ALBERT

Description: Vitamin D insufficiency is common globally and low levels are linked to higher cancer incidence. Although vitamin D insufficiency is related to inferior prognosis in some cancers, no data exist for chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). We evaluated the relationship of 25(OH)D serum levels with time-to-treatment (TTT) and overall survival (OS) in newly diagnosed CLL patients participating in a prospective cohort study (discovery cohort) and a separate cohort of previously untreated patients participating in an observational study (confirmation cohort). Of 390 CLL patients in the discovery cohort, 119 (30.5%) were 25(OH)D insufficient. After a median follow-up of 3 years, TTT (hazard ratio[HR] = 1.66; P = .005) and OS (HR = 2.39; P = .01) were shorter for 25(OH)D-insufficient patients. In the validation cohort, 61 of 153 patients (39.9%) were 25(OH)D insufficient. After a median follow-up of 9.9 years, TTT (HR = 1.59; P = .05) and OS (HR 1.63; P = .06) were again shorter for 25(OH)D-insufficient patients. On pooled multivariable analysis of patients in both cohorts adjusting for age, sex, Rai stage, CD38 status, ZAP-70 status, immunoglobulin heavy chain variable (IGHV) gene mutation status, CD49d status, and cytogenetic abnormalities assessed by interphase fluorescent in situ hybridization testing, 25(OH)D insufficiency remained an independent predictor of TTT (HR = 1.47; P = .008), although the association with OS was not significant (HR = 1.47; P = .07). Vitamin D insufficiency is associated with inferior TTT and OS in CLL patients. Whether normalizing vitamin D levels in deficient CLL patients would improve outcome merits clinical testing.

Description: Introduction.Graft versus host disease (GVHD) is a frequent and severe complication of allogeneic bone marrow transplantation (BMT). Novel and less toxic treatment regimens are needed to suppress GVHD and preserve graft versus tumor (GVT). Self-limited intestinal helminth colonization stimulates host regulatory T cell (Treg) subsets. The intestine is a prime organ in GVHD generation, with Tregs playing a pivotal role in controlling GVHD. Treg-mediated suppression of GVHD preserves GVT. We hypothesized that self-limited intestinal colonization with helminths protects BMT mice from lethal acute GVHD and preserves the GVT. Methods. Three weeks after administering 3rd stage adult Heligmosomoides polygyrus larvae into 5-6 week old male WT Balb/C recipient mice (H2d), we initiated H2b→H2d MHC I/II mismatch model of acute GVHD by total body irradiation (TBI; 850 cGy) and administration of total splenic T cells and T cell depleted (TCD) bone marrow (BM) cells from male C57BL/6 WT (MHC: H2b) donors into uninfected control and helminth-infected male wild-type (WT) Balb/C (MHC: H2d) bone marrow recipients. In certain experiments, we used donor T cells from TGFβ RII dominant negative (DN) (TGFβ RII DN) mice (MHC: H2b) whose T cells are unresponsive to TGFβ-mediated immune regulation due to over-expression of a truncated TGFβ receptor II. Th1 (IFNγ, TNFα) cytokine generation was analyzed by ELISA. Splenic and mesenteric lymph node (MLN) cells were stained for H2b, H2d, T cell surface markers and FoxP3. Tissues were analyzed for GVHD-related inflammation. GVT was assessed in uninfected and helminth-infected BMT recipients by administration of luciferase expressing A20 leukemia/lymphoma cells (A20-luc; H2d) and IVIS imaging. Statistical and survival difference between groups was determined by Student’s t-test and Kaplan Meier curves, respectively. Results. Helminths increased ~3-fold CD4 T cell and FoxP3+ Treg TGFβ generation (p

Description: Abstract 1705 Poster Board I-731 The CD16 (FcgammaRIIIa) has a functional polymorphism at position 158. CD16 with valine at position 158 (V) has higher affinity for human IgG1 than does CD16 with phenylalanine at position 158 (F). Follicular lymphoma patients with V have a higher response rate to single agent rituximab. In vitro, NK cells from subjects with V are activated at lower rituximab concentrations than NK cells from subjects with F. Little is known about the in vivo effects of rituximab on NK activation, and the influence CD16 polymorphisms have on that activation. The current studies were designed to explore the relationship between CD16 polymorphisms and NK cell activation in patients receiving rituximab for lymphoma. Subjects included 17 lymphoma patients with a variety of histologies scheduled to receive rituximab at the standard dose (375 mg/M2). Some had received prior treatment with rituximab, but not within the prior 6 months. Subjects did not receive recent or concurrent prednisone or immunosuppressive drugs. Blood was obtained immediately prior to initiation of the rituximab infusion, and 4 hours after the initiation of the infusion. CBC, NK cell number, NK cell CD16 and NK cell CD54 were determined at time 0 and time 4hrs, and changes in each parameter determined. All subjects were genotyped related to the CD16 position 158 polymorphism. Genotype testing revealed 9 subjects were FF, 7 were VF, and 1 was VV. NK cell numbers for the group as a whole were lower 4 hours after initiation of therapy when compared to pre-therapy. Phenotypic changes consistent with NK cell activation at 4 hours included downmodulation of CD16 and upregulation of the adhesion molecule CD54 (ICAM-1). Subset analysis demonstrated these changes were largely limited to subjects with VF/VV. No significant change in NK cell number or activation was seen in those with FF. There was no clear correlation between infusion reactions and genotype or NK activation. Ongoing analyses include evaluation of complement, rituximab and cytokine levels. All (n=17) p value VF/VV (n=8) p value FF (n=9) p value p value FF vs VF/VV D NK Cell # Post Rx/PreRx 0.72 +/- 0.81 0.05 0.41 +/- 0.31 0.008 1.00 +/- 1.03 0.62 0.31 D NK CD54 Post Rx/PreRx 2.14 +/- 1.69 0.0006 3.05 +/- 2.1 0.008 1.33 +/- 0.53 0.11 0.01 D NK CD16 Post Rx/PreRx 0.9 +/- 0.17 0.02 0.86 +/- 0.22 0.11 0.94 +/- 0.10 0.15 0.56 We conclude that NK activation occurs within 4 hours of infusion of rituximab. A decrease in the number of circulating NK cells occurs within 4 hours and is consistent with trafficking of NK cells out of the circulation. Subset analysis demonstrates changes in NK cell number and phenotype are statistically significant in subjects with the high affinity CD16 phenotype, but not the low affinity phenotype. This finding may help explain the enhanced efficacy of rituximab that has been observed for patients with high affinity CD16. Disclosures Link: Genentech: Consultancy.

