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Abstract 3922 Poster Board III-858 Abstract: Patients diagnosed with lymphoma are at high risk of developing osteoporosis. We have shown previously that the majority of newly diagnosed patients are osteopenic or osteoporotic prior to receiving chemotherapy. When treated with alkylating agents or corticosteroids, especially if the patient develops hypogonadism, the risk is further increased. Osteoporotic bone cannot easily be restored to normal levels of strength, thus prevention of bony loss is crucial. Pamidronate can reduce the risk of bone loss and vertebral fractures in lymphoma patients receiving chemotherapy. Zoledronic acid, a bisphosphonate approximately 100-fold more potent than pamidronate, has not been evaluated in lymphoma patients. We have conducted a phase III trial to evaluate the effect of zoledronic acid on the change in bone mineral density (BMD) in patients with untreated lymphoma undergoing chemotherapy. Urinary N-telopeptide cross-linked collagen type I (NTx), a marker of bone resorption, has early predictive value for long term bony outcomes in patients treated with bisphosphonates. Bone specific alkaline phosphatase (AP), a marker of bone formation, also correlates with response to bisphosphonate therapy. We report the change in urine NTx and bone specific AP levels in lymphoma patients treated with zoledronic acid. Methods All newly diagnosed lymphoma patients seen at our institution from 2005 – 2009 were evaluated for protocol eligibility. Exclusion criteria included bone fractures, BMD T-scores worse than –2.0, creatinine clearance less than 60 mL/min, dental problems, and recent steroid or bisphosphonate use. Patients on study were stratified as male, pre-menopausal female, or post-menopausal female. Accrued patients were randomized to receive either: 1) oral calcium and vitamin D (Ca+D) or 2) Ca+D and 4 mg zoledronic acid IV at baseline and at 6 months. Urine NTx and bone specific AP levels were measured at baseline, 3, 6, 9, and 12 months. Results To date, 33 patients have completed the study and have evaluable data. Patient characteristics include: 17 male, 4 pre-menopausal female, 12 post-menopausal female, median age 62 (range 33 – 80). Seventeen patients had osteopenia upon enrollment. Patients that received zoledronic acid had stable median T-scores at all locations during the 12 month observation, whereas the T-scores of the control group decreased at every location evaluated (Location: L1-4, p=0.004; L Neck, p= 0.001; L Hip, p=0.118; R Neck, p=0.009; R Hip, p=0.040). Each group had a similar baseline level of urine NTx and bone specific AP. At 6 months, the urine NTx decreased significantly in the zoledronic acid-treated group compared to urine NTx levels in the control group which increased (p Conclusions Treatment with zoledronic acid in newly-diagnosed lymphoma patients prevents the bone mineral density loss commonly seen in this population. Bone mass lost is difficult to restore, thus necessitating effective prevention strategies. Urine NTx and bone specific AP levels demonstrate early response to bisphosphonate therapy. The ability to predict response to bisphosphonates at 6 months may allow for early intervention, potentially preventing further bone loss. Our study demonstrates the bone markers urine NTx and bone specific AP are significantly associated with bone health in lymphoma patients receiving chemotherapy. Disclosures: Thompson: Novartis: Research Funding. Toth:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hagemeister:Novartis: Research Funding.

Abstract 1714 Poster Board I-740 Background Despite advances in therapy and a better understanding of the natural history of indolent non-Hodgkins lymphomas (NHL), the optimal treatment for newly diagnosed patients (pts) has not been determined. While several chemotherapy regimens have response rates approaching 90%, toxicity is common with standard genotoxic combinations, particularly with retreatment at relapse. Lenalidomide is approved for the treatment of relapsed/refractory multiple myeloma and myelodysplastic syndromes with del(5q) mutation. In pts with indolent relapsed or refractory NHL, treatment with lenalidomide resulted in durations of response lasting > 16.5 months (Witzig et al JCO in press). Rituximab has been shown to have clinical activity in indolent NHL, both as a single agent and in combination with chemotherapy. The aim of this phase II, single arm study is to evaluate the efficacy and safety of lenalidomide and rituximab in pts with untreated, stage III or IV, indolent NHL. Methods Previously untreated pts with indolent NHL and with measurable disease (>1.5 cm), were eligible for enrollment. For each 28-day cycle, pts received lenalidomide 20mg orally once daily on days 1-21 and rituximab 375mg/m2 intravenously on day 1, for up to 6 cycles of therapy. Response was assessed after 3 cycles and at the end of therapy using the International Working Group Response Criteria (Cheson et al 1999). Results At the time of this report, the planned accrual of 30 pts is complete. Response and adverse events are reported for the first 20 patients, which included 19 pts eligible for response assessment and 1 patient, who discontinued from study prior to response evaluation, secondary to leukocytoclastic vasculitis, which occurred during cycle 1. The median age was 55 yrs (range: 38-77) and 55% were male. The 20 currently evaluable patients include10 pts with follicular lymphoma, 8 pts with marginal zone lymphoma and 2 pts with small lymphocytic lymphoma. Of 19 pts eligible for response assessment, 18 completed 6 cycles of therapy and 1 pt, who was previously treated for Hodgkin's lymphoma, withdrew consent following cycle 3. The overall response rate was 84%, which included complete responses in 15 pts (79%; 58% CR/21% CRu) and 1 patient who achieved a partial response. An additional 3 pts (16%) remain with stable disease. By completion of 6 cycles of therapy, all 10 pts with follicular lymphoma achieved a complete response to therapy. No pt experienced progression of disease. The following grade 3/4 adverse events were reported; rash (6 pts), neutropenia (4 pts), myalgia (3 pts), neuropathy (1 pt), infection (1 pt), and fatigue (1 pt). Rashes, of all grades, occurred in 10 pts, which were mostly erythematous and transient, nonrecurring events. Response and toxicity assessment for the remaining 10 pts is ongoing and will be reported. Conclusion The biologic regimen of lenalidomide and rituximab as front line therapy produces excellent overall and complete response rates in pts with indolent B cell NHL. The combination was well tolerated with a manageable toxicity profile. Disclosures Fowler: Genentech: Honoraria, Speakers Bureau; BiogenIdec: Honoraria. Off Label Use: lenalidomide and rituximab for indolent B cell non-Hodgkin's lymphoma. McLaughlin:Genentech: Consultancy, Honoraria. Hagemeister:Genentech: Honoraria, Speakers Bureau; BiogenIdec: Honoraria, Speakers Bureau; Celgene: Consultancy. Kwak:Celgene: Research Funding. Samaniego:Celgene: Consultancy.

Abstract 105 Tumor lysis syndrome (TLS) is a potentially life threatening complication resulting from massive lysis of malignant cells and most frequently observed in patients with rapidly proliferating, bulky, and chemo-sensitive malignancies. The prevention and management of TLS includes hydration and reduction in the levels of serum uric acid by drugs that decrease production or increase excretion of uric acid. Rasburicase is a recombinant urate oxidase that rapidly reduces the levels of uric acid by enhancing the conversion of existing uric acid into allantoin which is more soluble in urine than uric acid. Rasburicase is approved in the USA for the treatment of pediatric patients at high risk for developing TLS, to be administered at a dose of 0.15 to 0.2 mg/kg once daily for 5 days. Although, activity has been seen with lower doses and shorter duration of treatment, the optimal dosing of rasburicase has not been established for adults. The purpose of this study was to evaluate the efficacy of rasburicase (0.15 mg/kg) administered as a single dose followed by as needed (max 5 doses over 5 days) as compared to standard fixed 5-days dosing in patients with hematologic malignancies at risk for TLS. The patients were stratified based on their risk for TLS and randomized 1:1 to rasburicase administered as a single dose followed by as needed (Arm A) or fixed daily dose x 5 days (Arm B). Sixty six patients were enrolled; 20 had > 2-fold LDH levels and 9 had above-normal serum creatinine. 64 patients received at least one dose of rasburicase and were evaluable for response [24 high risk with mean baseline uric acid levels, 9.8 (7.6–16.1 mg/dL) and 40 potential risk with uric acid, 5.3 (2.4-7.4 mg/dL)]. All patients normalized their uric acid levels at 4 hours after the first dose; 83% to an undetectable level (7.5 mg/dL. Serum creatinine normalized in all patients by day 5. The treatment was well tolerated, except for one incident of methemoglobinema and hemolytic anemia in a patient found to have G6PD deficiency. Since high uric acid levels have been associated with elevated cytokine levels, we examined the effects of treatment on plasma cytokines. At baseline, high risk patients showed increased TNF-α and IL-6 plasma levels. There was a marked decrease in the plasma levels of TNF-α (from mean 14.2 to 6.4 pg/ml, p Conclusion: Rasburicase is a highly effective uricolytic agent for prevention and management of TLS. This study demonstrates that it is feasible to decrease the duration of administration of rasburicase at the approved dose of 0.15 mg/kg. The majority of patients responded to a single dose of the agent, and only a small subset at high risk for TLS required a second dose. Disclosures: Vadhan-Raj: Sanofi Aventis: Honoraria, Research Funding. Off Label Use: Rasburicase is indicated for the initial management of plasma uric acid levels in pediatric patients with leukemia, lymphoma, and solid tumor malignancies who are receiving anti-cancer therapy expected to result in tumor lysis and subsequent elevation of uric acid. The approved administration dose, route, and schedule are 0.15-0.20 mg/kg IV infusion for 5 days, respectively.

Abstract 585 France SAR3419 (huB4-DM4)is an antibody–drug conjugate composed of a humanized IgG1 monoclonal antibody, huB4, which specifically targets the CD19 antigen, conjugated through a disulfide link to the maytansinoid derivative DM4, a potent tubulin inhibitor. After binding to the CD19 antigen, SAR3419 undergoes internalization, resulting in intracellular release of DM4 that binds to the vinca site. Based on encouraging in vitro and in vivo preclinical activity, a multi-dose phase I study was initiated to determine the maximum tolerated dose, to evaluate the safety, pharmacokinetic properties and anti-tumor activity of SAR3419 in patients with relapsed/refractory B-cell NHL expressing the CD19 antigen. Patients received SAR3419 by IV infusion once every 21 days. Patients were eligible if they had CD19+ relapsed or refractory B-cell lymphoma irrespective of the number of prior treatment regimens, including previous allogeneic stem cell transplant. To date, 29 patients have been enrolled on 7 dose levels ranging from 10-270 mg/m2. The histologic subtypes were follicular (FL;12), small lymphocytic (SLL;5), marginal zone (MZL;2) mantle cell (MCL;5) and diffuse large B-cell (DLBCL;4). The median number of prior treatment regimens was 4 (range 1-11), and 7 patients had prior autologous or allogeneic stem cell transplantation. The dose limiting toxicity (DLT) was reversible severe blurred vision, associated with microcystic epithelial corneal changes. This DLT was first observed in one patient at 208 mg/m2 during cycle 2 (grade 4). Consequently, patients having received the first dose of 270 mg/m2 continued on treatment at the dose of 208 mg/m2. 4 out of the remaining 8 patients treated with 208 mg/m2 experienced at least one episode of reversible grade 3 blurred vision during cycle 2 or a later cycle, requiring treatment with topical steroids and dose delay of 1-2 weeks to allow recovery. Therefore, an additional 15 patients will be dosed at the previous dose cohort of 160 mg/m2 to confirm the safety and to collect preliminary data on the efficacy of this dose. A total of 25 patients have completed at least 2 cycles of treatment with SAR3419 and are evaluable for tumor response. Seventeen (68%) patients demonstrated reduction in their tumor measurements ranging between 7% and 86%, of whom 5 patients achieved partial (n=2) or complete (n=3) remissions. Seven (53%) of 13 patients with rituximab refractory disease demonstrated reduction in their tumor measurement ranging between 15% and 86%. Pharmacokinetic profile of SAR3419 is characterized by linear kinetics, low clearance from 0.4 to 0.7 L/day/m2 and an elimination half-life in the range of 3 to 7 days. Low levels of free DM4 and DM4-methyl were detectable only at and above the dose of 80 mg/m2 (LOQ = 1ng/mL). Their exposure increased with the dose. Collectively, these results demonstrate evidence of acceptable safety and clinical activity of SAR3419 in patients with relapsed B-cell malignancies including those who were refractory to rituximab. Furthermore, the lack of significant hematologic toxicity may provide an opportunity for combining SAR3419 with other active regimens for the treatment of lymphoma. Enrolment continues in the extension cohort at 160 mg/m2. Disclosures: Younes: sanofi Aventis: Honoraria, Research Funding. Off Label Use: SAR3419. Lambert:ImmunoGen: Employment. Bagulho:Sanofi-Aventis: Employment. Morariu-Zamfir:Sanofi-Aventis: Employment.

