Ansell, Stephen M.

Abstract Background: Our previous work has shown that malignant B cells induce the development of intratumoral Treg cells that inhibit the host anti-tumor response. In contrast to an increase in Treg cells, we found that the number of effector T helper cells (TH1, TH2 and TH17) was low in B-cell NHL tumors, suggesting an imbalance between Treg and TH cells in the tumor microenvironment. Understanding the mechanism(s) of this imbalance is important to the development of treatments to enhance host immunity and in previous work we have shown that signaling through CD70, CD80 and CD86 plays a role. Since soluble factors, particularly TGF-β, have an important role in directing T-cell differentiation, we evaluated in this study the role of TGF-β in the lymphoma microenvironment. Goal: To determine the effect of TGF-β on the generation of intratumoral TH1, TH17 and Treg cells in human B-cell NHL. Results: Human B-cell NHL specimens were obtained from consenting patients and were used for all experiments. Using an ELISA assay, we found that malignant B cells variably secrete TGF-β - median 100 pg/ml per million cells (range: undetectable −229 pg/ml, n=7). Using flow cytometry, we showed that addition of exogenous TGF-β enhanced the expression of Foxp3+ in activated CD4+ or CD4+CD45RA+ or CD4+CD45RO+ nodal T cells, suggesting that TGF-β promotes the generation of Treg cells in tumor microenvironment. In contrast, TGF-β suppressed expression of IFN-g in activated CD4+ T cells and inhibited the up-regulation of IL-12 and IL-23-induced IFN-γ expression in CD4+ cells, indicating that TGF-β suppresses the generation of TH1 cells. TGF-β alone slightly inhibited IL-17 expression in CD4+ T cells; however, TGF-β, in the presence of IL-6 and IL-23, upregulated IL-17 expression in CD4+ T cells, suggesting proinflammatory cytokines are able to reverse the suppression induced by TGF-β. These results indicate that TGF-β plays an important role in the regulation of intratumoral TH17 cell generation. In additional experiments, TGF-β was found to exert a suppressive effect on the proliferation of both CD4+ and CD8+ intratumoral T cells. However, treatment with TGF-β enhanced IL-2 production by intratumoral CD4+ T cells detected by intracellular staining of flow cytometry. Interruption of IL-2 signaling by anti-IL-2 Ab abolished the upregulation of TGF-β-mediated Foxp3 expression and enhanced the production of IL-17 in CD4+ T cells. Furthermore, treatment with anti-IL-2 Ab reversed the inhibition of NHL B cell-mediated TH17 cell generation. Conclusion: These results suggest that TGF-β controls the generation of TH1, TH17 and Treg cells contributing to the imbalance of effector TH cells and inhibitory Treg cells in the tumor microenvironment of B-cell NHL through IL-2. Since malignant B-cells produce TGF-β, these results further support the important role of malignant B cells in the regulation of intratumoral T cell generation and the host immune response.

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

Abstract BACKGROUND: SGN-40 is a humanized monoclonal antibody that targets CD40. Upon binding to CD40, SGN-40 triggers pro-apoptotic signal transduction pathways and mediates effector cell functions (ADCC and ADCP). SGN-40 demonstrated preliminary anti-tumor activity in a phase 1 study of non-Hodgkin lymphoma, including diffuse large B-cell lymphoma (DLBCL) patients (ASH 2006, Abstract 695). METHODS: We conducted a multicenter, phase 2, open-label study to determine the overall response rate and toxicity profile of SGN-40 in patients with relapsed DLBCL. Tumor samples were assessed by a central lab for pathology confirmation and CD40 expression. Eligible patients had de novo or transformed DLBCL at diagnosis and were excluded if previously treated for indolent lymphoma. Required prior therapy consisted of combination chemotherapy with rituximab and, if eligible, autologous stem cell transplantation. Patients received 6 intravenous infusions of SGN-40 over 5 weeks (Cycle 1) with intra-patient dose loading (1 mg/kg on Day 1; 2 mg/kg on Day 4; 4 mg/kg on Day 8) and 8 mg/kg/wk thereafter. Responding patients and those with stable disease were eligible to continue therapy until disease progression or a maximum of 12 cycles. RESULTS: Forty-six patients (28 M, 18 F), with a median age of 72 years (range 17–85), were enrolled at 10 centers in the US. Patients were heavily pre-treated, with a median of 4 prior systemic chemotherapy regimens (range 1–10). Immunohistochemistry analysis of patient tumor samples demonstrated high expression of CD40 in all patients with the exception of 1 patient who was negative. Thirty-eight of the 46 patients met predefined criteria for evaluation (completed dose loading and had no major protocol violations). Best responses seen were CR (n=2, 5%) and PR (n=2, 5%), yielding an ORR of 10%. Furthermore, 9 (24%) patients had SD as best response. Reductions in tumor size were seen in approximately one-third of patients. Twenty (53%) patients had PD and 5 (13%) patients have yet to be assessed. Treatment with SGN-40 was generally well tolerated. Grade 3/4 events that occurred in more than 1 patient in the intent-to-treat population (N=46), regardless of drug attribution were thrombocytopenia (n=5); neutropenia (n=4); and anemia, fatigue, pneumonia, hyponatremia, DVT, and disease progression (n=2 pts each). Of these events, only fatigue (n=2) and neutropenia (n=2) were considered drug related by the investigator. CONCLUSION: Monotherapy with SGN-40 in DLBCL patients is well tolerated and resulted in objective responses consistent with the previous phase 1 experience. These data support further evaluation of SGN-40 in combination with chemotherapy in non-Hodgkin lymphoma; 3 combination clinical trials are ongoing. Retrospective correlation of clinical outcome and a gene signature that includes baseline CD40 activation status are planned.

