p=0.02), and MIG (p=0.05) all significantly decreased. All other cytokines/receptors remained unchanged. Higher pretreatment serum levels of IL-2R (p=0.008) and IL-12 (p=0.001) correlated with a shorter TTP. Furthermore, elevated serum IL-2R levels pretreatment correlated with a lack of response to rituximab (p=0.03). Differences in overall survival could not be determined due to the fact that only 8 patients enrolled in the study have died. Because serum IL-2R levels correlated with response and TTP, we measured the cell surface expression of IL-2R in the tumor by flow cytometry. IL-2R was detected on a subpopulation of CD4+ T-cells in all specimens tested (n=14), and on malignant B-cells in 5 cases (36%). Conclusions: Pretreatment serum levels of IL-2R and IL-12 are important prognostic factors for response to therapy or TTP in follicular lymphoma patients treated with rituximab. Furthermore, serum levels of BLyS, IL-1RN, IL-7, IL-10, IL-1β and MIG also change after rituximab treatment. Targeting these cytokines may therefore provide additional clinical benefit to patients treated with rituximab.

Abstract Serum BLyS levels have been found to be elevated in a number of immune disease models and there is increasing evidence that this may correlate with pathogenesis of various B cell related disorders including B cell malignancies. While it is clear that BLyS expression is required for normal B cell development and homeostasis, the exact source of BLyS in these scenarios and definition of the mechanisms that control BLyS expression remain to be fully elucidated. Serum BLyS levels are elevated in a number of B cell malignancies known to have a familial incidence. Therefore, we sought to determine if there was any correlation between serum BLyS levels and family history of B cell cancers. In our initial studies we examined the serum BLyS levels in patients with sporadic vs. familial B-cell chronic lymphocytic leukemia (B-CLL; near relative with B-CLL, multiple myeloma, or non-Hodgkin lymphoma), and normal age-matched controls. In the normal controls (n=50), the mean serum BLyS level was 12.7 ng/ml, and we found that 4/50 (8%) had BLyS levels exceeding 20 ng/ml. In the sporadic CLL cohort (n=52), the mean serum BLyS level was 18.3 ng/ml, and we found that 5/52 (10%) had BLyS levels exceeding 20 ng/ml. In striking contrast, in the familial CLL cohort (n=24), the mean serum BLyS levels was 33.4 ng/ml, and we found that 11/24 (46%) had BLyS levels exceeding 20 ng/ml. The percentage of patients with elevated BLyS levels, as well as the mean BLyS levels in the familial CLL cohort compared to the normal controls, was significantly higher (mean, p=0.002) and suggests that elevated BLyS levels may correlate with familial CLL. Because of the correlation between BLyS levels and family history B cell malignancies, we next wanted to determine if there was a common underlying genetic event influencing BLyS expression in this group of patients. We began by sequencing the BLyS promoter in 19 patients with B-CLL and 11 normal controls. We identified 2 sites that were polymorphic, −661 A/G and −871 C/T. The −871 T/T genotype has been previously reported to be expressed at increased frequency in SLE patients and is associated with elevated BLyS mRNA levels. Using RFLP analysis we examined the presence of the two polymorphisms in our control, sporadic, and familial CLL cohorts. In the control group (n=50) we found that at −871, C/C (wildtype) was expressed in 24%, C/T in 54%, and T/T in 22% of patients. Similarily, in the sporadic CLL group (n=35) we found that at −871, C/C was expressed in 29%, C/T in 57%, and T/T in 14%. In the familial group (n=24) we found that at −871, C/C was expressed in only 8%, C/T in 67%, and T/T in 25%. These data suggest a decreased representation of −871 C/C in the familial group. No significant changes were observed between the three groups at the −661 A/G locus. Additionally, we found that none of the patients with BLyS levels exceeding 20 ng/ml expresses C/C at −871. In summary, our data suggest that BLyS levels are elevated in patients with familial CLL relative to both normal controls and individuals with sporadic CLL and that elevated BLyS levels may correlate with the presence of a T at −871 in the BLyS promoter.