Abstract 2250 Poster Board II-227 Background: Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative treatment for multiple myeloma (MM) due to its graft-versus-myeloma effect. The use of reduced-intensity conditioning regimens with lower transplant related mortality has extended applicability of allo-HCT but is associated with a high risk of relapse. Active immunization of donors with a tumor-specific antigen prior to stem cell harvest and recipients following transplantation may enhance graft-versus myeloma effect without increasing the risk of graft-versus-host-disease (GVHD). Here, we evaluated the magnitude and quality of the vaccine-induced T-cell immunity in the donors and its transfer to recipients. Methods: Patient-specific idiotype (Id) protein was isolated from the plasma of the MM patient, conjugated to keyhole limpet hemocyanin (Id-KLH) and administered with GM-CSF as an adjuvant. Donors received 3 vaccine doses subcutaneously at 10, 8, and 4 weeks before the stem cell harvest. MM pts received 3 booster subcutaneous injections at 3, 4, and 6 months after allo-HCT following reduced-intensity conditioning regimen (Fludarabine/Cyclophosphamide) and wash out of GVHD prophylaxis. Th1 (IL-2, IFN-γ, TNF-β, and GM-CSF) and Th2 (IL-4, IL-5, IL-10, and IL-13) cellular immune responses against Id and the carrier molecule KLH were determined by multiplex cytokine assay on a Luminex platform. Th1 responses (IL-2, IFN-γ, TNF-β) were further quantified and characterized at the single cell level by intracellular cytokine assay (ICCA) in CD4+ T cells from unfractionated cryopreserved pre and postvaccine PBMC. Multicolor intracellular flow cytometry analysis was also used to determine the frequency of circulating regulatory T cells (Tregs), identified as CD4+CD25+CD127lowFOXP3+, at various time points in both donors and recipients. Results: Ten MM pts (median age: 54 years; IgG = 8, IgA = 2) and their respective donors were enrolled in this study. All 10 donors completed vaccinations without significant toxicity. Following vaccination, KLH-specific Th1/Th2 responses were observed in all 10 donors by multiplex cytokine assay and confirmed by intracellular cytokine assay (TNFβ and IL-2). Id-specific Th1 and Th2 responses were noted in 5 and 6 donors, respectively. All 10 MM pts underwent reduced intensity allo-HCT and 9 of these completed their post-transplant vaccinations. KLH-specific Th1 responses were detected in 6/7 evaluable recipients prior to post-transplant immunization and in 7/7 following booster immunizations by ICCA. Phenotypic characterization revealed that the transferred donor-derived KLH-specific CD4+TNF-a producing T cells mainly consisted of intermediate or late effectors (CD62L-CD27+ or CD62L-CD27-). Id-specific Th1 and Th2 responses were measured in 5 and 3 pts, respectively, prior to post-transplant booster injections. Two pts had Id-specific Th1 responses (IFNγ or TNFβ) detectable by ICCA after in vitro expansion from post-transplant immunizations PBMC with a frequency of 2.1% and 2.5% respectively. Since the use of GM-CSF has been associated with increased Tregs in mouse models, we determined the effects of Id-KLH+GM-CSF vaccination on the number of Tregs in the peripheral blood. The percentage of circulating Tregs did not change significantly following vaccination in the donors. In contrast, we observed a progressive increase in the number of Tregs in recipients reaching statistical significance at 7 months post transplant as compared to day 100 (median values 12.8% and 6.1 respectively, p Conclusions: Our results suggest that Id-specific and KLH-specific T cell immunity can be induced in sibling donors and transferred to recipients and may represent a strategy to enhance the graft-versus-myeloma effect. Regulatory T cells increased progressively in number in the recipients following transplantation and immunizations and may affect the quality and the magnitude of the vaccine-induced immune responses. Further investigations are needed to determine the reasons for the increased number of Tregs and to determine the optimal formulation and timing of booster immunizations in the recipients. Disclosures: No relevant conflicts of interest to declare.

8548 Background: Despite advances in therapy and a better understanding of the natural history of indolent non-Hodgkins lymphomas (NHL), the optimal treatment for newly diagnosed patients (pts) has not been determined. While several combination chemotherapy regimens have response rates approaching 90%, toxicity is common with genotoxic drugs and secondary malignancies is a concern. Lenalidomide has been shown to have single agent activity in indolent NHL, and is approved for the treatment of multiple myeloma and myelodysplastic syndrome. Rituximab is effective as a single agent and in combination with chemotherapy for indolent NHL. The aim of this phase II, single arm study is to evaluate the efficacy and safety of lenalidomide and rituximab in pts with untreated, stage III, or IV indolent NHL. Methods: Pts with indolent NHL who were previously untreated, with measurable disease (>1.5 cm), were eligible for enrollment. Pts received 20mg of lenalidomide orally once daily on days 1–21 and rituximab 375mg/m2 intravenously on day 1 of each 28 day cycle. Pts could receive up to 6 cycles of therapy. Response was assessed after 3 cycles and at the end of therapy using the International Working Group Response Criteria. Results: At time of this report 17 pts have been enrolled and 14 are eligible for safety evaluation. The median age was 55 (33–77) years and 53% were male. Therapy was well tolerated with the following grade 3 adverse events (AE) reported; myalgia (1 pt), rash (1 pt), peripheral neuropathy (1pt). There were no grade 4 AEs. There have been no reported grade 3/4 hematologic AEs. There has been no tumor flare observed. In the 5 pts eligible for response assessment, 4 pts (80%) attained a complete response (CR), 1 patient (20%) had stable disease (SD). After 3 cycles, one patient had unconfirmed stable disease who also was previously treated with combination chemotherapy for Hodgkin's lymphoma. Updates for response will be presented. Conclusions: The combination of lenalidomide and rituximab has activity and is well tolerated with minimal toxicity in patients with newly diagnosed indolent lymphoma. [Table: see text]

7024 Background: Relapse is the most common cause of treatment failure after reduced-intensity allogeneic hematopoietic cell transplantation (RI-alloHCT) for multiple myeloma (MM). To enhance graft-versus-myeloma effects, we investigated the use of patient-specific idiotype (Id) vaccine in both donors and recipients. Methods: Id protein was obtained by plasmapheresis and conjugated to keyhole limpet hemocyanin (Id-KLH) to generate vaccine. Donors received 3 SQ injections of Id-KLH + GM-CSF at 10, 8, and 4 weeks before stem cell donation. MM pts received a reduced-intensity conditioning regimen (Flu/Cy) and GVHD prophylaxis consisted of cyclosporine plus methotrexate. MM pts were vaccinated with the Id-KLH+GM-CSF at 3, 4, and 6 months post-transplant. Humoral responses were determined by ELISA and Th1 (IL-2, IFN-γ, TNF-α, and GM-CSF) and Th2 (IL-4, IL-5, IL-10, and IL-13) cellular immune responses against Id and KLH were determined by multiplex cytokine assay. Results: Ten MM pts (median age: 54 years; IgG = 8, IgA = 2) and their respective donors were enrolled onto study. All 10 donors completed vaccinations without significant complications. Following vaccination, KLH-specific antibody and Th1/Th2 responses were noted in all 10 donors. Id-specific antibody responses were noted in 6 donors; Th1 and Th2 responses were noted in 5 and 6 donors, respectively. All 10 MM pts underwent RI-alloHCT; the median potential follow-up post-transplant is 43.8 months. Id-specific Th1 and Th2 responses were noted in 5 and 3 pts, respectively, prior to post-transplant immunization; antibody response was noted in 1 patient. Nine evaluable MM pts completed their post-transplant vaccinations. KLH-specific antibody and Th1/Th2 responses were detectable in all 9 pts post-transplant. Id and KLH immune responses were further enhanced by post-transplant vaccinations. Median progression-free survival is 27.3 months; median survival has not been reached. Conclusions: The data suggest Id-specific immunity can be safely induced in normal stem cell donors and passively transferred to recipients of RI-alloHCT. Donor vaccination with tumor-specific antigen represents a tactic to potentially reduce relapse after RI-alloHCT. No significant financial relationships to disclose.

Follicular lymphoma (FL) is one of the most immune responsive of all human malignancies. However, immunoregulatory mechanisms in the tumor microenvironment may impair the efficacy of endogenous immunity as well as therapeutic vaccines and adoptive T-cell therapy. CD4+CD25+CD127loFoxp3+ regulatory T cells (Tregs) are one of the most potent suppressors of effector T cells and were recently shown to be increased in peripheral blood and tumors of patients with various malignancies. Here, we determined the percentage of Tregs, in the tumors and the peripheral blood mononuclear cells (PBMC) of patients with FL (n = 13) at the time of initial diagnosis and after induction of clinical remission with a uniform cyclophosphamide and doxorubicin containing chemotherapy regimen, PACE (Modified ProMACE without Methotrexate; Prednisone, Doxorubicin, Cyclophosphamide, and Etoposide). We observed that Tregs are markedly increased in number both in the tumors (median 19.45%, range 13.4%–44.5%) and PBMC (median 17.7%, range 7.9%–64.5%) of patients with FL at the time of initial diagnosis as compared with PBMC from normal donors (median 3.33%, range 1.88%–6.3%). Unexpectedly, Tregs persisted in high numbers in peripheral blood after induction of clinical remission with a cyclophosphamide and doxorubicin containing chemotherapy regimen (median 19.95%, range 7.6%–67.2%). To determine the immunosuppressive effects of Tregs, we assessed the proliferation of CFSE-labeled effector T cells following polyclonal stimulation with anti-CD3 and anti-CD28 antibodies. The presence of high numbers of Tregs was associated with significantly decreased proliferation of peripheral blood CD4+ T cells in patients with FL both at baseline (median 24.9%, range 3.7%–40.7%) and after chemotherapy induced clinical remission (median 15.8%, range 1.5%–23.5%) as compared with normal donors (median 38%, range 11.7%–58.8%). A similar decrease in proliferation was also noted in CD8+ T cells in the presence of high numbers of Tregs both at baseline and after induction of clinical remission in patients with FL as compared with normal donors. More importantly, intratumoral and peripheral blood Tregs significantly inhibited effector T cells proliferating in response to autologous tumor cell stimulation. In conclusion, these results suggest that CD4+CD25+CD127loFoxp3+ Tregs that inhibit the function of tumor-specific effector T cells are markedly increased in number in both tumor and peripheral blood of patients with FL and persist in high numbers despite induction of clinical remission with standard cyclophosphamide and doxorubicin containing chemotherapy. This observation may potentially explain the failure of two recent Phase III therapeutic vaccine trials in patients with FL. Furthermore, these results suggest that a state of peripheral tolerance continues to be present after induction of clinical remission and strategies that deplete or block function of Tregs may be necessary to enhance the efficacy of therapeutic vaccines and adoptive T-cell therapy in patients with follicular and possibly other non-Hodgkin’s lymphomas.

MCL has a poor outcome with current non-intense therapies. We present an update of our results utilizing an intense chemotherapy regimen comprising fractionated cyclophosphamide, doxorubicin, vincristine and dexamethasone to which rituximab has been added (R-HyperCVAD, one cycle), alternating with rituximab and high dose methotrexate/cytarabine (R-MA, one cycle) for a total of 6–8 cycles (J Clin Oncol 2006 Feb 1;24(4):724). Treatment was started at most within 2 months from the initial evaluation. Overall survival (OS) was defined as the time from start of treatment until recurrence or death from any cause. Failure-free survival (FFS) was defined as the time from start of treatment until recurrence or death from toxicity or death from treatment-related malignancy. Of 97 consecutive evaluable patients, 87% achieved a complete response (CR) or unconfirmed CR after 6 cycles and did not receive additional therapy. With a median follow up on 84 months (7 years), the overall survival and failure-free survival at 7 years was 60% and 43%, respectively. Among patients 65 years of age or younger, the 7-year OS and FFS was 68% and 52%, respectively. Important prognostic variables included age (65 yrs or less vs > 65 yrs; initial serum B2 microglobulin 3 or less mg/dL vs > 3); and initial serum lactate dehydrogenase (normal vs > normal). Further subset analysis will be presented at the meeting. Figure Figure

Resistance to Fas-mediated apoptosis in hematopoietic cancers interferes with the efficacy of currently available chemotherapy. Our prior studies of human herpesvirus 8 oncoprotein K1 showed that K1 binds to Fas and interferes with activation of Fas-mediated apoptotic signaling (Wang, W, et al, Blood2007; 109:5455–62). Herpesvirus proteins often mimic host proteins or their functions, we thus searched for cellular proteins associated with inactive Fas in order to identify potential regulators of Fas signaling. We identified CD74, the invariant light chain of the major histocompatibility class II complex, associated exclusively with inactive/activation resistant Fas in B-cell lymphoma-derived BJAB cell line. Interestingly, overexpression of CD74 has been previously reported in ~ 90% of hematopoietic cancers and derived cell lines (Stein, R, et al., Ciln Cancer Res.2007; 13: 5556s–63s), as well as in ~ 80% of non-small-cell lung carcinoma (Ioachim, H.L., Am J Surg Pathol.1996; 20: 64–71). The role of CD74 in carcinogenesis was thus suspected. Through siRNA suppression of CD74 expression in BJAB cells we have determined that CD74 positive cells are more resistant to agonistic antibody CH-11-induced Fas-mediated apoptosis then the CD74 siRNA transfected cells (38 ± 7.8 % vs. 54 ± 9.8 %; P = 0.045). In addition, a challenge with agonistic anti-Fas antibody showed a significant survival advantage of the mice expressing CD74 in their livers over the vector-transfected mice (83% vs. 0 %; P < 0.016). We have mapped domain of CD74 required for its association with Fas and for significant protection of mice to a membrane-proximal extracellular region of CD74. Treatment of BJAB cells for 24 hours with humanized anti-CD74 antibody (hLL1 + crosslinking antibody), FasL-Fc, or anti-Fas antibody CH-11 induced apoptosis in 19%, 25% and 13% of cells, respectively. Combination of hLL1+crosslinking antibody with FasL-Fc or CH-11 increased apoptosis to ~ 45% of cells in both cases (P < 0.01), while combination of FasL-Fc or CH-11 with hLL1 + irrelevant antibody had no significant effect on the extent of apoptosis. Our results support the idea of an endogenous regulatory system of Fas-mediated apoptosis that utilizes transmembrane proteins interacting with Fas. We anticipate that specific blocking of the CD74-Fas interaction will sensitize CD74 overexpressing cancer cells to Fas-mediated apoptosis and thus will lead to a more effective chemotherapy for hematopoietic cancers.

