ALBERT

Description: Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) are abnormally sensitive to complement. Two membrane proteins, the C8 binding protein (C8bp) and the decay accelerating factor (DAF), which are expressed on normal cells, function to restrict lysis by homologous complement, and both of these proteins are absent from PNH erythrocytes. DAF is anchored to the plasma membrane on normal cells by a phosphatidylinositol linkage. The investigators found that a purified phosphatidylinositol-specific phospholipase C cleaved C8bp from the surface of normal lymphocytes and monocytes. This finding indicates that the abnormal complement sensitivity of PNH erythrocytes arises from a common defect, the inability to attach the phosphatidylinositol- containing anchor that is necessary for the membrane expression of both membrane complement regulatory proteins, the C8bp, and DAF.

Description: Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) are abnormally sensitive to complement. Two membrane proteins, the C8 binding protein (C8bp) and the decay accelerating factor (DAF), which are expressed on normal cells, function to restrict lysis by homologous complement, and both of these proteins are absent from PNH erythrocytes. DAF is anchored to the plasma membrane on normal cells by a phosphatidylinositol linkage. The investigators found that a purified phosphatidylinositol-specific phospholipase C cleaved C8bp from the surface of normal lymphocytes and monocytes. This finding indicates that the abnormal complement sensitivity of PNH erythrocytes arises from a common defect, the inability to attach the phosphatidylinositol- containing anchor that is necessary for the membrane expression of both membrane complement regulatory proteins, the C8bp, and DAF.

Description: A distinct feature of multiple myeloma (MM) is the tight interaction between malignant plasma cells and their bone microenvironment, creating a niche suitable for MM growth. In particular, MM cells inhibit osteoblast (OB) differentiation and stimulate osteoclast (OC) function, resulting in imbalanced bone remodeling and osteolytic bone disease. Here we studied a novel cytokine, activin A, identified from a broad range of cytokines, in the development of MM bone disease. We next asked whether activin A inhibition could restore bone balance and suppress tumor growth. Activin, a member of the TNF-α superfamily, is a pleiotropic cytokine involved in bone remodeling. Here, we observed, that MM patients with multiple osteolytic lesions had a 4-fold increase in activin A expression levels in bone marrow plasma compared to MM patients with one or less osteolytic lesions and non-MM patients (average 123.6 ± 136 vs 26.4 ± 21.4 vs 30.6 ± 25.1 pg/ml respectively, p

