Novak, Anne J.

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 < 0.001 and glog2 difference > 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<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<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<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<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 (>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<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.

Abstract Waldenström macroglobulinemia (WM) is a monoclonal B cell disorder characterized by a circulating monoclonal IgM protein that may lead to serum hyperviscosity in association with an infiltration of lymphoplasmacytic cells into the bone marrow. Although proinflammatory and chemotactic cytokines can profoundly affect tumor cells and the tumor microenvironment, and many cytokines have been shown to have potent therapeutic efficacy in preclinical cancer models, the role of cytokine networks in WM is not fully understood. In this study, we used a high-throughput xMAP multiplex immunobead assay technology (Luminex Corp., Austin, TX) to simultaneously test 30 cytokines, chemokines, angiogenic factors as well as growth factors and soluble receptors in the sera of WM patients and compared them with other B cell malignancies including IgM monoclonal gammopathy of undetermined significance (MGUS), follicular lymphoma, chronic lymphocytic leukemia (CLL) as well as healthy controls. Using a Mann-Whitney U test to analyze the differences between the groups, 15 of the 30 cytokines tested had significantly different levels in WM compared to healthy controls. Of those 15 cytokines, 11 were elevated in WM patients and 4 were decreased. Cytokines were grouped into 3 groups; those with < 2-fold difference, 2–8 fold difference and those having > 8-fold difference in their cytokine levels compared to healthy donors. There was a greater than 8-fold increase in the serum levels of Rantes, G-CSF and IL-2R (p<0.0001) in WM patients. Furthermore, 3 cytokines had between 2–8-fold increase in WM patients including IL-4 (p<0.0001), IL-6 (p<0.0019) and IP-10 (p<0.0006). Five cytokines had statistically elevated levels in WM patients compared to healthy controls, however the fold increase was < 2 including HGF (p<0.0185), IL-10 (p<0.0002), MIP-1α (P<0.0484), IL-2 (P<0.0130) and IL-12 (P<0.0155). Of the cytokines that had significantly lower levels in the sera of WM patients, IL-8 (p<0.0001) and EGF (p<0.0001) were > 8-fold decreased, MCP-1 (p<0.0001) was 2–8 fold lower and Eotaxin (p<0.0004) was < 2-fold lower in WM patients. All of the cytokines that had the greatest fold difference (> 8-fold) in WM patients compared to healthy donors also differed significantly from the MGUS patients. Rantes, G-CSF, IL-2R and EGF had significantly different levels compared to other B cell malignancies. We tested for a correlation between the cytokines that had > 2-fold difference between the WM group and control group with clinical features of the disease and found the cytokines IL-6 and IL-2R had a significant correlation with β2-microglobulin levels (p<0.01). We analyzed cytokine levels in the bone marrow plasma of the same patients and found that high levels of IL-2R in the bone marrow microenvironment significantly correlated with anemia and elevated serum β2-microglobulin (p<0.01). In conclusion, we have simultaneously analyzed sera from WM patients for 30 cytokines and found the most significantly elevated cytokines are Rantes, G-CSF and IL-2R and the most significantly downregulated cytokines are IL-8 and EGF. Furthermore, we found that elevated serum levels of IL-6 and IL-2R correlated with β2-microglobulin levels, a measure of disease activity. Further analysis of the biological role of these cytokines in WM may offer insight into disease pathogenesis and provide a basis for novel targeted therapies.