Treatment of lymphoma with alkylating agents and steroids is known to cause bone loss and increased fracture risk. These effects result from multiple causes including systemic lymphoma-related cytokine activity, steroid-mediated bone loss, and therapy-induced hypogonadism. Furthermore, over half of all untreated lymphoma patients are either osteopenic or osteoporotic at the time of diagnosis.1,2 Pamidronate is known to reduce bone loss and risk of vertebral fractures in lymphoma patients undergoing chemotherapy. However, the effects of the more potent bisphosphonate zoledronic acid in this setting are unknown. Therefore, we report on an ongoing phase III trial designed to evaluate the effect of zoledronic acid on the change in bone mineral density (BMD) in patients with newly-diagnosed lymphoma undergoing chemotherapy. In total, 72 patients will be randomized to either the control arm consisting of therapy with calcium carbonate (1200 mg orally per day) plus vitamin D (400 mg orally per day), or the bisphosphonate arm consisting of therapy with calcium carbonate and vitamin D as in the control arm plus zoledronic acid (4 mg IV given at baseline and at 6 months). The primary endpoint of the study is to compare the absolute change between the baseline and 12-month measures of BMD at the lumbar spine and femoral necks. Thus far, 97 patients have been screened for enrollment. Twenty-four patients (24.7%) failed screening due to periodontal disease, a predetermined exclusion criterion of the study. Twenty-three patients have been randomized to the control arm, and 18 patients have been randomized to the bisphosphonate arm. To date, 11 patients in the control arm and 7 patients in the bisphosphonate arm have completed the one-year follow up period including baseline and one-year BMD evaluations. In comparing the change in BMD at one year of patients in the control arm to that of patients in the bisphosphonate arm, the average change in T scores at the lumber spine was −0.482 vs. 0, at the left femoral neck was −0.218 vs. 0.057, and at the right femoral neck was −0.309 vs. 0.243, respectively. There have been no therapy-related serious adverse events in either arm of the study. In addition, no occurrences of osteonecrosis of the jaw have been noted. In conclusion, treatment with the bisphosphonate zoledronic acid in combination with calcium carbonate and vitamin D supplementation appears to provide prevention of bone loss or improvement in the BMD of patients with lymphoma undergoing chemotherapy. Given the large number of patients with below-average BMD prior to therapy and the known deleterious effects of lymphoma therapy on bone density, baseline BMD evaluation may be warranted in all lymphoma patients. In addition, the unexpectedly high rate of periodontal disease in this patient population further supports the need for careful dental evaluation prior to the consideration of bisphosphonate therapy given the well-described, albeit rare, risk of osteonecrosis of the jaw from zoledronic acid.

Multiple myeloma (MM) remains fatal in the majority of patients with the disease, even though the application of new therapeutic agents has greatly advanced the treatment of the disease. There is an urgent need to develop novel therapeutic agents. We recently demonstrated that anti-β2-microglobulin monoclonal antibodies (anti-β2M mAbs) have remarkably strong apoptotic effects on myeloma in vitro and in SCID mice. However, whether the mAbs will be therapeutic and safe in treating myeloma patients, in whom every tissue expresses low densities of MHC class I molecules and elevated levels of soluble β2M are present, remains to be determined. In this study, we examined the antimyeloma activity and potential toxicity of the mAbs in a human-like situation. We developed and used a myeloma-HLA-A2-transgenic NOD/SCID mouse model. After myeloma establishment in NOD/SCID mice transgenic for HLA-A2 α-chain, we found that all tissues express human HLA-A2 and β2M, and detected high levels (2 mg/mL) of circulating human β2M. After one intraperitoneal (i.p.) injection of the mAbs (1 mg per mouse), the mAbs could be detected on the surface of tumors and murine organs. Furthermore, murine splenocytes were incubated with immobilized mAb W6/32 to crosslink MHC class I. Western blotting analysis showed that cross-linking human MHC class I triggers phosphorylation of Lyn, Syk, and PLC-γ2 in the treated cells, indicating that indeed human MHC class I are functional and able to transduce signals in murine cells. Thus, these findings demonstrate that the mouse model is suitable for our studies because myeloma-derived human β2M was highly elevated in the serum, and bound with the α-chain to form mature and functional human MHC class I on all tissues. By using the model, we first examined the therapeutic efficacy of the mAbs on established myeloma. Myeloma-bearing mice were i.p. injected with the mAbs (1 mg per mouse) twice a week for a total of 4 injections. The results showed that the mAbs effectively suppress myeloma growth, and activated caspase-9 and -3 and induced myeloma cell apoptosis in vivo. We also examined whether the mAbs damage human MHC class I-expressing normal cells and tissues. After treatment with the mAbs, murine organs were removed for histological examination. We found that although the mAbs were detected on different organs, no tissue damage or cell apoptosis was observed in the mice. Furthermore, we elucidated the mechanisms underlying the selectivity of the mAbs in killing myeloma cells without damaging normal cells. By using the QuantiBRITE beads, we quantified the numbers of surface β2M and found that myeloma cells have approximately 265,918 surface β2M molecules whereas normal blood lymphocytes have approximately 79,521, indicating that myeloma cells express 3-fold more surface β2M than lymphocytes. Myeloma cells treated with specific siRNAs for human β2M expressed a similarly low level of β2M (91,723) and became resistant to mAb-induced apoptosis. Thus, our findings indicate that the mAbs may be safe and that the tissue-expressed and soluble β2M may not compromise their therapeutic effects in myeloma patients. This study provides further support for the future application of the mAbs as therapeutic agents for MM.

Background: Though mantle cell lymphoma (MCL) outcome has improved over the last two decades - especially with dose-intense or high-dose therapy - MCL patients still relapse particularly in >65yo. Bortezomib has shown single agent activity of 33% in relapsed MCL and has been safely combined with conventional as well as intensified doxorubicin-containing regimens, but not with high doses of the methotrexate/cytarabine combination, where there is concern about lung toxicity. Materials and methods: MCL pts from ages 18 through 79, with adequate organ function unless due to lymphoma, and without HIV-1 infection or any significant medical/mental condition were treated under IRB-approved study using R-HCVAD alternating with R-M/A (JCO October 1, 2005) with doxorubicin given by bolus. Bortezomib was added to R-HCVAD as a fixed dose of 1.3 mg/m2 IV given at the end of the first infusion of cyclophosphamide on day 2 and after doxorubicin bolus on day 5. Bortezomib was added to R-M/A after rituximab on day 1 and on day 6, in increasing doses of 0.7, 1, and 1.3 mg/m2 in cohorts of 3 patients. As an additional precaution, the first cohort of patients received a −1 dose level of the cytarabine adjusted by age. Dose limiting toxicity was defined initially as a drop of 10% from baseline in diffusing lung capacity (DLCO) and later increased to 25% (grade 1 toxicity according to National Cancer Institute criteria) after the first cycle of R-MA with bortezomib. Results: Since April 2007, sixteen patients have been entered on the trial, ages 40 to 70 years (median 58). Currently 2/3 patients have been entered in the last cohort. There has been no DLT. After 59 cycles, toxicities according to NCI criteria were as follows: BR-HCVAD Phase I Toxicity in 59 cycles | Variable | Grade1 (%) | Grade2 (%) | Grade3 (%) | Grade4 (%) | |:-------------------------------------------------------------:| ---------- | ---------- | ---------- | ---------- | | *First R-M/A cycle of each patient. 15/16 patients evaluable. | | Thrombocytopenia | 7 (12) | 3 (5) | 8 (14) | 26 (44) | | Neutropenia | 3 (5) | 5 (8) | 4 (7) | 28 (47) | | Anemia | 11 (19) | 23 (39) | 11 (19) | 2 (3) | | Neutropenic fever | | | 5 (8) | 1 (2) | | Dyspnea | | | | | | DLCO* | 2 (3) | | | | | Neurological | 5 (8) | | | | | Sensory | | 1 (2) | | | As expected, responses are excellent with 11 complete responders and 4 partial responders out of 15 evaluable patients, of whom 3 are still on treatment. Conclusion: Bortezomib can be safely combined with R-M/A. Accrual is ongoing and more patients and dose levels will be reported on at the meeting.

Follicular lymphoma (FL) patients, (pts) with high-risk features using the FL International Prognostic Index (FLIPI) have an expected 5-year survival of only about 50% with conventional therapy. With the incorporation of anti-CD20 monoclonal antibody (mAb) therapy, results are improving (e.g., Buske, Blood2006; 108: 1504). Starting in 2003, we have treated high-risk (FLIPI ≥3) FL pts with R-FND (rituximab, fludarabine, mitoxantrone, dexamethasone) for 4 cycles, followed by radioimmunotherapy (RIT) with ibritumomab tiuxetan, and subsequent rituximab maintenance. Results for the first 35 pts are: complete (CR) and partial (PR) remission 83% and 14%; 3-year overall (OS) and failure-free survival (FFS) 89% and 74% (median follow-up 24 mo.). RIT converted 5 PR pts to CR. Toxicity was mainly hematologic. Five pts did not receive RIT, one because of neutropenia after R-FND. Following RIT, platelet and neutrophil nadirs were 28 and 0.3, occurring at 4–7 weeks. 16 pts required transfusions, and 27 received growth factors. 13 pts had infections, only 2 of which were grade 3. Recovery occurred by 3 weeks in most, with prolonged cytopenias in 6. There has been 1 case of myelodysplasia. In conclusion, the additional complexity of this RIT intensification strategy is warranted in this high-risk FL population, resulting in OS and FFS outcomes that are better than non-mAb therapies, and at least as good as published chemotherapy-rituximab combination therapy.

Multiple myeloma is still an incurable plasma malignancy because patients are prone to quickly relapse after conventional treatment or even high-dose chemotherapy. At the present time, monoclonal antibodies (mAbs) are being successfully used to treat cancers. We recently discovered that anti-β2-microglobulin (β2M) mAbs (IgG1 isotype) induced myeloma cell apoptosis (Yang et al., Cancer Cell2006; 10:295–307). The mAbs bind, cross-link, and recruit surface β2M/MHC class I molecules on myeloma cells to lipid rafts where downstream signaling pathways are activated. Therefore, it is possible that enhancing the capacity of the mAbs to cross-link surface MHC class I could further improve the efficacy of mAb-induced myeloma cell apoptosis. To examine this hypothesis, we generated anti-β2M mAbs of IgM isotype with a pentameric structure. By using Annexin V and TUNEL assays, we showed that, compared with monomeric IgG mAbs, IgM anti-β2M mAbs exhibit stronger tumoricidal activity on all six myeloma cell lines and primary myeloma cells isolated from five myeloma patients in dose- and time-dependent manner. About 80% to 90% of myeloma cells were apoptotic when treated with IgM mAb at a concentration as low as 20 mg/mL in a 12-hour culture. Furthermore, IgM anti-β2M mAbs, which at the same dose had stronger therapeutic efficacy than IgG mAbs in vivo, had greatly reduced tumor burdens and retarded tumor growth in SCID mice. To examine whether the pentameric structure plays an important role in IgM mAb-mediated tumoricidal activity, IgM mAbs were treated with β-mercaptoethanol (2ME), a specific agent that breaks the J-chain of IgM antibody leading to irreversible disruption of the IgM pentameric structure. By using native gel electrophoresis, we confirmed that without 2ME treatment, IgM mAbs were pentamers with one band of 950 kDa. After treatment with 2ME, IgM mAbs displayed a strong band in 175 kDa (monomer), indicating that 2ME completely broke pentamers to monomers. Furthermore, we found that the addition of 2ME-pretreated IgM mAbs to cell culture induced much weaker myeloma cell apoptosis than pentameric IgM mAbs. By using Western blot analysis, we further showed that 2ME-pretreated IgM mAbs induce weaker JNK activation and caspase-9, -8, -3, and PARP cleavage. By using confocal microscopy, we showed that 2ME-pretreated IgM mAbs are much less efficient at recruiting β2M/MHC class I molecules into lipid rafts on myeloma cells, although 2ME-pretreated IgM mAbs bound well to myeloma cell surface. Thus, our findings indicate that enhancing cross-linking of surface β2M/MHC class I molecules may be a novel approach to improve the antimyeloma efficacy of the mAbs. This study also suggests that our IgM anti-β2M mAbs may be a better therapeutic agent for future clinical application.

