Ziesmer, Steven C.

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 960 Diffuse large B cell lymphoma (DLBCL) is an aggressive lymphoma whose survival depends on various signaling pathways one of which is Signal transducer and activator of transcription 3 (STAT3)/Janus kinase 2 (JAK2). We have demonstrated that JAK2 is constitutively activated at the auto-phosphorylation site in many cases of DLBCL. The most common mechanism causing abnormal JAK2 activation is through dysregulated cytokine signaling. Cytokines are deregulated in several hematological malignancies and may play a role in tumor cell growth through receptor-mediated and ligand dependent activation of the JAK2 kinase. In this study we investigated the role of cytokine signaling in JAK2 activation in DLBCL and use of a novel JAK2 inhibitor (JAK2i) to inhibit IL-10 induced JAK/STAT signaling. We studied serum cytokines from 70 patients with new untreated DLBCL who participated in a clinical trial in the North Central Cancer Treatment Group (Micallef, IN et al Blood 2011). Compared to control sera, patients with DLBCL had higher levels of several JAK2 kinase related cytokines (IL-2, IL-6, IL-10, and EGF). IL-10 and IL-6 were significantly higher in DLBCL patients vs controls with p values >0.03 and >0.001, respectively. We next examined whether DLBCL cell lines produced IL-10. The phospho-JAK2 (pJAK2) positive DLBCL cell lines Ly10, DHL2 and HBL1 produced more IL-10 (40–80 pg/ml) than the pJAK2 negative cell lines Ly1, Ly19 and DHL6 (0–5 pg/ml). Analysis of the surface expression of IL-10 receptors (IL-10R) determined that most p-JAK2 positive DLBCL cells express either IL-10Ra or IL-10Rb or both. IL-10 had no effect on DLBCL cell survival in vitro; however, it did promote their proliferation. Only IL-10 (but not the other elevated cytokines) was specifically able to activate JAK2 and STAT3 phosphorylation in a subgroup of DLBCL cell lines. IL-10 was not able to activate JAK1, STAT1 and STAT5, which suggests a specific role of IL-10 in the JAK2 pathway. Moreover, neutralizing antibody to IL-10 inhibited IL-10-induced JAK2 and STAT3 tyrosine phosphorylation. We studied the effect of SAR302503 (SAR503, Sanofi, Cambridge, MA), a selective JAK2i currently in clinical trial for myelofibrosis on constitutive JAK2 signaling in DLBCL cells. SAR503 was able to inhibit JAK2 and STAT3 phosphorylation in a dose and time dependent manner in a variety of DLBCL cell lines and patient samples. JAK2 inhibition with SAR2503 caused a dose-dependent inhibitory effect on survival of pJAK2 positive DLBCL cells not observed in pJAK2 negative cells. Activation of STAT3 signaling up-regulates the expression of various genes involved in cell survival and proliferation such as bcl-xl, bcl-2, mcl-1 and c-myc. We observed a dose-dependent decrease in c-myc protein and mRNA levels in Ly3 and DHL2 cells with SAR503 treatment; however, SAR503 did not effect expression of bcl2, mcl-1 and bcl-xl proteins in DLBCL cells. Interestingly, JAK2 inhibition with SAR503 inhibited the autocrine secretion of IL-10 in a dose-dependent manner. Next, we determined if higher pre-treatment serum IL-10 correlates with the overall survival of 81 DLBCL patients. IL-10 by ELISA demonstrated that IL-10 levels were high in 51% (41/81) of patients (median, 57.7pg/ml; range, 26.1–503.7), and low in 49% (40/81) (median, 15.9 pg/ml; range, 0–25.9). Using a cut-off value of 26.1pg/ml, patients with a high serum IL-10 level had a significantly worse event-free survival (p=0.01). Clinical correlation of serum IL-10 with disease parameters showed that the IL-10 level was correlated with elevated serum LDH (p= 0.0085) and IPI score (p=0.01); there was no correlation with the number of extranodal sites, age, or B symptoms. DLBCL tumors from 40 patients were classified into germinal center B-cell type (GCB) and non-GCB type using the Hans algorithm. There was a clear trend towards a higher serum IL-10 in non-GCB DLBCL patients (p <0.06). In summary, our data identifies mechanisms of JAK2 kinase activation in DLBCL and supports the hypothesis that the IL-10-JAK2-cmyc pathway is activated in patients with DLBCL. Finally our data provide a mechanistic basis of selecting and targeting DLBCL tumor cells with high IL-10 levels and/or constitutive JAK2 activity with potent and novel JAK2 inhibitors such as SAR503. Disclosures: No relevant conflicts of interest to declare.

