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8052 Background: A significant proportion of patients with FL experience an early relapse and a subsequent poor outcome. While several prognostic indices have been developed, none were designed to predict early failure. Recently, we established that lack of intrafollicular CD4+ T-cell expression predicted risk of early failure, and integrating this microenvironment biomarker with the Follicular Lymphoma International Prognostic Index, termed BioFLIPI, further improved identification of FL patients at risk of early failure ( Blood 2019;134(suppl1):121). However, the microenvironment may be influenced by the genetic composition of tumor. We investigated whether the CD4 biomarker and BioFLIPI were impacted by genetic features of the tumor as assessed by a 23-gene expression prognostic score ( Lancet Oncol 2018;19:549-61). Methods: Of the 186 cases with FL grade 1-3A treated with immunochemotherapy (IC) in our prior study, 152 had digital expression quantification of 23 selected genes (23-GEP score), which used RNA from formalin-fixed, paraffin-embedded samples. Event-free survival (EFS) was defined as time from diagnosis to progression, relapse, retreatment, or death. Early failure was defined as failing to achieve EFS at 24 months. Risk of early failure was estimated using odds ratios (ORs) and 95% confidence intervals from logistic regression models. We also used Cox regression to assess associations with continuous EFS and overall survival (OS). Results: 28% of patients failed to achieve EFS24. Lack of CD4+ intrafollicular expression (38% of patients, OR = 2.33, p = 0.024) and high risk 23-GEP score (26% of patients, OR = 3.52, p = 0.001) each predicted early failure, and in a multivariable model that included FLIPI, both CD4+ (OR = 2.26, p = 0.046) and 23-GEP score (OR = 2.26, p = 0.0.057) remained predictors. Similarly, BioFLIPI modeled as a continuous score (1-4, OR per one point increase = 2.31, p < 0.001) predicted early failure, and the association remained (OR = 2.14, p < 0.001) when the high risk 23-GEP score (OR = 2.79, p = 0.013) was included in the model. When stratified on 23-GEP score, BioFLIPI was a stronger predictor of early failure in low risk (74%, OR = 2.51, p = 0.002) relative to high risk (26%, OR = 1.55, p = 0.27) patients. Similar patterns were observed for EFS and OS. Conclusions: CD4+ T-cell infiltrate and tumor gene expression appear to be independently predictive of early failure in newly diagnosed FL patients treated with IC. Future studies should integrate and validate these measures.

Abstract [Background]: MCL is an aggressive non-Hodgkin lymphoma (NHL) with a median survival of 3-4 years. New agents with mechanisms of action different than standard chemotherapy are needed to improve the outcome of MCL. Recent studies have documented single agent activity of bortezomib and rituximab in MCL. Analogs of rapamycin (temsirolimus) have also shown single agent activity in MCL (J Clin Oncol, June 27, 2005 epub). In order to provide rationale for a clinical trial combining rapamycin analogs with bortezomib or rituximab, we performed in vitro studies of the effect of combinations of these drugs on human MCL cell lines. [Methods]: Two human lymphoma cell lines - Granta 519 and M0258 - were incubated with various concentrations of rapamycin, bortezomib, and rituximab to determine the LD50 of each drug alone. For the experiments with rituximab a secondary goat anti-human (GAH) IgG Fc antibody was used to crosslink cell bound rituximab and induce cell killing. Percent viability was assessed after 48 hours using annexin/propidium iodide staining and flow cytometry. Tests for drug synergy used combinations of rapamycin and bortezomib and the Granta 519 cell line. Fixed ratios of the two drugs were tested and the results were evaluated for synergism (combination index &lt;1 in median effect analysis) in inducing cell death. The drug concentrations used spanned the LD50 of each drug determined from the single-agent studies. In the studies combining rituximab with bortezomib or rapamycin, a single fixed dose of rituximab and GAH Fc was tested with various concentrations of the other drugs to detect additive anti-tumor effects. Studies were performed at least in triplicate. [Results]: All three drugs exhibited single-agent activity in vitro against the two cell lines. The LD50 for rapamycin ranged from 17.5 – 25 micromolar for the Granta 519 cell line and 7.5–10 micromolar for M0258. The LD50 for bortezomib ranged from 2–5 ng/ml in both cell lines. Incubation of the cells with various concentrations of rituximab and GAH Fc had mild cytotoxic activity (reduced viability to 50–70% compared to GAH Fc control). This activity was not concentration dependent indicating saturation of CD20 by rituximab; therefore, a fixed concentration of rituximab was used for the combination experiments. When rituximab was added to bortezemib the % viability decreased by up to 37% (compared to bortezomib alone), and the bortezomib LD50 typically decreased to &lt;1–2 ng/ml (LD50 was 2–5 ng/ml with bortezomib alone). The addition of rapamycin to rituximab increased the cytotoxicity by 23% compared to either drug alone and decreased the rapamycin LD50. When rapamycin and bortezomib were combined as treatment of Granta 519 cells, rapamycin was synergistic with bortezomib with a combination index &lt;1. Sequencing the rapamycin either before or after the bortezomib treatment did not effect synergy in either direction. [Conclusion]: In vitro, rapamycin synergizes with bortezomib to enhance its cytotoxicity in MCL lines. Rituximab enhances the cytotoxicity of both of rapamycin and bortezomib. These in vitro studies provide the rationale to test combinations of these active agents in patients with MCL.