Background: The addition of rituximab to combination chemotherapy has improved treatment outcomes of indolent lymphomas. Combination therapy with purine analogs, akylating agents, and monoclonal antibodies is a promising approach for treating indolent B-cell lymphoma. Nucleoside analog-based regimens selectively target lymphoid cells, making them attractive drugs for lymphoid cancers. Pentostatin is a nucleoside analog that compared with other nucleoside analogs is reported to have less bone marrow cell toxicity. The combination of pentostatin, cyclophosphamide, and rituximab (PCR) is an effective regimen for relapsed chronic lymphocytic leukemia. Methods:In this study, we examined the efficacy of pentostatin, cyclophosphamide, and rituximab for the treatment of B-cell lymphoma. Pentostatin (4 mg/m2), cyclophosphamide (600 mg/m2), and rituximab (375 mg/m2) were given on day 1 of a 21-day cycle with planned 6 or 9 cycles and restaging after every 3 cycles. Patients received prophylaxis with acyclovir 400 mg/po bid and trimethoprim-sulfamethoxazole 3 times per week. Small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) tissues were stained for ZAP70 protein. Results: At the time of abstract submission, 80 patients with a median age was 60 years were treated, 43 patients with follicular lymphoma, 27 with SLL/CLL, and 9 with mucosa-associated lymphoid tissue. We observed an overall response of 77 out of 80 (96%), CR/CRu in 65 out of 80 (81%), PR in 12 out of 80 (15%), and mixed response or progression in 3 out of 80 (4%). Neutropenia (49% < 1000/uL), thrombocytopenia (2% < 100,000/uL), nausea (15% grade 3), fatigue (37% grade 3 and 4), and muscle pain (21% grade 3) was observed. ZAP70 staining and response will be reported. Two patients experienced prolonged pancytopenia that resolved and no cases of myelodysplasia were observed. Conclusions: PCR therapy is an effective regimen in indolent B cell lymphoma with tolerable toxicity.

8589 Background: Indolent lymphomas are the most common non-Hodgkin’s lymphomas (NHL). The cell adhesion molecules CD44, ICAM-1 and VCAM-1 present on lymphoma cells regulate cell-matrix interaction...

20505 Background: DLBCL is a common lymphoma of older adults, and a potentially curable disorder with the regimen RCHOP (Coiffier, et. Al. NEJM, 2002). Doxorubicin, a key drug in the regimen, has r...

Background: Relapsed/refractory MCL is difficult to treat. R targets CD20 antigen on the surface of MCL cells while Len may target the microenvironment of MCL cells and enhance the ADCC activity of R. To test this hypothesis, we initiated a single-center, phase I/II study. We now report the data from the completed phase I trial and the ongoing phase II trial. Methods: Eighteen eligible patients (pts) with MCL had 1–4 lines of prior therapies. None had prior thalidomide and all received R. Treatment consisted of Len given orally daily on days 1–21 of a 28-day cycle and R 375 mg/m2 by IV infusion weekly for 4 weeks only during the first cycle with the first dose on Day 1 in Cycle 1. A standard 3+3 dose escalation was used to determine MTD with Len doses at 10 mg, 15 mg, 20 mg, and 25 mg. DLT was defined as grade (G) 3 or 4 non-hematologic or G4 hematologic toxicity during the first cycle. Phase I has been completed with 15 pts. Phase II is ongoing with 3 pts enrolled at MTD. Total 80 cycles of therapy were given to 18 pts. In addition to the regular radiology evaluation, a separate radiology evaluation was performed independently. EGD and colonoscopy with random biopsies were performed to confirm all CRs. Results: In the phase I study, median age was 73 (62–84); median prior therapies were 3 (1–4); median cycles received to date (August 18, 2007) were 4 (range 1–14). Two DLT’s occurred at 25 mg. One G3 hypercalcemia and 1 G 4 non-neutropenic fever during the first cycle. After total 80 cycles of therapy in 18 patients, except for the 2 DLT’s, common non-hematologic toxic events included fatigue (23 G1-2), pruritis (18 G1-2), myalgias (5 G1-2, 1 G3) and rash (5 G1-2). There was no DVT/PE. Grade 3 hematologic toxic events included neutropenia (11), febrile neutropenia (2), and thrombocytopenia (2). The only grade 4 hematologic toxic events were neutropenia (5). There was no grade 3–4 anemia. There were no responses at 10 mg or 15 mg. Ten pts at MTD (20 mg) including 7 in phase I plus 3 in phase II were evaluable for response. Seven out of 10 pts achieved responses including 3 CRs (30%), 4 PR’s (40%), 1 SD and 2 PD’s. Therefore, the overall response rate was 70%. Media time to response was 2 cycles (range 2–4). Conclusions: The MTD for Len with R in relapsed/refractory MCL was 20 mg, orally daily on days 1–21 of a 28-day cycle. Early evidence of response is promising with a very favorable toxicity profile at 20 mg and is being further evaluated in the ongoing phase II trial.

Multiple myeloma (MM) is a B-cell malignancy dependent on the bone marrow microenvironment, where a number of cytokines contribute to myeloma cell growth, survival, migration and resistance to conventional chemotherapy. We have recently shown that β2M-specific mAbs have remarkably strong antimyeloma activities by binding to β2M and recruiting MHC class I to the lipid rafts, in turn activating JNK and inhibiting ERK and PI3K/Akt pathways, whereas, neither interleukin-6 (IL-6) nor insulin-like growth factor-I (IGF-I) affect mAb-tumoricidal effects. This study was undertaken to elucidate the mechanisms underlying anti-β2M mAb-induced PI3K/Akt and ERK inhibition and the inability of IL-6 and IGF-I to protect myeloma cells from mAb-induced apoptosis. In our experiments, β2M-specific mAbs were added to cultures of myeloma cells, including tumor cell lines and freshly isolated primary tumor cells from patients with MM, with or without addition of IL-6 or IGF-I. Cell apoptosis was examined 48 hours later by Annexin-V staining assay. The results confirmed that the addition of IL-6 or IGF-I significantly undermined dexamethasone-induced cell death, but did not abrogate mAb-induced apoptosis in myeloma cells. As lipid rafts are considered to function in part as a platform for signaling from the receptors, and ERK and PI3K/Akt are downstream of IL-6 and IGF-I receptor activation, we hypothesized that MHC class I relocation to lipid rafts by mAbs may disrupt IL-6 and IGF-I receptor signaling. For this purpose, myeloma cells were incubated with cytokines, with or without mAbs. After 30 minutes of treatment, lipid rafts were prepared and separated using a discontinuous sucrose gradient ultracentrifugation followed by immunoblotting with specific antibodies. The results showed that by recruiting MHC class I into lipid rafts, anti-β2M mAbs excluded IL-6 and IGF-I receptors and their substrates from the rafts. Likewise, upon immunoprecipitation, IL-6 and IGF-I receptors in cytokine-treated cells were physically associated with caveolin-1, a lipid raft-associated protein. Such an association was missing or less prominent in cytokine- and β2M-specific mAb-treated myeloma cells. Treatment with methyl-β-cyclodextrin (MCD), an agent that disrupts the structure of lipid rafts in the cell membrane, could abrogate both IL-6-mediated protection against apoptosis induced by dexamethasone and β2M-specific mAb-induced cell death of myeloma cells. Using Western blotting assay, we further found that β2M-specific mAbs were not only redistributed the receptors in cell membrane, but also abrogated cytokine-mediated JAK/STAT3, PI3K/Akt, and Ras/Raf/ERK pathway signaling, which are otherwise constitutively activated in myeloma cells. Thus, this study further defines the tumoricidal mechanism of the β2M-specific mAbs and provides strong evidence to support the potential of these mAbs as therapeutic agents for myeloma.

Dickkopf-1 (DKK1), a secreted protein and Wnt signaling pathway inhibitor, is highly expressed by the tumor cells of most multiple myeloma (MM) patients and contributes to osteolytic bone disease by inhibiting the differentiation of osteoblasts. DKK1 has been targeted as a myeloma-associated antigen by immunotherapeutical approaches based on DKK1-directed monoclonal antibodies and DKK1-specific cytolytic T lymphocytes (CTLs). The present study was conducted to develop a treatment strategy through adoptive transfer of DKK1-specific CTLs in a SCID-hu system. Using DKK1 peptide-pulsed dendritic cells (DCs), we successfully generated DKK1-specific CTL lines and clones from HLA-A2+ MM patients in vitro. These CTLs not only had cytolytic activity against DKK1 peptide-pulsed T2 cells, but also significantly lysed autologous myeloma tumor cells, HLA-A2+ and DKK1+ MM cell lines U266 and IM-9, and primary tumor cells in vitro. No killing was observed against HLA-A2+ normal lymphocytes, including B cells and HLA-A2+ DKK1- XG1 or HLA-A2- myeloma cell lines or primary myeloma cells from patients. To determine the in vivo antitumor activity, SCID-hu mice were engrafted with primary MM cells expressing high levels of DKK1 from patients and treated with DKK1-specific CTLs after one month of tumor injection. Control mice were treated with naïve CD8+ T cells or PBS alone. All mice were examined by X-ray, tumor burden was measured according to levels of circulating human IgG, and survival rates were determined. All mice established human myeloma within one month after tumor inoculation, at which time the level of circulating human IgG was greater than 600 ng/ml in mouse serum. After another 3 weeks, the level of circulating human IgG was greater than 2,500 ng/ml in the serum of all control mice, while only 40% mice treated with DKK1-specific CTLs had high levels of circulating human IgG. X-ray examination showed that osteolytic bone disease and a large tumor burden were found in all control mice, while the established tumor was eradicated in 60% mice treated with DKK1-specific CTLs. Results of immunofluorescence studies showed that only tumor-reactive DKK1-specific CD8+ T cells were able to infiltrate into the tumor and mediate apoptosis in tumor cells. All control mice succumbed to myeloma within one month of establishment of the myeloma, while 9 out of 15 mice treated with DKK1-specific CTLs were macroscopically disease-free at the end of the experiment. These results suggest that DKK1-specific CTLs are able to eradicate established patient-derived primary myeloma in the host and that adoptive transfer of DKK1-specific CTLs may be used for myeloma therapy.

Background: R-HCVAD (rituximab-cyclphosphamide, vincristine, dexamethasone, doxorubicin) alternating with R-MTX-ARAC (rituximab, methotrexate-cytarabine) is a chemoimmunotherapy regimen with activity in ALL, mantle cell, and Burkitt’s lymphomas. Histological differentiation between BL. atypical BL, and DLBCL with high-grade features can be difficult. We treated patients with DLBCL with high-grade histological features, and/or poor-risk IPI. Patients who received at least one dose of R-HCVAD were included in this cohort. Methods: Twenty-seven consecutive patients younger than 60 years, with information collected prospectively in the NCCN database, treated between 7/2002 and 10/05 at M.D. Anderson Cancer Center, 17 (63%) male, 15 (55%) HI and High IPI, 17 (63%) ↑LDH, 14 (52%) with Stage III/IV, 2 (5%) with PS>1, 60% had a Ki-67 higher than 80%. Results: ORR was of 100% with (95%) CR/CRu. With a median follow-up of 26 months, 2 patients failed: One died of disease, and another received a SCT when the patient was in PR. Four patients have died, three of disease, one of pulmonary embolism. The 3-year OS is 96% (95% CI 77%–100%); 3-year FFS is 86% (95% CI 64%–100%). The 3-year FFS for patients with L/LI risk (2-failures of 12 pts) was 69% and for patients with HI/H age-adjusted IPI (0 failures of 15 pts) 100%. Conclusions: R-HCVAD/R-MTC-ARAC is a very active regimen for DLBCL. Toxicity was mostly hematological and infectious. We are currently doing a prospective randomized phase II study comparing this regimen to standard R-CHOP in poor risk younger patients.

Introduction: Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with poor clinical outcome. Although frontline therapy induces a high rate of complete remission, relapse is inevitable and new regimens are needed for relapsed MCL. The proteasome inhibitor bortezomib (BTZ) induces apoptosis and sensitizes MCL cells to chemotherapy in relapsed MCL, but as a single agent, response rate is low, duration of response is short and side effects are severe. Here we evaluated whether BTZ is additive or synergistic with cyclophosphamide (CTX) and rituximab (RTX). Material and Methods: Four human MCL cell lines SP53, MINO, Grant 519, and Jeko-1 and freshly isolated primary tumor cells from three MCL patients were treated with BTZ, CTX, RTX individually or in combination of RTX and CTX (RC), or BTZ plus RTX and CTX (BRC regimen). Cell proliferation and apoptosis were evaluated to determine if there was additive or synergistic effect of the BRC regimen. Western blot analysis was used to elucidate the molecular mechanism by which BTZ, RTX, CTX, RC and BRC induces apoptosis in MCL cells. In addition, in vivo experiments using severe combined immunodeficiency mice with human mantle cell lymphoma xenografts were performed to examine the in vivo efficacy of the regimen to control the growth of and eradicate MCL cells. Results: BTZ and CTX as single agents inhibited the growth of MCL cell lines in a dose-dependent manner (P < 0.01). The IC50 (inhibitory concentration at 50%) for BTZ and for CTX were between 10 and 20 nM and between 5 and 20 mM, respectively. Increasing doses of BTZ with a fixed dose of RTX (10 μg/mL) and CTX (10 mM) resulted in markedly synergistic growth inhibition of MCL cells (P < 0.01). The BRC regimen induced apoptosis in about 69.7% of MCL cell lines and 92.6% of primary tumor cells (P < 0.05 and P < 0.01, compared with those induced by BTZ, RTX, CTX or RC). Furthermore, western blotting analysis showed that BRC induced apoptosis earlier via activation and cleavage of caspases-8, -9, and -3, and PARP as compared with BTZ, RTX, CTX or RC. The pan-caspase inhibitor z-VAD-FMK completely blocked apoptosis induced by BRC. In vivo studies demonstrated that BRC regimen eradicated subcutaneous tumors in MCL-bearing SCID mice and significantly prolonged the long-term event-free survival up to 10 weeks in 70% of the mice, whereas all tumor-bearing mice receiving BTZ, RTX, CTX or RC or PBS (control) died of aggressive MCL within 6 weeks. Conclusion: Cytoreductive chemotherapy with both BTZ and anti-CD20 antibody effectively inhibited the growth and induced apoptosis of MCL cells in vitro and in vivo. Bortezomib-rituximab-cyclophosphamide (BRC) regimen may offer a better therapeutic modality for MCL patients. Thus, our data lay the basis for a clinical trial in relapsed MCL using the BRC combination treatment.