Description: Background: Bortezomib (VELCADE®, Vel) and lenalidomide (Revlimid®, Rev) are both highly effective agents in multiple myeloma (MM). Preclinical studies show Rev sensitizes MM cells to Vel and dexamethasone (Dex), suggesting combination therapy may enhance clinical activity. This phase 1 dose-escalation study aimed to determine MTD and activity of Rev-Vel +/− Dex combination therapy in patients (pts) with relapsed and/or refractory MM. Methods: Eight cohorts (≥3 pts each) were planned, with dosing of Vel 1.0 or 1.3mg/m2 (d 1, 4, 8, 11) and Rev 5, 10, 15, or 20mg (d 1–14), in 21-d cycles. Dex 40mg (on day of and day after each Vel dose) could be added in pts with PD. NCI CTCAE v3.0 was used for toxicity assessment; DLT was defined as any grade (G) ≥3 non-hematologic toxicity, G4 neutropenia for ≥5 d and/or neutropenic fever, or platelets 1 occasion despite transfusion. Response was assessed by modified EBMT criteria. Results: 28 pts were enrolled in cohorts 1–6 (Rev 5–15mg, Vel 1.0–1.3mg/m2) plus 10 additional pts at the MTD (Dose Level 5), including 12 with relapsed and 26 with relapsed and refractory MM (n=38). Among 25 men and 13 women, median age was 60yrs (range: 37–79), and median no. of prior therapies was 5 (range: 1–13), including 23 pts with prior SCT, 23 with prior Vel, 6 with prior Rev, and 36 with prior thalidomide (Thal). One DLT was observed in cohort 4 (Rev 10mg–Vel 1.3mg/m2; transient G3 hyponatremia). DLT was reached in cohort 6 (Rev 15mg–Vel 1.3mg/m2) with 1 episode of G3 HZV reactivation (successfully treated with acyclovir) and 1 G4 neutropenia (reversed with GCSF support and dose reduction). MTD was therefore declared at Rev 15mg–Vel 1.0mg/m2. In total, 5 pts had dose reductions for Vel, 6 pts for Rev, and 5 pts for both Rev and Vel. No significant (G≥3) fatigue or peripheral neuropathy has been seen. No anticoagulant prophylaxis was required and only 1 pt had DVT while on Rev alone. In 36 evaluable pts, the overall response rate (CR+PR+MR) is 58% (90% CI: 46%, 75%), including 6% CR/nCR (Table) after a median of 6 cycles (range: 4–17). Responses were durable (median 6 months, range: 1–26), and 11 pts remain on therapy beyond 1 year. Dex has been added in 14 pts with PD, resulting in PR/MR/SD in 10 (71%), with just 1 pt experiencing Dex-related G2 diarrhea and fatigue, which prompted discontinuation of therapy. Conclusions: Rev-Vel +/− Dex is well tolerated and very active with durable responses seen in pts with heavily pretreated relapsed and/or refractory MM, including pts who have had prior Rev, Vel, Thal and SCT. An MTD of Rev 15mg–Vel 1.0mg/m2 has been defined, with Phase 2 studies now ongoing in both newly diagnosed and relapsed/refractory MM. Best responses by Rev-Vel cohort (EBMT criteria) Cohort Rev-Vel dose Vel 1.0mg/m2 Vel 1.3mg/m2 1–2 Rev 5mg 2PR, 1MR 1CR, 2PR 3–4 Rev 10mg 1nCR, 2PR 2PR, 2MR, 1SD, 1PD 5–6 Rev 15mg 2PR, 4MR, 7SD, 1PD 2 PR, 5SD

Description: E1496 is a phase III trial designed to evaluate the ability of 2 years (yr) of maintenance rituximab (MR) to prolong progression-free survival (PFS) after CVP (cyclophosphamide 1 G/m2 day [d] 1, vincristine 1.4 mg/m2 [max = 2 mg] d 1, prednisone 100 mg/m2 d 1–5) chemotherapy in stage III–IV follicular grade 1 and 2 and small lymphocytic lymphoma. After CVP treatment to maximum response, (6–8 cycles), stable and responding patients (pt) were randomized to MR (375 mg/m2 weekly x 4) every 6 months x 4 or observation (OBS). Stratification factors included histology, response and residual disease after CVP. With 3-yr median follow-up, survivals (from time of randomization, one-sided logrank p values) for all pt (n=304) favored MR for PFS (p = 3 x 10–8; hazard rate {HR} = 0.38 [0.28;0.54, 95% confidence intervals]) and OS (p = 0.09; HR = 0.66 [0.36–1.22]). Because the large majority of pt have FL and because rituximab efficacy is notably greater in FL, we focused in this report on the 237 FL pt. Median age was 58 yr, 65% were stage IV, 64% had marrow disease, 64% had high tumor burden and 37% had high-risk disease by the follicular lymphoma prognostic index. PFS after randomization was significantly longer for MR vs OBS (p = 3 x 10-7; HR = 0.39 [0.27;0.57]). The estimated PFS at 4 yr (~4.5 yr after start of treatment) was 56% for MR vs 33% for OBS. Differences in PFS were significant within the predefined strata and the differences were most significant in favor of MR for pt with high initial tumor burden and minimal residual disease after CVP. Overall survival was superior for MR (p = 0.03; HR = 0.51 [0.25;1.04]. Estimated OS at 4 yr (~4.5 years after start of treatment) was 88% for MR vs 72% for OBS. Of 33 deaths, 21 occurred on the OBS arm. These data demonstrate that maintenance rituximab not only significantly delays disease progression in FL compared with OBS but that a substantial proportion of patients treated with MR remain disease-free at 4 years after the completion of CVP. These are the first data to strongly suggest a survival benefit with a therapy that includes rituximab and CVP and the first to strongly suggest a survival benefit with maintenance rituximab in FL.