Abstract Abstract 3933 Poster Board III-869 Background Non-Hodgkin Lymphoma (NHL) is a malignancy of lymphocytes with few known risk factors identified to date. Members of the BCL2 and caspase gene families are known regulators of programmed cell death in these cells, and the t(14;18)(q32;q21) is the most commonly observed chromosomal translocation in NHL, a somatic event which results in constitutive BCL2 expression and inhibition of apoptosis. Further, recent pooled analyses of three case-control studies reported gene level associations for both BCL2L11 (Cancer Epidemiol Biomarkers Prev 2009;18:1259) and CASP9 (Blood 2009;114:264) with NHL risk. We therefore evaluated the hypothesis that germline variation in genes from the apoptosis pathway is associated with risk of developing NHL. Methods We genotyped 226 single nucleotide polymorphisms (SNPs) within 36 candidate BCL2-family and capsase-family member genes within the apoptosis pathway in a clinic-based study of 441 newly diagnosed NHL cases (within 9 months of first NHL diagnosis) and 475 frequency matched controls from Minnesota, Iowa, and Wisconsin who were seen at the Mayo Clinic from 2002-2005. Tagging SNPs were selected from HapMap for the following candidate genes: BCL2; BCLAF1; BCL2LA1; BCL2L1, 2, 10, 11, 12, 13, 14; BAD; BAX; BAG1, 3, 4, 5; BAK1; BID; BIK; BNIP2, 3; HRK; CASP1 – CASP10; APAF1; BIRC3; AIF1; and DFFB. Genotyping was completed using the ParAllele (now Affymetrix) Immune and Inflammation SNP panel, supplemented with 9 SNPs from a custom Illumina OPA. SNP call rate and per person call rate were >95% for all SNPs evaluated. The most prevalent homozygous genotype was used as the reference group, and each SNP was modeled individually as having a log-additive effect in a logistic regression model adjusted for age, sex, and residence. We used principal components analysis to assess the association with NHL risk at the gene level. Genes at p<0.05 were declared statistically significant, as were SNPs at p<0.05 from significant genes. Results The mean age at diagnosis for cases was 60.1 years, and 58% were male; among controls, the mean age at enrollment into the study was 61.7 years, and 55% were male. The most common NHL subtypes were chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL, 29%), follicular lymphoma (FL, 27%), and diffuse large B-cell lymphoma (DLBCL, 16%). In gene level analyses, BCL2L11 (also know as BIM) (p=0.0019), BLCAF1 (p=0.0097), BAG5 (p=0.026), and CASP9 (p=0.0022) were statistically significant at p<0.05. For BCL2L11, 2 of the 5 tagSNPs (both intronic) were significant at p<0.05 (rs6746608 and rs12613243). Both genotyped BCLAF1 tagSNPs (rs797558 and rs703193, both intronic) were significant, as was the single genotyped BAG5 tagSNP (rs7042474) from the non-coding region interval of an mRNA transcript. Three of the 7 genotyped CASP9 tagSNPs were significant (rs6685648, rs2020902, rs2042370, all intronic). Similar associations (as assessed by ordinal odds ratios) were observed for significant SNPs from BCLAF1, BAG5, BCL2L11 and CASP9 within the subtypes of CLL/SLL, FL, and DLBCL; however, caution is warranted in interpreting the subtype results due to small sample sizes. Conclusions Three genes from the BCL2 family (BCL2L11, BCLAF1, and BAG5) and one gene from the caspase family (CASP9) were associated with risk of NHL. This confirms gene level results for BCL2L11 and CASP9 in NHL overall and suggests a role for additional BCL2 family members in NHL etiology. In addition to extending these results to other populations, investigation of the functional significance of these genes and consideration of their upstream regulators and downstream targets should assist in clarifying the role of this pathway in lymphomagenesis. Furthermore, in the era of targeted therapeutics, identification of genetic variation in the apoptotic pathway may also assist in predicting prognosis as therapies targeting this pathway are developed. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3929 Poster Board III-865 In addition to providing prognostic information, gene expression profiling studies have helped to elucidate the putative cell of origin (e.g. activated or germinal center B cell) in diffuse large B-cell lymphoma. With the exception of angioimmunoblastic T-cell lymphomas, which are thought to derive from follicular helper T cells, little is known about the cell of origin for the most common peripheral T-cell lymphoma (PTCL), PTCL-unspecified (PTCL-U). Following appropriate antigenic stimulation, naïve CD4+ T cells may terminally differentiate into effector cells, defined by the production of specific cytokines, including IL-4/IL-10 producing Th2 cells. We have previously demonstrated that IL-10, produced by malignant T cells, is systemically elevated in the serum of patients with PTCL and contributes to the suppression of host anti-tumor immunity. Murine studies have demonstrated that the transcription factor GATA-3 is both critically important for IL-10 transcription and is required for the differentiation of Th2 cells. Therefore, we hypothesized that a subset of PTCL-U may be Th2-derived and express GATA-3. To examine this possibility, PTCL-U cases with adequate paraffin-embedded tissue were identified from a single institution (n=46) and immunohistochemically stained for GATA-3 expression. More than 10% of tumor cells expressed GATA-3 in 17 (37%) of the 46 cases examined. Follow up data sufficient to determine overall survival was available for 43 of these patients, in whom the expression of GATA-3 was associated with a poorer overall survival. Patients with GATA-3 negative tumors (n=27) had a median overall survival (OS) of 3.8 years, whereas patients with GATA-3 positive tumors (n=16) had a median OS of 0.7 years (p=.002). Among low-intermediate risk patients, as defined by the International Prognostic Index (IPI), GATA-3 expression was associated with inferior median overall survival (<12 months, p<.001). IL-10 production may explain the adverse impact of GATA-3 expression on OS in PTCL-U, as GATA-3 plays a central role in IL-10 transcription in normal T cells. To determine whether it may have a similar role in malignant T cells, we identified a PTCL cells line (SU-DHL-1) that highly expressed GATA-3 by intracellular flow cytometry. We next transfected these cells with a negative control or GATA-3 specific siRNA and demonstrated that transfection with GATA-3 siRNA significantly decreased GATA-3 expression in these cells. Following knockdown of GATA-3 expression, we measured both cell proliferation by 3H-TdR incorporation and IL-10 production by ELISA. No significant difference in cell proliferation was observed (n=4, p=0.2), whereas an approximately 50% reduction in IL-10 production was observed (n=3, p=.008) following inhibition of GATA-3 expression. Therefore, GATA-3 expression in malignant T cells may promote the production of IL-10, thus impairing host anti-tumor immunity. Immunomodulatory drugs (IMiDs), like lenalidomide, may downregulate GATA-3 expression in normal T cells. Therefore, we sought to determine whether lenalidomide may inhibit IL-10 production in normal and malignant T cells. T cells obtained from normal donors were activated with anti-CD3 and treated with either DMSO or lenalidomide (10 μM). Intracellular staining for both IL-10 and IL-2 was performed 3 days later. In DMSO control treated cells, 1.0% of cells expressed IL-10 and 12% expressed IL-2. In contrast, in lenalidomide treated cells, only 0.3% of cells expressed IL-10 and 17% expressed IL-2 (representative of at least 4 independently performed experiments). Similar results were obtained using T cells from a patient with Sezary Syndrome. We next treated SU-DHL-1 cells with DMSO or lenalidomide (0.1, 1.0 and 10 μM) and measured both cell proliferation by 3H-TdR incorporation and IL-10 production by ELISA. Significant inhibition of SU-DHL-1 proliferation was only observed when cells were treated with 10 μM lenalidomide. In contrast, significant inhibition of IL-10 production was observed in the presence of 1 μM and 10 μM lenalidomide (p<.01). Collectively, the data presented demonstrates that GATA-3 promotes the production of IL-10 by malignant T cells and is an adverse prognostic factor in PTCL-U. Given its immunomodulatory properties, including inhibition of IL-10 production, lenalidomide represents a promising therapeutic agent in PTCL-U. Disclosures: No relevant conflicts of interest to declare.