Abstract Background: Germinal center B cell-like (GCB) DLBCL, as determined by gene expression profiling or immunohistochemistry, is more likely to be cured by initial conventional chemoimmunotherapy than non-germinal center B cell-like (non-GCB) DLBCL. For patients with relapsed or refractory chemosensitive DLBCL, high-dose chemotherapy and autologous hematopoietic stem cell (HSC) transplant is considered standard-of-care treatment, but it is unknown whether the outcome of these patients is similarly influenced by the subtype of DLBCL. We therefore explored the differences between patients with GCB and non-GCB DLBCL as regards their clinical features at relapse after first line therapy and their outcome following salvage autologous HSC transplant. Methods: Patients undergoing BEAM conditioning and autologous HSC transplantation for relapsed or refractory chemosensitive DLBCL at Mayo Clinic, Rochester, MN between 2001 and 2006 were included. Immunohistochemical analysis was performed for CD10, BCL-6 and MUM1 allowing classification in GCB and non-GCB-like DLBCL, as well as for BCL-2. GCB and non-GCB groups were compared in terms of known prognostic factors at time of progression and outcome after HSC transplant Results: Fifty-nine patients were included and had retrievable tumor samples to allow immunohistochemical analysis. Median follow-up of survivors was 25 months; median age at the time of transplant was 60 years (range 17–77); All patients had failed at least one previous anthracycline-based regimen (15 had refractory disease). Overall, 25/59 cases (42%) were positive for CD10, 32/58 (55%) for BCL-6, and 19/58 (32%) for MUM1. Thirty-two patients (54%) were classified as having GCB and 27 (46%) non-GCB-like DLBCL. Patients in the GCB and non-GCB group had similar time to progression (TTP) (median 12.5 months vs. 11 months, Wilcoxon P=0.81) after first line therapy and similar IPI-R scores (Chi-square, P=0.38). In univariate analysis, GCB and non-GCB did not differ in time to relapse after HSC transplant (log rank test P=0.77) or survival (log rank test P=0.48; figure). The lack of demonstrable difference in survival persisted even after correction for IPI-R and TTP, factors know to affect transplant outcome (Cox regression, RR=0.80 for GCB; P=0.28). BCL-2 was highly expressed in both GCB (81%) and non-GCB (96%) and did not correlate with outcome in the entire population nor in any of the two groups. Conclusion: Patients with chemosensitive relapsed or refractory GCB and non-GCB-like DLBCL derive similar benefit from autologous HSC transplant. Figure Figure

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

Abstract Background: High-dose chemotherapy and hematopoietic stem cell (HSC) transplantation is curative in about 40% of patients with relapsed chemosensitive diffuse large B cell lymphoma (DLBCL). The international prognostic index obtained at the time of relapse (IPI-R) and the time from initial treatment to relapse (TTR) have been suggested in different series to be predictors of outcome after HSC transplantation. We explored the concomitant effect of IPI-R and TTR on therapeutic outcome in a modern and uniform cohort of patients with relapsed chemosensitive DLBC undergoing high-dose chemotherapy and peripheral HSC transplantation. Methods: Retrospective analysis of 80 consecutive patients treated at Mayo Clinic, Rochester, MN between 2001 and 2006 who received a peripheral HSC transplant (except for 3 patients who receive bone marrow) after being conditioned with BCNU, etoposide, cytarabine and melphalan (BEAM) for relapsed chemosensitive DLBCL. Results: Median follow-up of survivors was 31.4 months, Median age at the time of transplant was 62 (interquartile range 53–65; range 26–77), 65% were males; median number of prior chemotherapy regimens was 2. All patients had received at least one previous anthracycline-based regimen, 78% had been previously exposed to rituximab and all achieved at least CRu after the initial treatment. The median time from diagnosis to relapse was 19 months (interquartile range 10–43), 25% of patients had a high or high-intermediate IPI-R. The median survival from the time of transplant for patients with TTR>18 months v. ≤ 18 months was not reached and 50 months respectively (P=0.005). Median survival for patients with high or high-intermediate IPI-R at the time of relapse (IPI score ≥3) was 23.3 months and not reached for patients with low or low-intermediate IPI (P=0.01). These two factors were independent in multivariate analysis with RR for death of 1.8 (1.2–2.9; P=0.004) for TTR ≤ 18 months and 1.7 (1.1–2.5; P=0.017) for high or high-intermediate IPI and therefore combined in a prognostic system were patients with none (n=32), one (n=39) or two (n=9) of these prognostic factors had median disease free survival not reached v. 50 months v. 3.3 months (P=0.001) and overall survival not reached v. 50 months v. 5 months (P=0.0001) (Figure). Conclusion: TTR and IPI-R provide an easy and powerful prognostic system in patients with DLBCL undergoing modern high-dose chemotherapy with HSC support. Figure Figure