Background: Waldenstrom’s Macroglobulinemia (WM) is an uncommon subtype of B cell malignancy. The updated trend of its incidence in the United States has not been reported. Objective: To better understand the incidence and epidemiological features of WM in the United States. Methods: We conducted a population-based incidence study with 1463 patients (pts) with WM identified from the Surveillance, Epidemiology, and End Results (SEER) tumor registries over 17 years. From the SEER data, 78,558 pts of all ages diagnosed with non-Hodgkin’s lymphoma (NHL) were identified between 1988 and 2004 in 9 SEER areas. Of these pts, 1463 had pathologically confirmed WM, accounting for 2% of NHL. The incidence with 95% confidence intervals were generated from SEER*Stat Software and was age-adjusted to the U.S. year 2000 standard population. Annual Percent Change (APC)for the incidence was calculated and considered to be statistically significant if p value was less than 0.05. The crude 1-year survival rate was calculated as the ratio of the number of pts who survived over 1 year and the number of pts diagnosed with WM. Results: Of the1463 pts with WM, median age at diagnosis was 73. Overall incidence rate was 0.37/100,000/year, which increased with age from 0.03 in pts aged 0.05). Incidence of WM was higher in men (0.52) than in women (0.26) (P Conclusion: The overall incidence of Waldenstrom’s Macroglobulinemia did not change significantly from 1988 to 2004, and was significantly higher in male Caucasians. There were substantial geographical variations in the incidence of Waldenstrom’s Macroglobulinemia in the United States.

Background: FL3 is a subcategory of follicular lymphomas that is challenging in that it behaves aggressively like large cell lymphomas. If treated with CHOP, however it has a clinical course of relapse and treatment failure similar to grade 1–2 follicular lymphoma. We looked at the outcome of FL3 patients treated with RCHOP, combining rituximab with CHOP. There are no large study reports of this regimen’s results in FL3 to our knowledge. Patients and Methods: We retrospectively reviewed the records of 45 patients with follicular grade 3 lymphoma who were treated with rituximab and the combination of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) at The University of Texas MD Anderson Cancer Center (UTMDACC). Response rate (RR), failure-free survival (FFS), and overall survival (OS) were estimated and a historical comparison to 111 CHOP only treated patients was made. Results: The International Prognostic Index (IPI) distribution was: 47% Low, 36% Low-Intermediate, 13% High-intermediate, and 4% High-risk. The complete response rate was 96%. Forty-four out of 45 patients are still alive. Median follow-up is 3.5 years. The 3-year FFS rate according to IPI was 80% (95% CI: 64% to 100%) in low risk, 81% in low-intermediate (95% CI 64% to 100%), and was 50% (95% CI: 25% to 100%) in high-intermediate/high-risk patient group. The addition of rituximab to CHOP improved both 5-year FFS, 71% (95% CI: 58% to 87%) compared to 44% of CHOP alone (95% CI: 36% to 55%) with p-value of 0.019 and 5-year OS, 98% (95% CI: 93% to 100%) compared to 75% (95% CI: 67% to 84) with p-valule of 0.0034. The addition of rituximab to CHOP improve the FFS compared to CHOP alone when subgroups of IPI were analyzed and compared (p=.002) Conclusion: The addition of rituximab to CHOP provided a high response rate and excellent early survival. Poor risk patients continue to demonstrate a high rate of failure despite the use of rituximab.

Background: DLBCL is a common lymphoma of older adults, and a potentially curable disorder with the regimen RCHOP (Coiffier, et.al. NEJM,2002). Doxorubicin, a key drug in the regimen, has risk for cardiotoxicity, and thus of concern in older patients (pts) with higher incidence of cardiovascular (CV) disease. In breast cancer pts pegylated liposomal doxorubicin (D) has shown greater cardiac safety and similar efficacy to doxorubicin. We thus replaced doxorubicin with D in RCHOP (DRCOP). Methods: The regimen is: Rituximab 375 mg/m2 IV day (d) 1; D 40 mg/m2 IV d 1; cyclophosphamide 750 mg/m2 IV d 1; vincristine 2 mg IV d 1 total dose; prednisone 100 mg/d p.o. d 1–5. Eligibility criteria are: > 60 years of age; untreated confirmed DLBCL (no discordant histology); Ann Arbor stage II-IV; baseline LVEF 50%. CV disease is permitted, if symptoms controlled, but consult at baseline by Cardiologist and at follow-up required. Growth factor support recommended (not required). Results: 34 pts are evaluable for toxicity; 30 evaluable for response (1 pt off study after 1 cycle; 3 too early for response). Characteristics of pts: age 62–92 (median 75); 18 female; IPI 1=1pt; IPI 2 = 2 pts; IPI 3 = 16 pts; IPI >3 = 15 pts. Responses are: 28/30 (93%) CR; 2/30 (7%) PR. At this time, 3/30 failed: 1 progression; 1 death due to non-neutropenic pneumonia; 1 death due to disease. Grade 3–4 adverse events were: 1 drop in LVEF; 8 atrial arrhythmias (reversed); 1 hypotension (reversed); 1 chest pain (reversed); 17 neutropenia (13 febrile, with 5 infections); 3 hand-foot syndrome; 4 fatigue; 2 DVT; 2 neuropathy. Conclusions: DRCOP has a high CR rate in this study of older pts with DLBCL, despite intermediate/high risk IPI. We’ve noted only one case of low LVEF ; more common are atrial arrythmias (8 pts), reversible. Neutropenia is the most common grade 3–4 adverse event (17 pts), and thus we strongly recommend growth factor support.

Background The prognosis of patients with T-cell lymphoma (TCL) remains poor with conventional chemotherapy regimens and innovative approaches are needed. We evaluated the efficacy and safety of the regimen HCVIDDoxil, with pegylated doxorubicin substituting doxorubicin in the HCVAD regimen, alternated with MTX-Ara-C in patients with newly diagnosed TCL. Methods Previously untreated patients (pts) with Zubrod performance status 3 or less, age > 18 years, and adequate marrow, cardiac, liver and renal function were included in this study. Patients with ALK + anaplastic large cell lymphoma (ALCL), skin involvement alone, HIV positive serology, and evidence of CNS involvement were excluded. Patients received HCVIDDoxil (iv cyclophosphamide 300 mg/m2 iv Q 12 h X 6 doses days 1–3, mesna 600 mg/m2 iv daily over 24 hours by continuous infusion days 1–3, doxil 25 mg/m2/day iv on day 2, vincristine 1.4 mg/m2 (max. 2 mg) iv days 4 and 11, dexamethasone 40 mg iv or po daily X 4 days 1–4 and 11–14) alternated with MTX/Ara-C (MTX 200 mg/m2 iv over 2 hours on day 1, then 800 mg/m2 iv over 22 hours on day 1, Ara-C 3 g/m2 iv Q 12 hours X 4 doses on days 3–4). Courses were repeated every 3 weeks for a maximum of 8 cycles. Endpoints were response rate and progression-free survival. Results Between October 2003 and July 2007, 38 patients (pts) were included, 20 pts with PTCL, 6 pts with ALCL-ALK-, 4 pts with NK-TCL, 4 pts with HSL, and 4 pts with AIL. Median age was 54 (range, 23–70). Thirty-three (86%) pts had stage III/IV, 5 stage I/ II. Twenty-five (65%) pts had extranodal disease, and 14 (36%) pts had bone marrow involvement. A median of 5 cycles were delivered. To date 32 pts are evaluable for response. The ORR was 87%, with CR in 19 pts (59%) and PR in 9 pts (28%). Common Grade ¾ adverse events were thrombocytopenia in 31 pts (96%), neutropenia in 24 (75%), anemia in 23 (71%), and febrile neutropenia in 7 (32%). Six of the complete responders progressed (31%, median TTP 188 days), and 13 of the responses are ongoing (+6-+36 months). Conclusions In this high-risk population the regimen HCVIDDoxil alternated with MTX-Ara-C induced a high response rate. Further follow-up is needed to evaluate if the high response rate results in improved disease-free survival. As expected, the major toxicity was myelosuppression. Enrollment in this study continues.

Background: A previous single-arm Phase 2 study (Nature Med5:1171–7,1999), evaluated the ability of tumor-specific idiotype (ID) conjugated to keyhole limpet hemocyanin (KLH) administered with granulocyte-monocyte colony-stimulating factor - GM-CSF (BiovaxID vaccine) to induce complete remission (CR) and molecular remission (MR) in follicular NHL patients in first CR after chemotherapy. We reported (ASH 2005, Abstr 2441) that at 9.2 years follow-up, disease free survival (DFS) and overall survival (OS) were 45% and 95%, respectively; median DFS was 8.0 years. To date, there have been no additional reported mortalities in this cohort. Although these data are promising, improvements in the treatments and increasing survival for patients with follicular lymphoma have necessitated additional rigor in the evaluation of overall safety and overall risk-benefit. The ongoing Phase 3 study that opened in 2000 by the NCI is designed to evaluate the impact of this vaccine on DFS in previously untreated subjects who have attained CR/Cru. As of August 2007, 231 subjects have been enrolled to receive chemotherapy and 176 have been randomized to receive vaccine (ID-KLH or KLH-KLH). Methods: Since Phase 3 trial inception in 2000, SAEs have been reported through a pharmacoviligance group and identified according to time period on the study (i.e. during chemotherapy, during vaccine, after vaccine, or unknown), causality, and relatedness to study agent. Results: As of August 2007, 32 SAEs have been reported in the BV301 trial, 7 of which occurred during the vaccination segment of the study. At a blinded interim analysis of the Phase 3 safety data, of the 7 SAEs reported during the vaccine segment of the study, 3 are considered unlikely to be related to the study agent (chest pain, compression fracture, herpes zoster), 2 possibly related (arrhythmia supraventricular, chest pain), 1 unrelated (vomiting), and 1 unknown (febrile neutropenia). The 1 SAE, cerebral ischaemia, that occurred post vaccine is considered unrelated to the study agent. Conclusions: Additional follow-up on the Phase 2 patients is needed, as well as similar analyses in other cohorts. Blinded Phase 3 safety data shows a positive safety profile for BiovaxID. Unblinded safety data will be needed to determine the final efficacy and safety profile.

Introduction: Mantle cell lymphoma (MCL) has a poor outcome and is a therapeutic challenge. Preclinical evaluation of investigational agents for MCL has been limited by lack of suitable animal models that mimic the natural history of human MCL and provide the microenvironment in which MCL cells thrive. Since MCL usually involves the bone marrow, we developed an in vivo mouse model for primary human MCL cells in severe combined immunodeficient mice (SCID-hu), which have been implanted with human fetal bone. Materials and Methods: Human primary MCL cells were obtained and isolated from spleen, lymph nodes, bone marrow aspirates, or peripheral blood of six different MCL patients. Purified patient primary MCL cells were directly inoculated into human fetal bone chip or injected into mouse tail vein. Immunohistochemical staining with anti-human CD20 or cyclin D1 antibodies and detection of circulating human beta 2-microglobulin (B2M) in mouse serum were used to monitor the engraftment, growth, and immigration of human primary MCL cells in SCID-hu mice. Results: A total of 30 SCID-hu mice and 5 SCID mice were used. Twenty of SCID-hu mice received inoculation of 0.5 – 5 × 106 of patient primary MCL cells (2–5 SCID-hu mice/patient sample) into human fetal bone chips implanted in mice subcutaneously. Five of SCID-hu mice and 5 of SCID mice (without human fetal hone chips) were injected intravenously with 5 × 106 of patient MCL cells. The same number of cells were injected into human bone chips in 5 SCID-hu mice with equal volume of PBS as controls. Successful primary MCL cell engraftment was observed in 15 out of 20 SCID-hu mice after injection of these cells into human fetal bones. But only one out of 5 SCID-hu mice had successful engraftment after the intravenous injection of primary MCL cells into mouse tail vein. Importantly, none of SCID mice had successful engraftment after intravenous injection of 5 × 106 of primary MCL cells. These data indicated that human fetal bone provides a critical microenvironment for the survival and growth of primary MCL cells. Increasing levels of circulating human B2M in mouse serum were found after successful engraftment and growth of human primary MCL cells in SCID-hu mice. Immunohistochemical staining with anti-human CD20 and cyclin D1 antibodies confirmed that, similar to the human disease, primary MCL cells homed to mouse lymph nodes, spleen, bone marrow, and gastrointestinal tract but not to mouse liver. Treatment of MCL-bearing SCID-hu mice with atiprimod suppressed B2M secreted by functioning human MCL cells and induced tumor regression. Conclusion: Human primary MCL cells from patient were able to successfully engraft in SCID-hu mice, and mimiced the natural organ involvement of human disease homing to mouse lymph node, spleen, bone marrow, and gastrointestinal tract but not to liver. This in vivo model mimiced the natural biological features of human MCL and is therefore useful for preclinical evaluation of new therapeutic agents.