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 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 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.

Abstract Purpose: Flavopiridol is a cyclin-dependent kinase inhibitor with preclinical activity against multiple myeloma cells in vitro and ex vivo. It can induce apoptosis in non-cycling human cell lines, including RPMI-8226, a myeloma cell line. Suggested mechanisms of cytotoxicity include down regulation of anti-apoptotic regulators, direct binding to duplex DNA, disruption of transcription factor/DNA binding, upregulation of p53, inhibition of transcription, and inhibition of angiogenesis. A Phase II multicenter trial was conducted to determine the activity of flavopiridol in patients with relapsed or refractory multiple myeloma. Experimental Design: Eighteen patients were treated with 1 hour flavopiridol infusions (50 mg/m(2)/day) for 3 consecutive days every 21 days. Responses were assessed according to IBMTR criteria each cycle. Serial bone marrow aspirates were collected before and immediately after flavopiridol treatment to measure in vivo and ex vivo effects of flavopiridol on patient plasma cells. Immunoblotting for Mcl-1, Bcl-2, p53, cyclin D, phosphoRNA polymerase II and phosphoSTAT 3 was conducted on total cellular proteins isolated from sorted plasma/myeloma cells. Ex vivo assessment of flavopiridol sensitivity of freshly collected myeloma cells was performed for 5 patients pre-therapy. Results: Median age of patients ranged from 49 to 81 years, with a median of 65 years. Patients had received a median of 3 (range, 1–5) treatment regimens prior to enrollment. Sixty-one percent of patients were refractory to prior therapy and fifty-five percent had received prior high dose therapy with hematopoietic stem cell support. The immunoglobulin isotypes of the patients were as follows: IgG (10), IgA (4), light chain (3), and non-secretory (1). At enrollment, 13 patients had a beta-2 microglobulin greater than 3.5. Seven had a plasma cell labeling index greater than or equal to 1%. Four had an elevated LDH. The trial was stopped at time of interim analysis due to a lack of clinical efficacy. Of eighteen treated patients, 15 progressed (including 1 death on study) and 3 discontinued due to toxicity. All patients have ended the active treatment phase, and the mean number of cycles administered for all enrolled patients was 1.6 cycles (range 1–3). No objective responses were observed. The most frequent adverse events were leukopenia and diarrhea (83% each), followed by thrombocytopenia, nausea, and fatigue (61% each). Ex vivo flavopiridol treatment of pre-therapy patient plasma/myeloma cells led to cytotoxicity, but only after longer exposure times at higher flavopiridol concentrations than were anticipated to be achieved in vivo. Further, immunoblotting demonstrated no indication that known in vitro cellular effects of flavopiridol were recapitulated in vivo. Conclusions: Flavopiridol has little single-agent activity in relapsed or refractory multiple myeloma when administered at this dose and schedule, which is likely due to the inability to achieve biologically relevant concentrations in patients. .

Abstract Background The role of intratumoral PD-1+ T cells and their contribution to patient outcome in follicular lymphoma (FL) has been controversial. While some studies have found that increased numbers of PD-1+ T cells correlate with an inferior prognosis or have no impact on patient outcomes, other studies observed that the numbers of intratumoral PD-1+ T cells are an indicator of a favorable outcome in FL. In previous work, we observed that PD-1 can be expressed by different subsets of intratumoral T cells in FL including T follicular helper (TFH) cells and exhausted T effector cells. However, the function, prevalence, distribution and clinical importance of the respective intratumoral PD-1+ T subsets in FL patients are largely unknown. Goal To characterize PD-1+ T cell subsets and to determine the biological and clinical relevance of these T cell subsets in FL. Results In an analysis of biopsy specimens from 33 previously untreated FL patients, we found that PD-1 is abundantly expressed on intratumoral T cells. A median of 54.9% (range: 17.2-92.6, n=33) of CD4+ or 45.8% (range: 12.8-81.7, n=33) of CD8+ T cells express PD-1 on the cell surface. We found by flow cytometry that PD-1 is expressed on intratumoral CD4+ T cells with bright or dim intensity that represents two different subpopulations in FL. By immunohistochemistry, we identified that CD4+PD-1bright T cells predominantly reside in the lymph node follicles while PD-1dim T cells are mainly located in an inter-follicular pattern. Intratumoral CD4+PD-1bright T cells have a TFH cell phenotype and express CXCR5, secret IL-21 and are BCL-6 positive with no TIM-3 expression. In contrast, CD4+PD-1dim T cells have an exhausted phenotype, express TIM-3 and do not express BCL-6 and CXCR5. Functionally, CD4+PD-1dim T cells displayed reduced proliferation, cytokine production and cell signal transduction. When the CD8+ T cells were analyzed, we found that intratumoral CD8+ T cells, primarily reside in the inter-follicular regions, express PD-1 with low intensity and exhibit similar phenotypic and functional changes to inter-follicular CD4+PD-1dim T cells. These cells express TIM-3 but lack CXCR5 and BCL-6. They also displayed reduced proliferation and cytokine production. Clinically, we observed that both the numbers of CD4+PD-1dim and CD8+PD-1dim exhausted T cells significantly correlate with a reduced progression-free survival in FL patients treated with single agent rituximab (p=0.007 and p=0.04 respectively; n=31). Although it did not reach statistical significance, CD4+PD-1bright TFH cells appeared to correlate with a favorable survival, suggesting that TFH and exhausted T cells differentially impact patient outcome in FL. Conclusion Taken together, these results indicated that PD-1 expression defines two subpopulations with distinct functions that differentially impact patient outcome in FL. This finding provides not only an explanation for the previously discrepant clinical observations, but is also important in the design of future anti-PD-1 antibody-based therapy in FL. Disclosures: No relevant conflicts of interest to declare.