Abstract The proposed immune-mediated mechanisms of action of Rituximab (ADCC or “vaccinal” effect) suggest that the efficacy of rituximab therapy depends on the immunocompetency of the host. Therapies, such as IL-12, that stimulate the immune system may increase the efficacy of rituximab. Recently, we reported that absolute lymphocyte count (ALC), as a surrogate maker of immune recovery, is a survival prognostic factor in autologous stem cell transplantation for non-Hodgkin’s lymphoma (NHL). We hypothesize host immune competence (i.e, normal ALC counts) would predict a superior survival in NHL patients treated with rituximab-based regimens. The primary end-point of the study was to assess whether the ALC count prior to rituximab therapy predicted progression-free survival (PFS) and the second end-point was to identify which lymphocyte subset was responsible for affecting PFS. We studied 41 NHL patients that participated in our Phase I interleukin-12 plus rituximab study. These patients were selected because the Phase I study required baseline ALC count and flow cytometry analysis providing lymphocyte subset data prior to treatment. There were 20 females and 21 males in the study. The median age of the cohort was 53 years (range: 34–84). PFS was assessed from the initial date of treatment. The median follow-up was 42 months (1–84 months). Patients with an ALC ≥ 0.9 × 109/l experienced a superior PFS versus an ALC &lt; 0.9 × 109/l prior to rituximab therapy (31 months vs 4 months, p &lt; 0.0001). The relapse rate in the ALC ≥ 0.9 × 109/l group was 55% (11/20) versus 95 % (20/21) in the ALC &lt; 0.9 × 109/l (p &lt; 0.0016). Patients with a CD4 count ≥ 500 cells/μl also experienced a superior PFS versus a CD4 count &lt; 500 cells/μl (21 months vs 9 months, p &lt; 0.0062). The relapse rate in the CD4 count ≥ 500 cells/μl group was 56% (9/16) versus 88 % (22/25) in the CD4 count &lt; 500 cells/μl (p &lt; 0.0216). No association with PFS was identified for CD3, CD8, CD19, CD16/56, the use of IL-12, or lymphoma type by WHO classification. CD4 count was found to be an independent prognostic factor for PFS. In the subgroup of patients with follicular lymphoma, patients with an ALC ≥ 0.9 × 109/l experienced a superior PFS versus an ALC &lt; 0.9 × 109/l prior to rituximab therapy (35 months vs 9 months, p &lt; 0.0076). The relapse rate in the ALC ≥ 0.9 × 109/l group was 45% (5/11) versus 90 % (9/10) in the ALC &lt; 0.9 × 109/l (p &lt; 0.0243). Patients with a CD4 count ≥ 500 cells/μl also experienced superior PFS versus a CD4 count &lt; 500 cells/μl (not reached vs 9 months, p &lt; 0.0098). The relapse rate in the CD4 count ≥ 500 cells/μl group was 29% (2/7) versus 86 % (12/14) in the group with a CD4 count &lt; 500 cells/μl (p &lt; 0.0216). CD4 count was found to be an independent prognostic factor for PFS when compared to the Follicular Lymphoma International Prognostic Index at the time of treatment. Our data suggest the significance of an immunocompetent host in predicting the efficacy of rituximab therapy. These data support our previous report that patients with higher number of activated CD4+ cells at the lymphoma site have a better prognosis (Ansell et al, JCO2001; 19: 720–6). These findings warrant further studies to understand the role of ALC (i.e, CD4 count) in the mechanism of action of rituximab. Supported in part by CA69912