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

Abstract Background: In Hodgkin’s Disease (HD), G-CSF has been used to maintain maximum dose intensity by avoiding neutropenia and treatment delays. Pre-clinical data and anecdotal reports have shown an association between G-CSF administration and bleomycin pulmonary toxicity (BPT). This complication may cause access morbidity and have a detrimental impact on survival in this otherwise good prognosis population. We embarked on a retrospective analysis to determine if G-CSF increased the incidence of this complication or had an impact on outcomes. Methods: We reviewed all patients managed for HD at Mayo Clinic, Rochester, from January 1986 to February 2003. BPT was defined by the presence of pulmonary symptoms, bilateral interstitial infiltrates, and no evidence for an infectious etiology. Patients who received G-CSF support with one or more of their bleomycin-containing chemotherapy cycles were considered as being treated with G-CSF. Results: A retrospective review identified 141 patients with HD treated with a bleomycin-containing regimen. The mean age of patients at diagnosis was 34 years. Fifty four percent were males. The histology was nodular sclerosing in 85%. The Hasenclever index was 0 in 15%, 1 in 35%, 2 in 24%, 3 in 16%, 4 in 9%, and ≥ 5 in 1%. The G-CSF and non G-CSF groups were balanced for poor prognosis risk factors including Hasenclever index. Frontline chemotherapy included ABVD in 57%, MOPP-ABV(D) in 33%, COPP-ABV in 8%, BEACOPP in 3%, and Stanford V in 2%. Forty one percent of patients received radiation. The 5-year overall survival (OS) and progression free survival (PFS) for all patients were 87% and 79% respectively. G-CSF was administered to 52% (74/141) of patients. BPT occurred in 18% of patients (25/141). A higher rate of BPT was observed in patients receiving G-CSF, 26% (19/74) versus 9% (6/67) in non G-CSF patients (p=0.0141). In this population, G-CSF administration was the only factor associated with an increased risk of BPT. There was no difference between the two groups for BPT associated risk factors including radiation, bleomycin dose, renal insufficiency, smoking history, and underlying lung disease. Additionally these risk factors showed no correlation with the development of BPT. The mortality from BPT was 4.2% (6/141) in all patients and 24% (6/25) in patients who developed the pulmonary syndrome. Of the 6 patients who died acutely, 83% (5/6) received G-CSF. CR rates were equal between the G-CSF and non G-CSF groups at 92% and 94% respectively. Five year OS and PFS were equal between the G-CSF and non G-CSF groups at 82% versus 89% (p=0.473) and 74% versus 84% (p=0.152) respectively. Conclusion: G-CSF administration increases the incidence of pulmonary toxicity in HD patients treated with bleomycin based chemotherapy. The major impact was on treatment related morbidity as 5-year OS and PFS were equal between the G-CSF and non G-CSF groups.