Abstract Background IL-10 polarized macrophages promote tumor growth and survival. The T-cell transcription factor GATA binding protein-3 (GATA3) epigenetically regulates IL10 expression. Therefore, we investigated the extent to which GATA-3 regulates IL-10 production and macrophage polarization in T-cell lymphomas. Methods Monocyte-derived macrophages were generated and polarized with T-cell lymphoma conditioned media ((TCL-CM). Immunophenotypic and functional characteristics associated with IL10-polarized macrophages were examined. GATA3-dependent cytokine production was evaluated in T-cell lymphoma lines by shRNA knockdown. Alternatively polarized macrophages and GATA3 expression were examined in a cohort of patients with peripheral T cell lymphoma, not otherwise specified (PTCL, NOS) (n=76). Comparisons among groups were evaluated using a Student's t test. Progression-free and overall survival (PFS & OS) was estimated using the Kaplan-Meier method and two-tailed log-rank test based on Cox proportional hazards model. Results To investigate the ability of T-cell lymphomas to alternatively polarize macrophages, monocytes from healthy donors were cocultured with TCL-CM. Macrophages were shown to have immunophenotypic characteristics of IL10-polarized macrophages (pSTAT3+/CD16hi/CD163hi/HLA-DR-/lo) by flow cytometry, which was abrogated by IL10, but not IL6, neutralization. TCL-CM polarized macrophages produced abundant IL-10 and were impaired in their ability to stimulate T-cell proliferation. Collectively, these results demonstrate that T-cell lymphomas promote alternative macrophage polarization in an IL-10 dependent manner, and both CD163 and pSTAT3 may aid in their identification in clinical T-cell lymphoma specimens. Therefore, CD163+/pSTAT3+ macrophages were visualized by immunofluorescence in PTCL, NOS specimens. IL-10 polarized macrophages were not identified in normal tissues, but were abundant constituents of the tumor microenvironment in 68% of the PTCL, NOS specimens examined (n=31). STAT3 phosphorylation in response to IL-10 is dependent upon the Janus kinases (JAK) JAK1 and TYK2, both of which are inhibited at nanomolar (and clinically achievable) concentrations by the “JAK2” inhibitor ruxolitinib. Therefore, we sought to determine whether ruxolitinib may prevent IL-10-induced macrophage polarization. As expected, ruxolitinib inhibited IL-10-induced STAT3 phosphorylation. More importantly, macrophages polarized in the presence of ruxolitinib lost the immunophenotype characteristics of IL-10 polarized macrophages, and were CD16-/loCD163-/loHLA-DRhi. Furthermore, IL-10 production was significantly impaired in macrophages polarized in the presence of ruxolitinib, while their ability to stimulate T-cell proliferation was significantly increased. In a separate initiative, we identified GATA3 as the key regulator of IL-10 and Th2 cytokine (IL-4, IL-13 and IL-5) expression in T-cell lymphoma lines by shRNA knockdown assays. Therefore, we examined GATA3 expression by immunohistochemistry in PTCL, NOS specimens. GATA-3 expression was observed in most of the T-cell lymphoma lines and in 47% of PTCL, NOS specimens (n=76). In the cohort of the 76 PTCL, NOS patients, the median PFS and OS observed in GATA3+ PTCL, NOS was 5 months (4-8 months, 95% CI) and 8 months (5-14 months, 95% CI), respectively. In contrast, the median PFS and OS in GATA3- PTCL, NOS was 1.3 years (0.6-1.6, 95% CI) and 1.6 years (0.7-6.9 95% CI), respectively. Conclusions Alternatively polarized macrophages are abundant in T-cell lymphomas and are generated in an IL-10- and STAT3-dependent manner. GATA-3 regulates IL-10 production in these lymphomas, and its expression identified a high-risk subset of PTCL, NOS with distinct clinicopathological features. As IL-10-dependent STAT3 phosphorylation and macrophage polarization were impaired by ruxolitinib, the JAK2 inhibitors may warrant closer scrutiny as immunomodulatory agents. Disclosures: Off Label Use: Ruxolitinib, a JAK2 inhibitor, may warrant closer scrutiny as an immunomodulatory agent in TCL therapeutics.