8030 Background: MCL remains a therapeutic challenge. R targets CD20 antigen on MCL cells while Len may target the microenvironment of MCL cells and enhance the ADCC activity of R. To test this hypothesis, we initiated a single-center; open label, phase I/II study. We now report the completed phase I. Methods: Eligible patients (pts) had 1–4 lines of prior therapy including prior thalidomide or R, regardless of resistance. Each cycle of treatment consisted of Len given orally daily on days 1–21 of a 28-day cycle and R 375 mg/m2 by IV infusion weekly for 4 weeks. A standard 3+3 dose escalation was used to determine MTD with Len doses at 10 mg, 15 mg, 20 mg, and 25 mg. DLT was defined as grade (G) 3 or 4 non-hematologic or G4 hematologic toxicity during the first cycle. Results: The phase I portion completed enrollment with 15 pts (4 at 10 mg, 3 at 15 mg, 6 at 20 mg and 2 at 25 mg). Thirteen pts were evaluable. Median age was 73 (62–84); median prior lines of therapy were 3 (1–4); median cycles received to date were 2 (1–7). Two DLT's occurred at 25 mg. One pt had G3 hypercalcemia. The other had G4 neutropenic fever and died of sepsis (G5) during the first cycle. Three additional pts were therefore enrolled at 20 mg. Common non-hematologic toxic events included pruritis (21 G1–2), hypercalcemia (9 G1–2, 1 G3), fatigue (9 G1–2), constipation (8 G1), diarrhea (6 G1–2), fever (6 G1–2), myalgias (4 G1–2, 1 G3) and elevated LDH (4 G1–2, 1 G3). Hematologic events included neutropenia (20 G1–2, 4 G3), thrombocytopenia (6 G1–2, 2 G3) and anemia (6 G1). There were no responses at 10 mg or 15 mg. At 20 mg after 2 cycles, 5 out of 6 pts achieved responses including 1 CR, 1 PR, 3 minor responses (MRs) and only 1 pt progressed. The 1 pt with PR went on to achieve a CR after 6 cycles. The 3 pts with MRs had tumor reductions by 43%, 40% and 38% respectively. These 3 patients with MRs continue to receive Len and might later achieve PR or CR. Conclusions: The MTD for Len with R in relapsed/refractory MCL was 20 mg, orally daily on days 1–21 of a 28-day cycle. Responses observed at 20 mg are promising with a favorable toxicity profile and are being evaluated further in an ongoing phase II study. [Table: see text]

Recent studies showed that infiltrating T regulatory cells inhibit effector T-cell function in B-cell non-Hodgkin lymphomas. Therefore, it was suggested that strategies that inactivate or remove regulatory T-cell populations should be done in conjunction with established clinical protocols in B-cell lymphomas bearing patients. In the present study, we purified CD4+CD25−, CD4+CD25highICOS+ and CD4+CD25highICOS- cell subsets by flow cytometry from a follicular lymphoma patient. We examined their intracellular FoxP3 expression and determined their IL-10 secretion in cell supernatants under different conditions. The cell subsets were stimulated with anti-CD3 and anti-ICOS antibodies in the presence of IL-2 on parental L cells or OX40L-L cells. Cytokine production in supernatants was measured by ELISA. Both CD4+CD25highICOS+ and CD4+CD25highICOS- cells showed FoxP3+ expression by FACS analysis, while it was negative in CD4+CD25−. After 3 or 6 days of culture, only ICOS+ cells produced large amounts of IL-10 (470 and 3,250 pg/ml respectively) and OX40L dramatically inhibited this production (87 and 94% of inhibition). Immunohistochemistry staining demonstrated simultaneous expression of FoxP3 and ICOS on T-cells infiltrating B-cell lymphoma tissues. These results suggest that OX40L could be potentially used as a therapeutic agent to modulate T regulatory cells function in B-cell lymphoma patients.

Human C-reactive protein (CRP) is an acute-phase protein, and elevated levels of CRP are present in patients with infections, inflammatory diseases, necrosis such as myocardial infarction, or malignancies including multiple myeloma (MM), lymphoma, and carcinoma. CRP is able to bind a variety of ligands and receptors, activating the classical complement pathway. Accumulating evidence strongly suggests that in cardiovascular disease CRP is not only a marker of inflammation, but also contributes to pathogenesis of the disease. These findings led to our hypothesis that CRP may have a functional effect on tumor cells. The present study was undertaken in a myeloma setting to determine whether CRP might affect tumor cell growth and survival. When added to the culture of primary myeloma cells isolated from patients, CRP promoted cell proliferation and reduced cell apoptosis in a dose-dependent manner. To confirm this result, we examined the effects of CRP on myeloma cell lines under stressed conditions and showed that CRP protected myeloma cells from apoptosis induced by serum starvation or IL-6 deprivation. More importantly, CRP also protected myeloma cells from apoptosis induced by dexamethasone or melphalan, two common chemotherapy drugs for MM. The protection was significant since CRP reduced cell death by 50 to 60%, and may be clinically relevant because the results were reproduced in myeloma-SCID mouse models. Injection of CRP prior to treatment of myeloma-bearing mice with dexamethasone or melphalan significantly undermined the therapeutic effects of these chemotherapy drugs. Mice receiving CRP with dexamethasone or melphalan had significantly larger tumor burdens compared with mice treated with dexamethasone or melphalan alone, whereas treatment with CRP alone had no effects on the tumor cells. CRP protected tumor cells from apoptosis by downregulating Bax expression, inhibiting phosphorylation of Bcl-2, and upregulating phosphorylation of Bad, which led to inhibited caspase-9, caspase-3 and PARP activation induced by dexamethasone. We next examined cell surface receptors for CRP and found that CRP bound Fcγ RII CD32A and/or CD32C, but not CD32B. Specific siRNAs that inhibited CD32A or CD32C but not CD32B expression, and antibodies against CD32A/C that blocked receptor-ligand interaction abrogated CRP-mediated protection of cell apoptosis. These results indicate that CRP mediated its effects via activating immunoreceptor tyrosine-based activation motif (ITAM)-containing Fcγ RII. By binding to these receptors, CRP increased the level of phosphorylated Akt, ERK1/2, and IkBa; relocalized NFkB p65 to the nucleus; and inhibited p38 kinase activity. Inhibitors against these signaling molecules blocked the activity of these pathways and abrogated CRP-mediated protection of myeloma cell apoptosis induced by dexamethasone. Therefore, our results provide strong evidence for a novel effect of CRP on myeloma cells. This study also implicates CRP as a potential target for MM therapy.

Previous studies have demonstrated that circulating dendritic cells (DCs) in myeloma patients are functionally abnormal. However, the phenotypic and functional properties of monocyte-derived DCs (MoDCs) from myeloma patients, which are commonly used for immunotherapy in patients, are still poorly defined. This study was undertaken to examine the quality of MoDCs from myeloma patients compared with cells from healthy donors. We analyzed and compared cells from 12 newly diagnosed, stage II or III myeloma patients and 10 healthy donors. MoDCs were generated by culturing adherent cells or purified CD14+ monocytes in medium containing GM-CSF and IL-4. A cocktail of IL-1β, IL-6, TNF-α, and PGE2 was used to induce DC maturation. We found that patient-derived MoDCs are phenotypically and functionally defective. Compared with their normal counterpart, patient-derived, immature and mature MoDCs expressed significantly lower levels of CD1a, CD40, CD80, and HLA-DR. The mature cells were also poor at activating alloreactive T cells, presenting recall antigen and activating autologous antigen-specific T cells, and inducing myeloma-specific cytotoxic T-lymphocyte response. These abnormalities may be attributed to elevated production of autocrine cytokines such as IL-6, constitutively activated p38 and STAT3, and inhibited MEK/ERK signaling pathways in the progenitor cells. Treatment with neutralizing IL-6-specific antibody and more importantly, p38 inhibitor, or both, could correct these abnormalities. Treating patient-derived cells with these agents not only significantly increased cell yield, but also produced MoDCs that were as functional as their normal counterpart. We further showed that the p38 inhibitor inhibited the activity of p38 and drastically increased MEK1/2 and ERK1/2 phosphorylation and activation, and a combination of IL-6 neutralizing antibody with the p38 inhibitor further upregulated phosphorylation of MEK and ERK. By examining the role of STAT3 in MoDC differentiation, we found that STAT3 activation did not impair differentiation of patient-derived cells. These results indicate that the p38 inhibitor improved the generation and function of the MoDCs via inhibiting p38 and activating Raf/MEK/ERK signaling pathways, independent of the STAT3 pathway. Thus, this study has delineated the mechanistic defects of MoDCs from myeloma patients, and identified ways for restoring the function of the cells to improve the efficacy of DC-based immunotherapy against multiple myeloma.

The addition of rituximab to combination chemotherapy has improved treatment outcomes of indolent lymphomas. Combination therapy with purine analogs, akylating agents, and monoclonal antibodies is a promising approach for treating indolent B-cell lymphoma. Nucleoside analog-based regimens selectively target lymphoid cells, making them attractive drugs for lymphoid cancers. Pentostatin is a nucleoside analog that compared with other nucleoside analogs appears to have less bone marrow cell toxicity. The combination of pentostatin, cyclophosphamide, and rituximab (PCR) is an effective regimen for relapsed chronic lymphocytic leukemia and relapsed indolent B-cell lymphoma. In this study, we examined the efficacy of pentostatin, cyclophosphamide, and rituximab for the treatment of B-cell lymphoma. Pentostatin (4 mg/m2), cyclophosphamide (600 mg/m2), and rituximab (375 mg/m2) were given on day 1 of a 21-day cycle with restaging after every 3 cycles. Patients received prophylaxis with acyclovir 400 mg/po bid and trimethoprim-sulfamethoxazole 3 times per week. Small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL), was stained for ZAP70 staining and immunoglobulin heavy chain gene mutations. At the time of abstract submission, 29 patients are eligible for response and toxicity. There were 12 patients with follicular lymphoma, 13 with SLL/CLL, and 4 with mucosa-associated lymphoid tissue (MALT), and the median age was 63 years. This regimen was used as first-line therapy for 26 patients and second-line therapy in 3 patients. We observed an overall response of 27 out of 29 (93%), CR/CRu in 19 out of 29 (65%), PR in 8 out of 29 (27%), and mixed response in 2 out of 29 (7%). The main toxicities were nausea and vomiting. No prolonged pancytopenia or myelodysplasia were observed. The correlation of ZAP70 and immunoglobulin heavy chain gene mutations with response is ongoing. Our preliminary data demonstrate that PCR therapy is an effective regimen in previously untreated indolent lymphoma.

Background: Follicular lymphoma grade 3 has a natural history that is more akin to that of diffuse large B-cell lymphoma. The addition of rituximab to standard CHOP has resulted in improved response and survival in diffuse large B-cell lymphoma. Information about outcomes in follicular lymphoma grade 3 is lacking. Methods: A single institution retrospective review of patients with follicular grade 3 lymphoma evaluated at the UTMDACC from 1999 to 2004. Patients were located from the UTMDACC lymphoma database. All patients were initially treated with R-CHOP. Results: Forty-five patients were identified: 51% male, 47% ≥60 years, and 87% follicular grade 3b. The LDH was elevated in 24%, ECOG performance status was >1 in 2%, and >1 site of extranodal involvement was present in 10%. Stage distribution was 11% stage I, 11% stage II, 42% stage III, and 36% stage IV, bulky disease (>7cm) was present in 11%, and B symptoms occurred in 13%. Beta-2 microglobulin was elevated in 57% with values >3 μg/dL in over 50%. IPI distribution was: 46% IPI Low, 38% LI, 11% IH, and 4% IPI High. Overall response rate was 100% with 96% complete responses. Relapse rate by IPI category was 24% for Low IPI, 18% for IPI LI, and 40% for IPI IH, and 100% for the two patients with High IPI. With median follow-up of 33 months, three year failure-free survival (FFS) is 73% (95% CI: 59 to 87%). One patient died (2%) with an overall survival (OS) at three years of 97% (95% CI: 93 to 100%). Conclusion: The addition of rituximab to CHOP provided a high response rate and excellent early survival in this group of mostly good prognosis patients. Relapses were still seen; longer follow-up is needed.

Adoptive T-cell therapy using donor lymphocyte infusions is a promising approach for treating hematological malignancies. But, efficacy is limited by the induction of graft-versus-host disease. Transfer of tumor-specific T-cell clones could enhance the graft-versus-tumor effect and eliminate graft-versus-host disease. However, isolating antigen-specific T-cell clones by the traditional limiting dilution approach is a time-consuming and laborious process. Here, we describe a novel strategy for rapidly cloning tumor-specific T cells. Lymphoma-specific T-cell lines were generated from two follicular lymphoma patients by repeated in vitro stimulation of lymphocytes isolated from tumor or blood with autologous soluble CD40 ligand-activated tumor cells. After four in vitro stimulations at 10-day intervals in the presence of IL-2 and IL-15, T-cell lines were found to be predominantly CD4+ T cells and produced significant amounts of TNF-a, GM-CSF, and IFN-γ in response to autologous tumor cells. The tumor reactivity was MHC class II restricted suggesting that it was mediated by CD4+ T cells. Staining with a TCR Vb antibody panel, a set of monoclonal antibodies against 24 human TCR Vb families, revealed that certain Vb families were overrepresented in each CD4+ T-cell line. In patient 1, 51% of CD4+ T cells were Vb1 positive, and in patient 2, 27% of CD4+ T cells were Vb8 positive. To clone lymphoma-specific T cells, CD4+ T-cell lines were labeled with CFSE and stimulated with autologous tumor cells. After 9 days of in vitro expansion in the presence of IL-2 and IL-15, CD4+ T-cell lines were stained with an anti-human CD4-APC monoclonal antibody and an anti-human TCR Vb-PE monoclonal antibody for each CD4+ T-cell line. Proliferating Vb1 cells from patient 1 and Vb8 cells from patient 2 were identified by their reduction in CFSE staining, and CD4+TCRV b +CFSEdim cells were sorted by flow cytometer. Monoclonality of the sorted cells was confirmed by PCR using a panel of optimized primers specific for 24 TCR Vb families, by TCR Vb spectratype analysis, and finally, by sequencing the TCR Vb gene used by each T-cell clone. Sorted tumor-specific T-cell clones could be expanded to large numbers using a 14-day rapid expansion protocol with allofeeder PBMCs, and confirmed to retain specificity against autologous tumor cells in a cytokine induction assay. This approach was also successfully used to isolate melanoma-specific CD8+ T-cell clones from two patients. We conclude that this approach is highly reproducible, rapid, and efficient for generating antigen-specific T-cell clones for adoptive T-cell therapy against human malignancies in the autologous or allogeneic setting.