Abstract MicroRNAs (miRNAs) are small noncoding RNAs that are approximately 20–22 nucleotides with critical functions in cell growth, survival, and differentiation. These conserved sequences can regulate expression of multiple genes and are often tissue specific and dysregulated in malignancies. Thus, miRNA profiling has been used to create signatures for many solid tumors. These profiles have been used to classify tumors and to help predict survival and outcome. In the present study, we utilized the DiscovArray miRNA profiling service (Asuragen Services, Austin, TX) which utilizes a custom-manufactured Affymetrix GeneChip® from Ambion that covers miRNAs derived from the Sanger miRBase (http://microrna.sanger.ac.uk/sequences/index.shtml) and over 11,000 predicted miRNAs derived from published reports. The signal processing implemented was a multi-step process involving probe-specific signal detection calls, background estimation and correction, constant variance stabilization and global normalization. For each probe, an estimated background value was subtracted derived from the median signal of a set of G-C-matched anti-genomic controls. Arrays within a specific experiment were normalized together according to variance stabilization method. Detection calls were based on a Wilcoxon rank-sum test of the miRNA probe signal compared to the distribution of signals from GC-content matched anti-genomic probes. For statistical hypothesis testing, a two-sample t-test, with assumption of equal variance, was applied. One-way ANOVA was used for multiple group comparison. Probes were considered to be differentially expressed based on two criteria: a p-value of &lt; 0.001 and glog2 difference &gt; 1. miRNA expression was analyzed in all malignant B lineage cells (CD19+ CD138+) (n=8), malignant B cells alone (CD19+) (n=6) and plasma cells alone (CD138+) (n=3) from Waldenström macroglobulinemia (WM) patients. The expression was compared to malignant CD19+ B cells from chronic lymphocytic leukemia (CLL) patients (n=5), malignant plasma cells (CD138+) from multiple myeloma (MM) patients (n=5) and to B lineage cells (CD19+ CD138+) (n=4), CD19+ B-lymphocytes (n=3) and CD138+ plasma cells (n=6) from healthy donors. Data analysis based on a total of approximately 11,000 miRNAs analyzed shows that CD19+ CD138+ cells (double sorting) from WM patients did not cluster as a unique group. Some samples had a pattern similar to CLL, some similar to MM and others similar to CD19+ CD138+ cells from healthy controls. This lack of clear signature was observed by others in gene expression profiling and CGH arrays. We therefore hypothesized this lack of clustering was due to the lymphoplasmacytic nature of WM cells and therefore we analyzed B cells (CD19+) and plasma cells (CD138+) separately. miRNA expression in B cells (CD19+) identifies a signature in normal B cells that is absent in both WM B cells (CD19+) and CLL cells. There is also a set of miRNAs that are absent in normal B cells that are expressed in WM B cells and CLL. In addition, WM B cells had a unique miRNA signature that is unique compared to CLL and normal B cells. An additional set of miRNAs were expressed and clustered only in CLL patients. Similar to B cells, plasma cell (CD138+) analysis in WM, MM and healthy donors shows a clustering pattern that identifies normal plasma cells from MM plasma cells. WM plasma cells had a miRNA signature that is unique only to WM patients, however, a subset of miRNAs shared an expression pattern with MM plasma cells. While miRNAs can target multiple genes, some of the genes that are targets of the miRNAs identified in this analysis include XBP-1, Blimp-1, IRF-4, Bcl-6 and TACI. These target genes are known to be important in B cell and plasma cell development. In summary, we have analyzed miRNA expression in malignant B cells (CD19+) and malignant plasma cells (CD138+) from WM patients and compared their expression pattern to their normal counterpart as well as malignant counterpart in CLL B cells and MM plasma cells. Our analysis shows that WM B cells have a miRNA signature unique to WM only and one that is shared by CLL cells. Similarly, WM plasma cells have a unique miRNA signature but also has some miRNAs that are shared by malignant plasma cells in MM. These miRNAs target genes involved in B cell differentiation. Analysis of the functional roles of these miRNAs will and their regulation will further our understanding of the regulation of B cells development in normal and malignant conditions.