Abstract Abstract 2642 BAFF is essential for B cell maturation, and a lack of either BAFF or its primary receptor, BAFF-R, results in a severe depletion of T2 marginal zone and follicular B cells. Elevated serum BAFF levels have been correlated with an increased risk of developing non-Hodgkin's lymphoma (NHL), along with a more aggressive phenotype. A growing body of genetic evidence points toward an association between the development of human disease and variation in genes encoding BAFF and its receptors. Recently, we characterized a novel lymphoma-associated mutation in TNFRSF13C, the gene encoding BAFF-R. This mutation (BAFF-R H159Y) encodes a His159Tyr substitution in the C-terminus of BAFF-R adjacent to the TRAF3 binding motif. Signaling through BAFF-R H159Y results in increased NF-κB activity, elevated immunoglobulin production and increased association with TRAF2, TRAF3 and TRAF6 compared to wild type (WT) BAFF-R. We have detected this mutation in 6% of total NHL cases (n=129), and in 10% of follicular lymphoma (FL) cases (n=41) evaluated thus far. We previously reported that BAFF-R H159Y expressing mouse B cells exhibited significantly more resistance to Fas ligand (FasL) induced apoptosis compared to their cells expressing BAFF-R WT, and we propose here that BAFF-R H159Y mediated increases in PI3K activity may explain such an enhanced anti-apoptotic response. In this study we now show that BAFF stimulated HEK 293 cells stably expressing BAFF-R H159Y not only display significantly increased Akt phosphorylation when compared to BAFF-R WT expressing cells, but also demonstrate robust Akt phosphorylation in the absence of BAFF. BAFF-R H159Y-dependent Akt activation also led to activation of the downstream Akt targets mTOR and GSK3β and their phosphorylation was inhibited following treatment with the PI3- kinase inhibitor wortmannin. We next examined the impact of the BAFF-R H159Y mutation on expression of BAFF-target genes. Quantitative PCR analyses revealed that BAFF-R H159Y cells exhibited a pattern of gene expression indicative of promoting cell survival, displaying significantly higher levels of BCL2, BCL2L1 and PIN1, while down-regulating expression of the pro-apoptotic gene BIM. We recently reported that TRAF6 associates with BAFF-R, and that such binding is more pronounced in cells expressing BAFF-R H159Y. In order to investigate the role TRAF6 plays in mediating BAFF-R-dependent PI3K activity, we silenced TRAF6 expression in HEK 293 and Karpas 422 lymphoma cells using TRAF6 shRNA. Reduced TRAF6 protein expression resulted in a parallel decrease in BAFF-R WT mediated phosphorylation of mTOR in Karpas 422 cells and phosphorylation of both Akt and GSK3β was markedly reduced in BAFF-R H159Y expressing HEK 293 cells. Interestingly, TRAF6 knock-down did not affect NF-kB2 activation in either Karpas 422 or HEK BAFF-R expressing cells suggesting that Akt does not play a role in BAFF-R mediated activation of non-canonical NF-kB. Finally, preliminary co-precipitation studies indicate that Akt can be recruited to BAFF-R itself, and our initial observations suggest that such an association is significantly reduced in cells expressing BAFF-R H159Y. Taken together, these studies suggest that the BAFF-R H159Y mutation confers enhanced BAFF-R-dependent PI3K signaling and pro-survival gene expression independent of BAFF. Moreover, such enhanced P13K activation is partly dependent upon TRAF6, and decreased recruitment of Akt to BAFF-R H159Y may function to increase the amount of this PI3K target for activation. Thus, BAFF-R H159Y likely contributes to BAFF signaling irregularities in NHL patients harboring this mutation, and may predispose individuals to developing lymphoma regardless of their serum BAFF concentration. Disclosures: No relevant conflicts of interest to declare.