Abstract Background: Bleomycin-containing chemotherapy regimens have become the mainstay of treatment in Hodgkin’s Disease (HD). The risk of bleomycin pulmonary toxicity (BPT) ranges from 0–46% and mortality rates range from 0–27%. Although there is no excepted standard treatment for BPT, corticosteroid treatment, withholding bleomycin from subsequent chemotherapy, and proceeding with a non-bleomycin containing regimen is the most common approach. We reviewed our experience to better delineate outcome and appropriate treatment in these patients. Methods: We reviewed all patients managed for HD at Mayo Clinic, Rochester, from January 1986 to February 2003. BPT was defined by the presence of pulmonary symptoms, bilateral interstitial infiltrates, and no evidence for an infectious etiology. Results: A retrospective review identified 141 patients with HD treated with a bleomycin-containing regimen. The mean age of patients at diagnosis was 34 years. Fifty four percent were males. The histology was nodular sclerosing in 85%. The Hasenclever index was 0 in 15%, 1 in 35%, 2 in 24%, 3 in 16%, 4 in 9%, and >5 in 1%. Frontline chemotherapy included ABVD in 57%, MOPP-ABV(D) in 33%, COPP-ABV in 8%, BEACOPP in 3%, and Stanford V in 2%. Forty one percent of patients received radiation. The 5-year overall survival (OS) and progression free survival (PFS) for all patients were 87% and 79% respectively. BPT was observed in 18% of patients (25/141). There was a significant decrease in OS, 63% in patients with BPT versus 90% in non-BPT patients at 5 years (p=0.0013). The mortality from BPT was 4.2% (6/141) in all patients and 24% (6/25) in patients who developed the pulmonary syndrome. These 6 patients all died within 9 months of their HD diagnosis from BPT. The Hasenclever index was 3 or less in 5 of these 6 patients. CR rates were equal at 91% and 93% in BPT and non-BPT patients respectively (p=0.299). Twenty two percent (31/141) of patients, either symptomatic or asymptomatic, had bleomycin omitted at any time from their regimen with no difference in OS or PFS, 83% versus 88% (p=0.369) and 82% versus 78% (p=0.853) respectively. The mean bleomycin dose was 84 mg/m2 (range of 20–180 mg/m2). Bleomycin dose had no impact on OS or PFS. In BPT patients subsequent non-bleomycin therapy included AVD 44%, MOPP-AV 31%, or no further chemotherapy 25%. The five-year OS was equal between the AVD and MOPP-AV groups at 91%. However in patients who received no further chemotherapy 5-year OS was 30% (p=0.0001). Conclusion: Bleomycin pulmonary toxicity (BPT) results in a significant decrease in OS survival at 5 years in patients being treated for Hodgkin’s Disease. In patients who do not die acutely from pulmonary toxicity both OS and PFS appear equal despite the omission of bleomycin.

Abstract Background: MDX-060 is a fully human anti-CD30 IgG1k monoclonal antibody that induces Fc-receptor mediated killing of CD30 expressing cell lines and inhibits the growth of CD30-expressing tumor cells in a murine xenograft model. We previously reported data from the first 31 patients in a Phase I/II open-label, dose escalation study of MDX-060 in patients with relapsed or refractory CD30+ lymphomas. The purpose of the study is to determine the safety and tolerability profile of the drug, and to preliminarily evaluate efficacy. We now update the findings of this study, including reporting the results of a new higher dose cohort. Methods: In the Phase I portion of the study, MDX-060 was administered intravenously at dose levels of 0.1, 1, 5, or 10 mg/kg weekly for 4 weeks to cohorts of 3–6 patients. In the Phase II portion of the study additional patients were administered MDX-060 intravenously at 10 (n=10) or 15 (n=17) mg/kg weekly for 4 weeks. Responses were assessed at month 2. Results: To date, MDX-060 has been administered to 48 patients; 40 with Hodgkin’s disease (HD), 6 with anaplastic large cell lymphoma (ALCL) and 2 with other CD30 positive lymphomas. MDX-060 has been well tolerated, and a maximum tolerated dose has not been identified. There were 2 drug-related serious adverse events, 1 patient in the 1 mg/kg dosing cohort had Grade 3 elevated liver transaminase, and 1 patient in the 15 mg/kg cohort had Grade 3 pneumonia/Grade 4 ARDS. Objective clinical responses have been observed in 5 patients. Two patients had complete responses; 1 patient with ALCL in the 1 mg/kg cohort lasting 100 days, and 1 patient with HD in the 15 mg/kg cohort lasting 117 days. Retreatment of the ALCL patient with MDX-060 resulted in a second complete response. Three patients had partial responses; 1 patient with HD in the 5 mg/kg cohort lasting 92 days, and 1 patient with HD in the 15 mg/kg cohort, which is continuing at 176 days. In addition, 1 patient with ALCL in the 15 mg/kg cohort had a partial response. Stable disease was observed in 17 patients following MDX-060 administration with a median duration of 132 days (range:15–387 days). Baseline soluble CD30 levels in the plasma were evaluated, but did not appear to correlate with clinical outcome. Conclusions: Preliminary evidence indicates MDX-060 to be well tolerated in heavily pre-treated patients with CD30+ lymphomas, with minimal toxicity, and with clinical activity observed at 1 mg/kg, 5 mg/kg and 15 mg/kg. Additional patients are being accrued at 1 mg/kg and 5 mg/kg to more accurately assess efficacy at these dose levels.