Abstract Diffuse large B cell lymphoma (DLBCL) is an aggressive form of non-Hodgkin lymphoma and the treatment of these patients has improved with rituximab-based chemo-immunotherapy. However, approximately 40% of patients die of their disease and new agents with novel mechanisms of action are needed for this disease. The mammalian target of rapamycin (mTOR) has emerged as an important therapeutic target in cancer cells and rapamycin and its derivatives, which specifically inhibit mTOR, are now being actively evaluated. Recent clinical trials of mTOR inhibitors have demonstrated that a fraction of patients (35%) with relapsed DLBCL respond to single-agent temsirolimus (Smith S et al J Clin Oncol 26; May 20 Supplement abstract 8514) or everolimus (Reeder et al 2007 ASH Annual Meeting Abstracts 110 (11) abstract 121) in relapsed DLBCL, however most patients are resistant to this therapy. The mechanism of this resistance remains a subject of major therapeutic significance. Herein, we report that cells from DLBCL lines (DHL-6, Ly7 and Ly3) and primary tissues from DLBCL patients (n=10) display persistent activation of the mTOR pathway as determined by phosphorylation of mTOR targets S6 ribosomal protein (S6rp) and 4E-binding protein 1 (4E-BP1). Treatment of human DLBCL cells with various doses of rapamycin predictably demonstrated a decrease in proliferation but less than 10% reduction in overall cell survival. However, rapamycin suppressed the phosphorylation of S6rp and 4E-BP1, indicating an inhibition of raptor-mTOR (mTORC1) signaling. Paradoxically, rapamycin also concurrently increased, through a negative feedback mechanism, the phosphorylation of Akt that may contribute to drug resistance. Interestingly we found that rapamycin treatment also increased the phosphorylation of eIF4E, a survival protein downstream of mTOR, which may also be responsible for resistance of rapamycin along with Akt. To determine whether the observations found in vitro are clinically relevant, we obtained peripheral blood samples from patients with aggressive lymphoma treated with the rapamycin derivative everolimus. The levels of phosphorylated Akt and eIF4E were increased in 3 of 3 patient samples at 48 hrs and after 1 cycle compared to untreated control. Our observations therefore suggest that rapamycin derivatives potently activate Akt and eIF4E activity via activation of the mTORC2 assembly, in addition to its well-characterized ability to suppress the mTORC1 pathway. Treatment of DLBCL cells with histone deacetylase (HDAC) inhibitor LBH589 induced growth inhibition of DLBCL cells at nanomolar concentrations in a dose dependent manner in association with hyperacetylation of histones H3 and H4. LBH alone was able to inhibit the phosphorylation of S6rp and 4E-BP1, while combined treatment with rapamycin inhibited the phosphorylation of S6rp and 4EBP1 to a greater extent than either agent alone. Surprisingly LBH inhibited constitutive as well as rapamycin-induced activation of Akt and eIF4E in a time and dose dependent manner. Our co-immunoprecipitation data suggest that LBH alone was able to alter the level of intact mTORC2 by reducing the amounts of rictor bound to mTOR, which is further decreased when combined with rapamycin. In support of this model, rapamycin combined with LBH exhibited enhanced synergistic inhibitory effects on survival and proliferation of DLBCL cells. The mTOR pathway is also considered to control the translation of specific mRNA species, some of which are involved in cell cycle control and angiogenesis (e.g. cyclin D1, c-Myc and HIF-1a). LBH alone down-regulated expression of c-Myc and HIF-1a, while have no effect on cyclin D1 expression. Combination of LBH with rapamycin further decreased expression of c-Myc and HIF-1a. Overall these results indicate that pharmacological inhibition of the mTOR pathway by rapamycin and LBH interferes with essential survival and proliferating pathways in DLBCL cells. We propose that the rapamycin-induced functional blockade of Akt and eIF4E is inhibited by LBH, and the combination increased the anti tumor activity of rapamycin. In summary, our data provide a mechanistic basis for enhancing mTORtargeted cancer therapy by combining an mTOR inhibitor with a HDAC inhibitor such as LBH. A phase I/II trial of this combination is planned.