Description: Rituximab (RTX) is an accepted therapy for B cell malignancies, but there is still much to learn about the mechanisms responsible for the observed responses and the potential interactions between various mechanisms of action. Some studies suggest that complement fixation followed by lysis through the membrane attack complex contributes to the anti-tumor effects of RTX. Other investigations indicate that antibody dependent cellular cytotoxicity (ADCC) mediated by NK cells is central to the response of therapy. In prior studies, we found that RTX-coated target cells activate NK cells as indicated by NK cell modulation of CD16, upregulation of CD54 and CD69, and production of IFNγ. NK activation induced by RTX-coated target cells was dependent on the affinity of multivalent interactions between Fc γ receptors III (CD16) of the NK cell and Fc regions of cell-bound RTX molecules. We used these in vitro assays to assess the relationship between complement fixation, and the ability of RTX-coated target cells to activate NK cells. Normal human serum inhibited the modulation of NK cell CD16, and also blocked upregulation of CD54, induced by RTX-coated target cells. The ability of serum to inhibit NK activation was dose dependent and was abrogated upon heat inactivation. Serum depleted of C1q or C3 also failed to inhibit NK cell activation. The inhibitory activity of serum depleted of these complement components was restored when purified C1q or C3 were added back respectively. In addition, the level of NK cell inhibition was dependent on the amount of C3b deposited on the target cells. An antibody that stabilizes C3b on the target cell surface (3E7, DiLillo et al., Molec Immunol 2006) further enhanced the inhibition of NK cell activation induced by RTX-coated target cells. One possible explanation for these findings is that complement-mediated lysis destroyed the RTX-coated target cells before they had the opportunity to induce activation of the NK cells. To assess this possibility, lymphoma cells were killed, fixed with formaldehyde, and washed prior to their use as target cells. These RTX-coated and fixed target cells were able to induce modulation of CD16 on the NK cells, which was again inhibited by normal human serum. These findings indicate that the observed inhibition of NK activation by complement is unlikely to be a consequence of complement mediated lysis of the target cells. Instead, these data suggest that C3b deposition on RTX-coated target cells inhibits the interaction between the Fc portions of RTX and CD16 on the NK cells, and so limits the ability of RTX-coated target cells to induce NK activation. These results could have implications in our understanding of the relationship between complement fixation and ADCC, and their relative roles in potentiating destruction of malignant cells in the blood and tissues.

Description: Immunostimulatory oligodeoxynucleotides containing the CpG motif (CpG ODN) can activate various immune cell subsets and induce production of a number of cytokines. Prior studies have demonstrated that both CpG ODN and granulocyte-macrophage colony-stimulating factor (GM-CSF) can serve as potent vaccine adjuvants. We used the 38C13 murine lymphoma system to evaluate the immune response to a combination of these two adjuvants. Immunization using antigen, CpG ODN, and soluble GM-CSF enhanced production of antigen-specific antibody and shifted production towards the IgG2a isotype, suggesting an enhanced TH1 response. This effect was most pronounced after repeat immunizations with CpG ODN and antigen/GM-CSF fusion protein. A single immunization with CpG ODN and antigen/GM-CSF fusion protein 3 days before tumor inoculation prevented tumor growth. CpG ODN enhanced the production of interleukin-12 by bone marrow-derived dendritic cells and increased expression of major histocompatibility complex class I and class II molecules, particularly when cells were pulsed with antigen/GM-CSF fusion protein. We conclude that the use of CpG ODN in combination with strategies involving GM-CSF enhances the immune response to antigen and shifts the response towards a TH1 response and that this approach deserves further evaluation in tumor immunization approaches and other conditions in which an antigen-specific TH1 response is desirable.

Description: Human plasmacytoid dendritic cells (pDCs) are crucially involved in the modulation of adaptive T-cell responses in the course of neoplastic, viral, and autoimmune disorders. In several of these diseases elevated extracellular levels of the serine protease granzyme B (GrB) are observed. Here we demonstrate that human pDCs can be an abundant source of GrB and that such GrB+ pDCs potently suppress T-cell proliferation in a GrB-dependent, perforin-independent manner, a process reminiscent of regulatory T cells. Moreover, we show that GrB expression is strictly regulated on a transcriptional level involving Janus kinase 1 (JAK1), signal transducer and activator of transcription 3 (STAT3), and STAT5 and that interleukin-3 (IL-3), a cytokine secreted by activated T cells, plays a central role for GrB induction. Moreover, we find that the immunosuppressive cytokine IL-10 enhances, while Toll-like receptor agonists and CD40 ligand strongly inhibit, GrB secretion by pDCs. GrB-secreting pDCs may play a regulatory role for immune evasion of tumors, antiviral immune responses, and autoimmune processes. Our results provide novel information about the complex network of pDC–T-cell interactions and may contribute to an improvement of prophylactic and therapeutic vaccinations.

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