Hepatosplenic T-cell lymphoma is a rare peripheral T-cell lymphoma, and less than 100 cases have been described in the literature since the entity was first proposed in 1990. This condition is characterized by malignant T-cell proliferation in the sinusoids of the liver, sinuses and red pulp of the spleen, and sinuses of the bone marrow. The commonly reported T-cell phenotype is CD2+, CD3+, CD4−, CD5−, CD7±, CD8−, with either gamma-delta or alpha-beta T-cell receptor expression. Associated cytogenetic abnormalities include isochromosome 7q with or without trisomy 8. Approximately 10–20% of patients have a history of immunocompromise. Treatment to date with standard anthracycline containing chemotherapy regimens has been disappointing with variable response rates, high relapse rates, and a short median survival. An optimal treatment strategy for this small patient population has not yet been determined. Methods: Eleven cases of pathologically confirmed hepatosplenic T-cell lymphoma seen at M.D. Anderson Cancer Center between January 1990 and June 2006 were identified in the institution’s database. Clinical characteristics and treatment results were retrospectively reviewed. Results: The mean age at the time of diagnosis was 35.3 years (range 8 – 58 years). Six patients were male and five were female. Seven patients had the gamma-delta T-cell receptor phenotype, and three patients had the alpha-beta T-cell receptor phenotype. Cytogenetic abnormalities observed included t(7;14)(q34q13), 46,idem,del(2)(q32q37), 45XY,der(13;14)(q10;q10), 45XY,i(7)(q10),der(13;14)(q10;q10), del(22q).ish22(WCP22x2). One patient had lymphomatous involvement of the skin, a finding not previously reported in hepatosplenic T cell lymphoma. Two patients had a history of significant immunocompromise, including one patient with Sjogren’s disease treated intermittently with prednisolone and methotrexate, and one patient with Crohn’s disease on chronic treatment with mercaptopurine. Complete responses (CR) were achieved in four out of eight patients who received chemotherapy and whose complete records were available. CRs were achieved with the following regimens: (1) Sequential IDSHAP/AMDBIDCOS/MINE, (2) CHOP/methotrexate and autologous stem cell transplant, (3) CHOP, (4) pentostatin/campath and allogeneic stem cell transplant. PET scans in 3 patients and Gallium scans in 5 patients did not correlate with disease activity or clinical response. Mean duration of CR was 8 months (range 2 – 14+ months). Mean overall survival was 11.4 months (range 3 – 25 months), with one patient still alive and in remission at 20 months. Conclusion: In one of the largest series of hepatosplenic T-cell lymphoma patients, we confirm the aggressive clinical course of this subtype of peripheral T-cell lymphoma occurring in a young patient population. We discovered a near-equal incidence of this disease in males and females, contrary to results in earlier series which suggested this disease is usually diagnosed in males. Standard chemotherapy treatment strategies resulted in poor outcomes. However, survival may be improved upon with newer strategies such as the use of alemtuzumab, pentostatin, and high dose chemotherapy with stem cell rescue. New therapeutic strategies are needed to improve the outcome of patients with hepatosplenic T-cell lymphoma.

7582 Background: A single-arm Phase 2 study (Nature Med 5:1171–7,1999), evaluated the ability of tumor-specific idiotype (Id) conjugated to keyhole limpet hemocyanin (KLH) administered with granulocyte-monocyte colony-stimulating factor - GM-CSF (BiovaxID vaccine) to induce complete remission (CR) and molecular remission (MR) in follicular NHL patients in first CR after chemotherapy. We reported (ASH 2005, Abstr 2441) that at 9.2 years follow-up, disease free survival (DFS) and overall survival (OS) were 45% and 95%, respectively; median DFS was 8.0 years. Previous studies indicated that the t(14;18) chromosomal translocation is associated with follicular NHL. We tested the hypothesis that absence of t(14;18) in peripheral blood (PB) of patients after Id vaccination is predictive of DFS. Methods: A 20-patient cohort in first CR treated with the vaccine was evaluated. t(14;18)- (MR) is defined as 5 cells using PCR of the MBR breakpoint cluster on DNA from tumor biopsies or PB. Of the 20 patients, 11 had t(14;18)+ tumors and PB before chemotherapy. Following chemotherapy all 11 patients continued to have t(14;18)+ PB despite being in CR. However, at 1 month following final vaccine treatment, 8 of these patients had converted to t(14;18)- . In this ad hoc analysis, the proportion of patients remaining in DFS was stratified by t(14;18) status; Kaplan-Meier analysis compared median DFS of patients across t(14;18) status following vaccination. Results: At median follow-up of 9.2 years, 55% of patients had clinically relapsed. Although not statistically significant, of the 17 patients t(14;18)- post-vaccine, only 47.1% had clinically relapsed while 100% of the 3 t(14;18)+ patients had relapsed [Chi-square test; P=0.089]. Median DFS in t(14;18)+ patients was 60 months (5.0 years) and had not yet been reached in t(14;18)- patients at 91 months (7.6 years) [Log-rank test; P=0.069]. Conclusions: These data show a possible association between t(14;18) negativity following vaccine and prolonged DFS. Additional follow-up on these patients is needed, as well as similar analyses in other cohorts, to further explore this association. [Table: see text]

7597 Background: T cell lymphomas (TCL) generally are more aggressive and have a poorer prognosis than the corresponding B cell lymphomas. A chemotherapeutic regimen that is effective in aggressive lymphoid malignancies is HCVAD alternating with methotrexate (MTX) and cytarabine (Ara-C). Pegylated liposomal doxorubicin (DOXIL) is associated with greater tumor penetration and less toxicity. We initiated a phase II study to evaluate the efficacy of the regimen HCVIDDOXIL, using DOXIL as a substitute for doxorubicin in the HCVAD regimen, alternated with MTX-Ara-C . Methods: Previously untreated patients (pts) with Zubrod performance status ≤3, age > 18 years, and adequate organ function were eligible. Pts with skin involvement alone, CD30+, ALK +, T-anaplastic large cell lymphoma (ALCL), HIV-1 positive serology, and evidence of CNS involvement were excluded. Pts received HCVIDDOXIL ( cyclophosphamide 300 mg/m2 iv Q 12 h × 6 doses d 1–3, mesna 600 mg/m2 iv daily over 24 hours by continuous infusion d 1–3, DOXIL 25 mg/m2/day iv on d 2, vincristine 1.4 mg/m2 (max. 2 mg) iv days 4 and 11, dexamethasone 40 mg iv or po daily × 4 d 1–4 and 11–14) alternated with MTX/Ara-C ( MTX 200 mg/m2 iv over 2 hours on d 1, then 800 mg/m2 iv over 22 hours on d1, Ara-C 3 g/m2 iv Q 12 hours × 4 doses on d 3–4) every 3 weeks for a maximum of 8 cycles. Endpoints were progression-free survival and response rate. Results: Between October 2003 and November 2005, 23 pts were enrolled. Median age 53 years (range, 23–68). Twelve pts had stage IV, 6 stage III, 3 stage I, and 2 stage II disease. Fourteen (60%) pts had extranodal disease, and 8 (36%) pts had an elevated LDH. In 21 evaluable pts, the ORR was 90% [CR n=12 (57%); PR n= 7 pts (33%)]. Common Grade 3–4 adverse events were thrombocytopenia in 17 pts (77%), neutropenia in 6 (27%), anemia in 5 (23%), and febrile neutropenia in 7 (32%). With a median follow-up of 13 months, the median progression-free survival is 8.3 months (95% CI: 6.5 to 14.2 months). Conclusions: In this high-risk population the regimen HCVIDDOXIL alternated with MTX-Ara-C induced a high response rate. As expected, the most common toxicity was myelosuppression. The efficacy of this regimen should be confirmed in a larger cohort of patients. Enrollment in this study continues. No significant financial relationships to disclose.

Mantle cell lymphoma (MCL) has a poor prognosis. Relapsed/refractory patients must respond to salvage chemotherapy in order to receive potentially curative stem cell transplantation (SCT). A salvage regimen with higher rate of response will offer the patient a better chance of survival. We have previously reported the results of R-HCVAD alternating with R-M-A in frontline therapy of MCL (Blood, 104:40a, 2004, (Abstract #128). The current trial looked at relapsed/refractory MCL patients treated also with R-HCVAD alternating with R-M-A. Since August 2001, the trial has accrued 24 out of a planned number of 41 patients of whom 21 are evaluable for response and survival. Median age was 63 years (range 45–78) and male:female ratio was 5:1. Three patients had received previous R-HCVAD alternating with R-M-A and three patients had failed an autologous stem cell transplant. Immediate therapy prior to the study for the 21 patients included R-HCVAD/SCT (1), CHOPw/wo rituximab (8 patients), cyclophosphamide, vincristine and rituximab (1), fludarabine (1), fludarabine, mitoxantrone, dexamethasone and rituximab (2), fludarabine and cyclophosphamide (1), radiotherapy (2), gemcitabine, mitoxantrone and dexamathasone (1) ifosfamide, carboplatin, etoposide and rituximab (1), Velcade (1), gemcitabine (1), and rituximab (1). The median number of prior regimens was one (range 1–6). Responses to the previous treatment included complete response (CR; 8 patients, 38%), partial response (PR; 6 patients, 29%), and no response or progression (7 patients, 33%). Results of the trial are as follows: Median number of cycles received = 4 (range 1–7), with an overall response rate (ORR) of 95% (43% CR/Cru; 52% PR). 5/5 patients who had progressed through the previous treatment responded (1CR, 4 PR), and 2/2 patients who had no change to the prior therapy responded (2 PR’s). We evaluated 12 cases whose response in our trial was classified as PR and found that in 4 of them it was the best response achieved but another 4 were referred to transplant while the tumor was still responding and in another case treatment was still ongoing. In 3 cases toxicity precluded continuation of therapy. Five (24%) of the patients were consolidated with non-myeloablative allogeneic stem cell transplantation. Sixteen were not transplanted for the following reasons: age (2 patients), lack of donor (5), Progressive disease (2), patient refusal (4), physician’s choice (1), waiting for match (1), and lost to follow up (1). Toxicity after 81 cycles included neutropenic fever (14%), grade 4 neutropenia (58%) and grade 4 thrombocytopenia (53%). The were no deaths due to toxicity. With a median follow-up of 21 months range 5–45 months), the median failure-free survival is 18 months as compared to a median FFS of 9 months response duration with the previous therapy, with no plateau in the curve. Patients who underwent stem cell transplant were censored at the time of transplant. The high response rates achieved R-HCVAD alternating with R-M-A makes this regimen an excellent choice for induction therapy prior to stem cell transplantation.

We have used the combination of fludarabine/cyclophosphamide/rituximab (FCR) as a conditioning regimen for NMT in patients (pts) with relapsed NHL (Blood989:3595, 2001). Seventy-eight consecutive pts were treated. Median age was 53 yrs (range, 21–68). Median # of prior chemoregimens was 3. Twenty-one (27%) had failed a prior autologous. Histologies included follicular (FL)=47 pts (60%), diffuse large cell (DLCL)=16 pts (21%) and mantle cell (MCL)=15 pts (19%). Thirty-nine (50%) were in partial remission (PR), five (6%) had stable or progressive disease (SPD) and 34 (44%) were in complete remission (CR) at study entry. Sixty-nine (88%) had a matched sibling, 6 a matched unrelated, and three a mismatched sibling donor. Ten pts had either PET or Gallium-avid disease at transplant: six were in clinical PR and 4 were in SPD. Peripheral blood was the source of graft in 71 pts (91%). Tacrolimus/Methotrexate were used for GVHD prophylaxis. Median time for ANC >500 was 10 days. Sixty pts did not require any platelet transfusion. All but 2 pts who had SPD, converted to CR post transplant. All pts engrafted donor cells. Six had a secondary graft failure (GF), two of which occurred in the setting of disease progression. With a median follow-up time of 34 months (mos) (range, 3 to 70 mos), only 6 pts (8%) relapsed: two pts were in SPD/PET or Gallium(+), 3 were in PR/PET or Gallium(−) and one was in CR at transplant. None of the 6 pts who were PR/PET or Gallium (+) at study entry relapsed. Overall the risk of relapse in PET/Gallium (+) or (−) disease was not significant (P=0.15). Five pts received donor lymphocyte infusion (DLI) for disease progression: one achieved CR; the 4 other pts (two were in GF) did not respond. DLI was also given successfully to two other pts because of cytopenia related to a viral infection (1), and because of decreasing chimerism (1). Overall survival (OS) for all pts was 88%, 82% and 74% at 1,2 and 3-yr, respectively. By univariate analysis, OS was 95% at 3-yr for those who had failed a prior autologous transplant (P = 0.04). Patients with FL histology had also a better 3-yr survival than DLCL [88 % vs 51% (P = 0.008)]; the difference with MCL (65% at 3-yr) was not statistically significant (P = 0.2). Non-relapse related mortality was 11%, 13%, and 19% at 1, 2 and 3-yr, respectively. The incidence of acute II-IV GVHD was 17%. Patients with FL disease had lower incidence of acute II-IV and III-IV GVHD than DLCL [11% vs 31%, P = 0.04; and 2% vs 25%, P = 0.02; respectively] which may have contributed to the higher OS rate in FL. Both FL and DLCL pts had similar age and donor type distribution. Two pts developed chronic GVHD post DLI. The incidence of chronic extensive and limited GVHD was 51% and was not statistically different between various histologies. We conclude that NMT with FCR for NHL is an effective treatment even in pts who failed a prior autologous transplantation and is associated with a low incidence of acute GVHD. The lower than expected relapse rate observed may be indicative that NMT could be curative for these diseases.