Abstract Waldenström macroglobulinemia (WM) is a B-cell malignancy characterized by the aberrant production of a monoclonal IgM protein that may lead to hyperviscosity. Although this is a major factor causing significant morbidity in patients, little is known about the mechanisms that regulate monoclonal protein synthesis. Cytokines are protein mediators that are known to be involved in many biological processes, and can profoundly affect tumor cells and the tumor microenvironment. Many cytokines have been shown to have potent therapeutic efficacy in preclinical cancer models; however, the role of cytokine networks in WM is not fully understood and few studies have described the precise functional roles of cytokines in WM. To address this issue we performed a multiplex ELISA analysis to test cytokine levels in sera from patients with WM. We found that Rantes/CCL5 is significantly elevated in WM patients and correlates with disease activity. Elevated expression of RANTES in the serum was confirmed by ELISA and was also detected in the bone marrow of WM patients by ELISA and immunohistochemistry. RANTES expression serves as a marker for recruitment of immune cells and is associated with a wide range of immune-mediated diseases. However, the impact of RANTES in WM is not known. We analyzed the expression of receptors for Rantes by flow cytometry and found that WM B cells and stromal cells express CCR1 and CCR3, but not CCR5. Using a standard chemotaxis assay, we determined that Rantes had no effect on B cell or stromal cell recruitment. Rantes also had no effect on B cell or stromal cell survival, however it did promote stromal cell proliferation (p&lt;0.04). The interaction between Rantes and IL-6 has been described in an autoimmune disease model. Since stromal cells secrete significantly more IL-6 than WM B cells, we treated stromal cells with Rantes for 24 hr and found that Rantes increases IL-6 secretion (p&lt;0.03). To characterize the mechanism of Rantes-mediated IL-6 secretion, we transfected stromal cells with an IL-6 promoter construct and treated with Rantes for 12 hr and found a significant increase in IL-6 promoter activity (p&lt;0.0162) indicating Rantes can regulate IL-6 transcription. Bioinformatics analysis of the IL-6 promoter indicates the presence of multiple candidate binding sites for transcription factors that have been previously shown to play a role in the biology of B cells, including NFkB, AP1, and GLI. Co-transfection of stromal cells with an IL-6 reporter construct and a plasmid expressing GLI1, GLI2 or GLI3 demonstrates that GLI2 and GLI3 proteins can regulate the IL-6 promoter. We then transfected stromal cells with a reporter construct containing 8X-GLI binding sites and demonstrate that Rantes can regulate GLI transcription further supporting a role for the interaction between Rantes and IL-6 through GLI transcription factors. IL-6 rich tumor microenvironment supports malignant cells. Elevated IL-6 levels have no effect on survival of BCWM.1 cells or CD19+ 138+ cells from WM patients, but leads to upregulation of IgM secretion by BCWM.1 cells and CD19+ CD138+ cells from WM patients, and increases their proliferation (p&lt;0.0039). IL-6 activates Erk1/2 and Jak/ Stat in WM and stimulation of the IgM secreting cells BCWM.1 with IL-6 in the presence of PD98059 MAPK inhibitor had no effect on IgM secretion. However, stimulation of BCWM.1 cells with IL-6 in the presence of JakI inhibitor abolished the IL-6 mediated IgM secretion suggesting IL-6 mediated increase in IgM secretion occurs through Jak/ Stat signaling pathway. Analysis of Rantes levels in other B cell malignancies including follicular lymphoma, chronic lymphocytic leukemia, monoclonal gammopathy of undetermined significance and multiple myeloma indicates that Rantes is elevated in other B cell lymphoproliferative disorders and suggest Rantes may play a similar role in other malignancies. In summary, our data identifies a novel Rantes-GLI-IL-6 interplay in the stromal microenvironment that promotes IgM production by malignant B cells. This therefore provides multiple new potential therapeutic avenues, targeting both malignant cells and the microenvironment to control malignant cell growth, and immunoglobulin secretion in WM and Ig-mediated diseases.