Abstract Rituximab is a chimeric murine/human monoclonal antibody that binds to CD20 on B-lymphocytes. Binding of the Fab domain to B-cells directly induces apoptosis, while the Fc domain recruits immune effector functions to mediate cell lysis. Because rituximab therapy alone does not result in durable responses for all patients, combination therapies have been explored. We have previously shown in a phase I study that interleukin-12 (IL-12), which facilitates cytolytic T-cell responses and enhances the lytic activity of NK cells, can be safely combined with rituximab and that IL-12 significantly upregulated gamma interferon, CXCL10 (inducible protein-10) and NK cell activity in the peripheral blood. To confirm whether IL-12 could augment the immune mediated cell lysis induced by rituximab, a subsequent randomized phase II study of the combination was performed in patients with B-cell lymphoma. While the combination of IL-12 and rituximab was well tolerated with acceptable toxicity, only moderate disease activity was seen and the response rate to the combination was similar to that seen with rituximab alone. Additionally, the sequential administration of IL-12 at the time of disease progression after treatment with rituximab did not result in any clinical responses. This study was therefore performed to determine potential biologic reasons for the lack of increased clinical efficacy when IL-12 was added to rituximab therapy in patients with B-cell non-Hodgkin lymphoma. Of the 52 patients treated on the phase II study, 8 patients had matched tumor biopsies and peripheral blood specimens obtained prior to therapy and again 2 weeks after treatment was started. Six of the patients were receiving IL-12 plus rituximab at the time the specimens were obtained while 2 were receiving rituximab alone. Gene expression array analysis using the Affymetrix U133 plus chip was performed on RNA isolated from cells from involved lymph nodes and from peripheral blood mononuclear cells. Specimens from the peripheral blood of patients who received IL-12 in combination with rituximab showed a greater than 5-fold increase in the expression of multiple genes known to be upregulated by IL-12 signaling including interferon gamma, CXCL10, IFIT2 and 4 (interferon-induced protein with tetratricopeptide repeats 2 and 4), IL-8 and CXCL2 (macrophage inflammatory protein-2). These increases in gene expression were not seen in the peripheral blood of patients who received rituximab alone. Furthermore, the significant changes seen in cells obtained from the peripheral blood were not seen in cells obtained from lymph nodes involved by lymphoma, despite the samples being obtained from the same patient on the same day. In the tumor specimens, the changes in gene expression involved none of the genes downstream of IL-12 signaling. Instead, many of the upregulated genes were associated with cell cycle and spindle checkpoint proteins suggesting ongoing tumor cell proliferation. In conclusion, while IL-12 significantly upregulated gene expression in the peripheral blood, the same changes were not seen in the tumor. This would suggest that systemically administered IL-12 may not be effectively delivered to the site of tumor involvement. This finding may explain the lack of additional clinical benefit when IL-12 was added to rituximab as therapy for patients with non-Hodgkin lymphoma.

8538 Background: Follicular lymphoma (FL) is a heterogeneous disease with a variable prognosis. Transformation to an aggressive lymphoma is associated with a poor prognosis. Previous studies have identified tumor microenvironment cells such as CD68+ macrophages, PD-1+ and FOXP3+ cells as prognostic in FL. However, results have been conflicting and infrequently address the risk of transformation. In this study we analyzed various components of the microenvironment and their association with transformation. Methods: Patients with FL that later transformed were identified through Mayo Clinic lymphoma database. Tissue specimens at diagnosis were stained with CD68, CD11c, CD21, CXCL13, FOXP3, PD-1 and CD14 and characterized by pattern of location and semi-quantitative cell content. Time to transformation (TTT) and overall survival (OS) were assessed by Kaplan-Meir analysis. Significant variables were further analyzed by cox proportional hazards model. Results: 58 patients were included with a median TTT of 4.7 years (range, 0.4-20). Presence of CD14+ cells vs absence and their location (follicular vs non-follicular) were associated with shorter TTT (4.5 vs 6.2 yrs., p=0.037 and 3.8 vs 5.9 yrs. p= 0.027, respectively). The quantity of PD-1+ cell content was not associated with TTT or OS. However, localization of PD-1 to the follicle compared to diffuse was associated with a longer TTT (6.1 vs 3.6 yrs. p=0.033) and superior OS (9.7 vs 4.6 yrs. p=0.009). On multivariate analysis, the pattern of CD14 and PD-1 staining remained significantly associated with shorter TTT (Table). Conclusions: In follicular lymphoma, a diffuse pattern of PD-1+ cells is associated with inferior TTT and OS. CD14+ cells localized to the follicle is associated with a shorter TTT. After accounting for FLIPI score, both these factors remained significant. These results identify two independent predictors of the rate of transformation in follicular lymphoma and suggest it is location rather than quantity of CD14+ or PD-1+ cells that influence outcomes. [Table: see text]