We discovered that monoclonal antibodies (mAbs) specific to human β2-microglobulin (β2M) induce programmed death of myeloma and other hematological tumor cells. The mAbs exhibited potent in vitro tumoricidal activity on all 14 β2M/HLA-ABC-bearing myeloma, lymphoma, and leukemia cell lines and primary myeloma cells isolated from eight patients. Cell death occurred rapidly, without the need for exogenous immunological effector mechanisms. Although the expression of β2M on normal hematopoietic cells is a potential safety concern, the mAbs seem to be selective to tumor-transformed cells and did not induce apoptosis of normal cells, including T and B lymphocytes and CD34+ bone marrow stem cells. Furthermore, the mAbs were able to selectively kill myeloma cells without damaging normal stromal cells in their cocultures. After binding to cell surface, the mAbs mediated internalization and down-modulation of surface β2M and HLA-ABC molecules. The mAbs induced cell death via inhibiting PI3K/Akt and ERK, activating JNK, upregulating Bad and Bax protein expression, inducing phosphorylation of Bcl-2, and decreasing phosphorylation of Bad, all of which compromised mitochondrial integrity, leading to cytochrome c release into cytosol and activation of the caspase-9-dependent cascade. Inhibitors to pan-caspases or caspase-9, but not to caspase-8, or to JNK, and knockdown of surface β2M and HLA-ABC expression by β2M-specific siRNA, but not control siRNA, abrogated apoptosis of tumor cells induced by the mAbs. Furthermore, anti-β2M mAbs were also active and therapeutic in vivo; After administration subcutaneous or intraperitoneal, the mAbs substantially reduced tumor burdens and retarded tumor growth in SCID mice subcutaneously implanted with human myeloma cell lines ARP-1 or MM.1S. Therefore, such mAbs offer the potential for a novel therapeutic approach to hematological malignancies and possibly to other cancers that express surface β2M. This study also suggests that differential expression of β2M/MHC class I molecules by normal and cancer cells might be a crucial point in the sensitivity and selectivity of anti-β2M mAb-induced apoptosis.

Background: BLyS is a TNF family member which was recently shown to play a critical role for survival of normal and malignant B cells. BLyS is expressed by monocytes, macrophages, dendritic cells as well as lymphoma cells, where its expression levels increase as tumors transform to a more aggressive phenotype. Previous small studies suggested that serum BLyS level is elevated in some proportion of patients with aggressive non-Hodgkin lymphoma (NHL). Little is known if serum BLyS level has prognostic value in patients with lymphoma. Thus, we determined serum soluble BLyS levels in 191 samples obtained from patients with various forms of lymphoma, and correlated them with the clinical characteristics and the prognosis. Materials and Methods: We analyzed serum BLyS level with ELISA in normal individuals and patients with newly diagnosed or relapsed lymphoma. Patients’ histopathological diagnosis, stage, presence of bulky disease, serum LDH, serum beta-2 microglobulin (b2m), IPI score, FLIPI score, progression free survival (PFS) and overall survival (OS) were correlated with serum BLyS levels. For survival analysis. Results: The sensitivity of the ELISA assay was 0.4 ng/ml. Serum samples from 93 normal individuals,135 newly diagnosed patients with lymphoma, and 56 patients with relapsed lymphoma were examined for serum soluble BLyS levels. Median serum BLyS levels were =2ng/ml) were observed more frequently in patients with relapsed lymphomas (42/56, 75%) than in patients with newly diagnosed lymphomas (46/136, 45%, p Conclusion: Serum BLyS levels are frequently elevated in patients with lymphoma, especially HL. Serum BLyS level is particularly higher in patients with relapsed disease, and is associated with poor prognosis in those patients group. Our study suggests the promising role of BLyS in the prognostication of patients with lymphoma, particularly aggressive NHL and HL.

Dendritic cells (DCs) from cancer patients are functionally defective, however, molecular mechanisms underlying are still poorly understood. In this study, we used the murine 5TGM1 myeloma model to examine the effect and mechanism of tumor-derived factors on the differentiation and function of DCs. Myeloma cells (5TGM1) or tumor culture conditioning medium (TCCM) were shown to inhibit differentiation and function of BM-derived DCs (BMDCs), evidenced by the downregulated expression of DC-related surface molecules, decreased IL-12 but increased IL-10 secretion, and compromised capacity of the cells to activate allospecific T cells in vitro. Similar results were obtained with other murine myeloma cells MOPC-315 and MPC-11. Moreover, TCCM-treated BMDCs were inferior to normal BMDCs at priming tumor-specific humoral and cellular immune responses in vivo (in the 5TGM1 mouse model). Neutralizing antibodies against IL-6, IL-10, and TGF-β partially abrogated the effects. TCCM treatment activated p38 mitogen-activated protein kinase (MAPK) and JNK but inhibited extracellular signal-related kinase (ERK). Inhibiting p38 MAPK by three different specific inhibitors was found to restore the phenotype, cytokine secretion, and function of TCCM-treated BMDCs. Vaccinating mice with BMDCs obtained from cultures in which both TCCM and p38 inhibitor were added was as efficacious as normal BMDCs at inducing tumor-specific antibody, type-1 (IFN-γ) T-cell, and CTL responses. Thus, our results suggest that tumor-induced p38 MAPK activation and ERK inhibition in DCs may be a new mechanism for tumor evasion, and regulating these signaling pathways in vivo or during DC differentiation may provide new strategies for generating potent DC vaccines for immunotherapy of multiple myeloma and other tumors.

Active immunization of follicular lymphoma patients with idiotypic vaccines elicits antigen-specific antibody responses, T-cell responses, and antitumor effects (Bendandi, M. et al., Nat Med5, 1171). We hypothesized that these vaccinated patients could generate tumor specific immune responses, not only against idiotype, but also against other tumor associated antigens (TAA) by a mechanism of epitope spreading. We further hypothesize that these antigens may represent tumor rejection targets for immunotherapy. To identify potential antigens, a phage surface expressed cDNA library derived from primary tumor cells was screened with sera from idiotype-vaccinated patients. Consistent with our hypothesis, we identified two immunogenic peptides (FL-aa-7 and 18), unrelated to idiotype, which were recognized by postvaccine sera but not by prevaccine or normal human sera. These peptide sequences derived from the 5′-untranslated regions of the human GTPase, IMAP family member 7 gene (FL-aa-7) and an alternative reading frame of U1-snRNP 70 (FL-aa-18), respectively, suggesting that epitope spreading had occurred. To further determine the specificity of these patients’ post vaccine sera, the corresponding FL-aa-7 and FL-aa-18 peptides were synthesized and tested for serum binding. As expected, post-vaccine but not pre-vaccine or normal, specifically bound the FL-aa-7 and FL-aa-18 peptides, respectively. Furthermore, both peptides specifically inhibited the binding of post-vaccine sera to the respective FL-aa-7 and FL-aa-18 phage clones in a dose-dependent manner. These data suggest that Id vaccinated patients generated specific antibodies to novel peptides FL-aa-7 and FL-aa-18, unrelated to Id. Overall, these data indicate that Id-vaccinated B cell lymphoma patients mounted antibody responses specifically for FL-aa-7-1 and FL-aa-18 peptides, which may represent a potential immunotherapeutic tumor antigen. The clinical and biological relevance of the response is to be elucidated.

The malignant Hodgkin and Reed-Sternberg (HRS) cells of Hodgkin lymphoma (HL) rarely survive outside their microenvironment, which is illustrated by the difficulty in establishing HRS-derived cell lines. Thus, we hypothesized that destroying the HL microenvironment may deprive HRS cells from critical survival factors leading to their death. The majority of the cells in this microenvironment are benign T and B lymphocytes. Both B and T cells have been reported to express growth and survival factors such as CD30 ligand, CD40 ligand, and RANK ligand that may support HRS cell survival. To determine the contribution of benign B-cells to the survival of HRS cells in vivo, we recently conducted a pilot study using rituximab alone in patients with relapsed classical HL. Rituximab produced an overall response rate of 23% in heavily pretreated patients with relapsed classical HL, presumably by depleting benign B cells from HL lesions. In this study, we evaluated the safety and efficacy of the novel combination of rituximab and ABVD (R-ABVD) chemotherapy in newly diagnosed patients with classical HL. Rituximab was given at 375 mg/m2 weekly for 6 weeks to rapidly deplete B lymphocytes, while ABVD was given at a standard dose and schedule. The first dose of rituximab was given concurrently with the first dose of ABVD (schedule A) or 3 weeks before the first dose of ABVD (schedule B). Patients with lesions larger than 5 cm received involved field radiation therapy at the end of R-ABVD. Patients were eligible if they were older than 16 years of age and had biopsy-confirmed classical HD irrespective of CD20 expression on RS cells, bidimensionally measurable disease, adequate bone marrow reserve (ANC > 1,000/uL, Platelet > 100,000/uL) and adequate cardiac and renal functions. They were excluded if they had HIV infection, or were pregnant women. To date 72 newly diagnosed pts are enrolled. Analysis of the first 52 pts is provided. Analysis of the entire 72 patients will be presented at the meeting. The median age was 30 years (range; 18–63 years). Patients had stage II (50%), stage III (31%), stage IV (19%) disease. Using the German prognostic score model, 32 patients (62%) had a score of 2 or higher. With a median follow up of 32 months, the estimated event-free survival (EFS) is 82% and overall survival 100%. EFS for patients with a prognostic score of 0–1 is 92%, score 0–2 is 86%, and score 3–5 is 73%. Treatment was reasonably well tolerated with the majority of toxicities being grade 1 and 2. One patient developed PCP pneumonia proven by bronchalveolar lavage. In two patients, fine needle aspiration of a peripheral lymph node was performed before the first and after the third dose of rituximab (schedule B-before starting ABVD). Rituximab induced complete depletion of B cells from HL node. We conclude that in patients with classical HL, the addition of 6 weekly doses of rituximab to standard dose and schedule of ABVD chemotherapy is effective in terms of remission rate and remission duration irrespective of CD20 expression on HRS cells. Furthermore, patients with prognostic score of 3 or higher had a 73% EFS suggesting that this strategy may improve treatment outcome in this patient population.

Advanced stage follicular lymphoma remains an incurable disease with a median survival of 8 to 10 years that has not significantly changed over the last four decades. Therefore, novel treatment options are necessary to improve the clinical outcome in these patients. The observation of spontaneous regressions in a small percentage of patients suggested that augmenting the host immune response could potentially control this malignancy. Strategies using active specific immunotherapy with idiotype vaccines led to induction of clinical and molecular responses in a few patients but have met with only limited success possibly due to the low frequency of antigen-specific T cells induced in the patients. In contrast to active immunization, T cells of a given specificity and function may be selected and expanded in vitro to the desired number for adoptive cell transfer. Towards this goal, we stimulated tumor infiltrating lymphocytes (TILs) or peripheral blood mononuclear cells (PBMCs) from five follicular lymphoma patients with CD40 ligand-activated autologous tumor cells at approximately ten-day intervals in the presence of IL-2 and IL-15. After four rounds of stimulations, T cell lines generated from 3/5 patients recognized autologous unmodified tumor cells by producing significant amounts of TNF-α, GM-CSF and/or IFN-γ. By phenotypic analysis, the T cell lines were predominantly CD4+ T cells (> 70%), and intracellular cytokine assay showed that up to 40% of the CD4+ T cells were tumor-reactive. The inhibition of cytokine production by anti-HLA class II but not class I blocking antibodies confirmed that the CD4+ T cells were tumor-reactive. Further characterization revealed that the T cells from one patient recognized autologous tumor but not autologous normal B cells suggesting that they were tumor-specific. While in a second patient CD4+ T cell clones generated from the T cell line by limiting dilution recognized autologous tumor and autologous normal B cells but not autologous monocytes suggesting that they were B cell lineage-specific. We conclude that follicular lymphoma-specific T cells exist and can be efficiently expanded in vitro from both TILs and PBMCs using CD40 ligand-activated autologous tumor cells for adoptive T cell therapy. Additionally, identification of antigens recognized by these T cells could lead to development of novel immunotherapeutic strategies for lymphomas.