Abstract The tumor microenvironment in B-cell non-Hodgkin’s lymphoma (NHL) plays an important role in supporting the growth and survival of malignant lymphocytes. In contrast, the extent to which this is true in T-cell NHL is less clear. While professional monocytederived antigen-presenting cells (MDC) are required for T-cell activation and survival, a growing body of evidence demonstrates that MDC may promote the survival of malignant T cells. Immunohistochemical staining of skin biopsies from cutaneous T-cell lymphoma (CTCL) patients (n=11) demonstrated a rich infiltrate of CD68+ macrophages and CD1a+ dendritic cells (DC). As these cells are derived from CD14+ monocytes, we examined the role of monocytes in promoting survival in malignant T cells obtained from CTCL patients (n=9). Initially, peripheral blood mononuclear cells (PBMC) were either depleted of CD14+ monocytes (&gt;85% depleted) or mock-depleted prior to being cultured. Viability of malignant CD4+CD7− T cells was determined by Annexin V/7-AAD staining. While 90.9% (S.D. +/− 0.4%) of mock-depleted malignant T cells were viable, only 73.9% (S.D. +/− 1.3%) of monocyte-depleted cells remained viable, suggesting that monocytes may promote the survival of malignant T cells. In a similar fashion, HuT-78 cells (Sézary Syndrome cell line) were cocultured with CD14+ monocytes from normal donors. Compared with HuT-78 cells alone, the presence of CD14+ monocytes resulted in a 2.7 fold increase in thymidine incorporation (n=5; S.D. +/− 0.6). This is largely due to cytokine production, as the same experiment performed with a transwell insert resulted in a 2.1 fold increase in HuT-78 thymidine incorporation (n=5; S.D. +/− 0.4). Similar results were obtained using additional Sézary Syndrome (i.e. SeAx) and Mycosis Fungoides (i.e. MyLa) cell lines. Monocytes obtained from normal donors were subsequently cultured and cell-free conditioned media was obtained 48 hours later. Media supplemented with monocyte-conditioned media (monocyte-CM) prevented the induction of apoptosis in CTCL cell lines as measured by thymidine incorporation, trypan blue exclusion and Annexin V/propidium iodide staining. This affect was monocyte-specific, as neither T-cell nor B-cell conditioned media were observed to promote survival of CTCL cell lines. Furthermore, monocyte-CM had no affect on resting or anti-CD3 activated normal T cells. We next cultured malignant T cells obtained from PBMC (n=6) or skin biopsies (n=3) from CTCL patients in the presence or absence of monocyte-CM and assessed cell viability by Annexin V staining. 32.1% (S.D. +/− 14.7%) and 60.5% (S.D. +/− 21.5%) of malignant T cells isolated from PBMC remained viable in the absence or presence of monocyte-CM, respectively; 13.0% (S.D. +/− 10.8%) of malignant T cells isolated from skin biopsies remained viable, compared with 26.5% (S.D. +/− 8.2%) of cells grown in the presence of monocyte-CM (p=.03). Given the prevalence of MDC within CTCL-involved skin and their potential role in promoting the survival of malignant T cells, one may predict that malignant T cells may recruit monocytes in a chemokinedependent fashion. Therefore, cell-free conditioned media from CTCL cell lines (HuT- 78, SeAx, MyLa) was collected and used to promote monocyte chemotaxis in a standard chemotaxis assay. Only neutralization of CCL5 (RANTES) was capable of abrogating monocyte chemotaxis, demonstrating that the production of CCL5 by malignant T cells promotes monocyte migration. Therefore, we examined serum CCL5 levels by ELISA from both normal donors (n=24) and CTCL patients (n=23) and found an approximately 3-fold elevation in serum CCL5 in CTCL patients (normal donors: mean CCL5 10.0 ng/mL; 95% CI 7.1–13.0; CTCL patients: mean 29.2 ng/mL; 95% CI 20.6–37.8; p&lt;0.0001). Serial serum samples were available for 6 patients, 4 of whom had progressive disease. Serum CCL5 increased from a mean of 17.9 ng/mL before progression (S.D. +/− 7.0) to 44.8 ng/mL (S.D. +/− 20.0) after progression (p=.04). In contrast, serum CCL5 decreased by at least 50% in both patients with responsive disease. Our findings demonstrate that monocytes, recruited by malignant T cells in a CCL5-dependent fashion, promote the survival of lymphoma cells in CTCL, thus raising the possibility that targeting tumor-associated monocytes and MDC may represent a novel therapeutic strategy in CTCL.

Abstract Background: Our previous work has shown that malignant B cells induce the development of intratumoral Treg cells that inhibit the host anti-tumor response. In contrast to an increase in Treg cells, we found that the number of effector T helper cells (TH1, TH2 and TH17) was low in B-cell NHL tumors, suggesting an imbalance between Treg and TH cells in the tumor microenvironment. Understanding the mechanism(s) of this imbalance is important to the development of treatments to enhance host immunity and in previous work we have shown that signaling through CD70, CD80 and CD86 plays a role. Since soluble factors, particularly TGF-β, have an important role in directing T-cell differentiation, we evaluated in this study the role of TGF-β in the lymphoma microenvironment. Goal: To determine the effect of TGF-β on the generation of intratumoral TH1, TH17 and Treg cells in human B-cell NHL. Results: Human B-cell NHL specimens were obtained from consenting patients and were used for all experiments. Using an ELISA assay, we found that malignant B cells variably secrete TGF-β - median 100 pg/ml per million cells (range: undetectable −229 pg/ml, n=7). Using flow cytometry, we showed that addition of exogenous TGF-β enhanced the expression of Foxp3+ in activated CD4+ or CD4+CD45RA+ or CD4+CD45RO+ nodal T cells, suggesting that TGF-β promotes the generation of Treg cells in tumor microenvironment. In contrast, TGF-β suppressed expression of IFN-g 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, upregulated IL-17 expression in CD4+ T cells, suggesting proinflammatory cytokines are able to reverse the suppression induced by TGF-β. These results indicate that TGF-β plays an important role in the regulation of intratumoral TH17 cell generation. In additional experiments, TGF-β was found to exert a suppressive effect on the proliferation of both CD4+ and CD8+ intratumoral T cells. However, treatment with TGF-β enhanced IL-2 production by intratumoral CD4+ T cells detected by intracellular staining of flow cytometry. Interruption of IL-2 signaling by anti-IL-2 Ab abolished the upregulation of TGF-β-mediated Foxp3 expression and enhanced the production of IL-17 in CD4+ T cells. Furthermore, treatment with anti-IL-2 Ab reversed the inhibition of NHL B cell-mediated TH17 cell generation. Conclusion: 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 through IL-2. 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 generation and the host immune response.

Abstract MALT lymphoma is a genetically unique disorder and five mutually exclusive chromosomal translocations have been identified thus far in this disease: t(11;18), t(14;18), t(1;14), t(1;2) and t(3;14). Despite this genetic heterogeneity, all but one of the translocations affect the NF-κB signaling pathway, which is critical if not essential for antigen receptor mediated B- and T-cell activation and likely enhances MALT lymphoma growth. However, the known translocations are present in only 25% of cases suggesting that additional uncharacterized translocations exist. We used long-distance inverse polymerase chain reaction (LDI-PCR) technique to clone a novel IGH translocation partner in a 60-year-old female with a history of Sjogren’s syndrome and primary MALT lymphoma involving the parotid gland. The breakpoint on chromosome 14 occurred within the IGHSA2 switch region while the breakpoint on chromosome X fell within Xp11.4. The breakpoint on chromosome Xp11.2 fell between two genes, GPR82 and GPR34, both of which code for orphan G-protein coupled receptors (GPCRs). The breakpoint also fell within and disrupted a larger gene called CASK (a membrane-associated guanylate kinase-MAGUK). To determine the prevalence of this translocation in MALT lymphoma we performed interphase FISH studies on 64 MALT lymphomas using a breakapart probe for GPR82. Only the index case had an abnormal split signal pattern. We next designed primers to perform real-time quantitative RT-PCR for the genes located on Xp11 and found that GPR34 RNA was highly expressed, a 49-fold increase, in the patient carrying the t(X;14)(p11.4;q32) translocation compared to a normal splenic B cell control. Expression of GPR82 and CASK RNA was similar between normal B cells and the patient carrying the t(X;14)(p11.4;q32) translocation. These data suggest that the GPR34 gene is dysregulated upon its translocation to the IGHSA2 switch region. We then measured GPR34 RNA expression in a panel of MALT lymphomas (n=12) and found that GPR34 was expressed at levels higher than that seen in normal B cells with an average increase of 11 fold, 9/11 of which had an expression lever greater than 3-fold over normal splenic B cells. In a gastric MALT lymphoma specimen arising in a 67-year-old male, we saw a 64 fold increase in GPR34 expression. Interphase 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. GPR34 RNA was also detected in other normal B cell populations and histologic subtypes of NHL but not to the extent seen in MALT lymphoma; values represent the expression of GPR34 normalized to β-actin and a normal B cell control (value of 1.0): t(X;14)(p11.4;q32) specimen, 49.0; MALT lymphoma, 11.0 (n=12); peripheral blood B cells, 0.48 (n=2); normal bone marrow B cells, 0.97 (n=3); follicular lymphoma, 2.47 (n=3); marginal zone B cell lymphoma, 2.47 (n=3); diffuse large cell lymphoma, 0.36, (n=3); mantle cell lymphoma, 2.67 (n=3); and multiple myeloma 0.52 (n=6). The receptor encoded by GPR34 is most similar to the PY2 receptor subfamily of GPCR and GPR34 mRNA transcripts are particularly abundant in mast cells while lower levels were detected in other immune cells including B cells. However, little is known about its natural ligand, biologic function, or the signaling cascades activated by its engagement. Because the NF-κB signaling pathway has been shown to be a common downstream target of MALT lymphoma translocations we first examined the impact of GPR34 expression on phosphorylation of Iκ-Bα. Transient expression of a YFP-GPR34 expression plasmid in HeLa cells results in increased phosphorylation of Iκ-Bα compared to the YFP control. Additionally, we observed increased phosphorylation of ERK1/2 in GPR34-expressing cells, however no change in phosphorylation of GSK3β was detected. In summary, these data identify a novel IGHS translocation partner in MALT lymphoma and suggest that dysregulation of GPR34 is commonly found in MALT lymphoma. Overexpression of GPR34 results in activation of the NF-κB and MAP kinase pathways and may be a novel mechanism by which MALT lymphoma occurs.

Abstract B cell non-Hodgkin lymphoma (NHL) is a serious and frequently fatal illness. Some of the genetic changes that lead to lymphoma have been discovered, but many of the other contributing mechanistic details underlying transformation events are not yet known. Members of the TNF super-family have been shown to be key mediators in the formation and regulation of both normal and malignant cell growth. APRIL (A Proliferation Inducing-ligand), a TNF family member expressed by monocytes, macrophages, and tumor cells, stimulates B-cell NHL growth and transgenic over-expression of APRIL in mice results in B cell hyperplasia and lymphoma. The ability of APRIL to bind to TNFR related receptors, BCMA (B Cell Maturation Antigen) and TACI (Transmembrane Activator and CAML Interactor), and with lower affinity to heparan sulfate proteoglycans (HSPG) has complicated studies to better understand APRIL mediated signaling. It has been shown that NHL cells variably express TACI and BCMA, with TACI being most predominant, however, the specific mechanisms of how APRIL induces NHL cell growth remain to be fully defined. Therefore, we sought to specifically characterize APRIL-mediated signaling events through TACI to better understand the role of this receptor-ligand interaction in B cell NHL biology. Using a TACI-expressing NHL cell line (Karpas) we found that APRIL consistently increased NHL cell proliferation, but had very little effect on cell survival. Since the PI-3K/Akt pathway has been shown to be important in regulating cell cycle progression, we explored the possibility that APRIL-TACI interactions resulted in activation of this pathway. We found that stimulation of TACI-expressing NHL cells with APRIL results in phosphorylation of Akt but had no effect on NF-kB classical pathway activation. Because APRIL can also bind to cells via proteoglycans, we also tested the ability of APRIL to activate Akt in the presence of heparin and found that APRIL-mediated Akt phosphorylation was still detectable. These results suggest that APRIL-mediated signaling occurs independent of proteoglycan binding. In addition to activating Akt, we also found that stimulation of NHL cells with APRIL resulted in up-regulation of cyclin D1 and p21, but not c-myc and p27. Inhibition of the PI3K/AKT signal transduction pathway with the pharmacological inhibitor wortmannin prevented APRIL-induced proliferation as well as up-regulation of p21 and cyclin D1 levels suggesting that PI-3K kinase is required. To further substantiate the contribution of TACI in APRIL-mediated signaling, we selectively targeted TACI expression using a siRNA approach. When TACI expression was inhibited, APRIL no longer induced NHL-B cell proliferation, Akt phosphorylation or up-regulation of p21 and cyclin D1. Taken together, these studies suggest that APRIL-TACI interactions result in activation of the PI3K/Akt pathway and up-regulation of cyclin D1 and p21, and help define the mechanism by which APRIL enhances B cell NHL growth. The inhibition of APRIL by blocking APRIL/receptor or interruption of downstream signaling may provide potential therapeutic option for patients with B-cell non-Hodgkin’s Lymphoma.