Description: This multicenter, open-label, randomized phase 2 study evaluated 2 dose regimens of lenalidomide for relapsed, refractory myeloma. Seventy patients were randomized to receive either 30 mg once-daily or 15 mg twice-daily oral lenalidomide for 21 days of every 28-day cycle. Patients with progressive or stable disease after 2 cycles received dexamethasone. Analysis of the first 70 patients showed increased grade 3/4 myelo-suppression in patients receiving 15 mg twice daily (41% versus 13%, P = .03). An additional 32 patients received 30 mg once daily. Responses were evaluated according to European Group for Blood and Marrow Transplantation (EBMT) criteria. Overall response rate (complete, partial, or minor) to lenalidomide alone was 25% (24% for once-daily and 29% for twice-daily lenalidomide). Median overall survival in 30-mg once-daily and twice-daily groups was 28 and 27 months, respectively. Median progression-free survival was 7.7 months on once-daily versus 3.9 months on twice-daily lenalidomide (P = .2). Dexamethasone was added in 68 patients and 29% responded. Time to first occurrence of clinically significant grade 3/4 myelosuppression was shorter in the twice-daily group (1.8 vs 5.5 months, P = .05). Significant peripheral neuropathy and deep vein thrombosis each occurred in only 3%. Lenalidomide is active and well tolerated in relapsed, refractory myeloma, with the 30-mg once-daily regimen providing the basis for future studies as monotherapy and with dexamethasone.

Description: Background: Outcome of patients with immunocompetent primary central nervous system lymphoma (PCNSL) has improved since the introduction of high-dose (HD) methotrexate (MTX). However, treatment may be perturbed by considerable toxicity. Acute toxicity comprises hematologic, renal, hepatic, pulmonary events and mucositis while neurologic impairment is essentially restricted to long-term survivors. MTX acts via inhibition of 5,10-methylentetrahydrofolate reductase (MTHFR) as well as thymidylate synthase (TYMS) in target cells what eventually results in decreased DNA synthesis. Intracellular uptake of folates is mediated by the reduced folate carrier (RFC). Two common MTHFR single nucleotide polymorphisms (SNPs) affect enzyme activity: 677 C→T and 1298A→C. A 28 base pair (bp) tandemly repeated sequence in the TYMS enhancer region (5′-UTR) containing either 2 or 3 repeats alters enzyme activity as does a 6 bp deletion (del) polymorphism in the 3′-UTR of TYMS. An RFC SNP (80G→A) is known to influence folate and MTX transport. We hypothesized that these polymorphisms may be directly linked to toxicity in PCNSL patients receiving high-dose MTX. Patients and methods: Genomic DNA was prospectively collected from patients (pts) with immunocompetent PCNSL who received HD MTX 4 g/m² every 2 weeks after enrolment onto a German multicenter trial. They had to have histological confirmation of their disease as well as adequate organ and bone marrow function. Genotyping of the two MTHFR and the RFC SNPs was performed by melting point analysis using the Light Cycler® technology. Genotyping of the TYMS 28bp polymorphism was performed by conventional PCR followed by agarose gel electrophoresis. The TYMS 6bp del polymorphism was analyzed by PCR, restriction enzyme digest and agarose gel electrophoresis. Hematologic (Hb, WBC, ANC, platelets) and non-hematologic toxicity (renal, hepatic, pulmonary, mucositis) were assessed prospectively and correlated with polymorphisms with respect to presumable functional relevance. Results: 123 pts with a median age of 62 (range, 27 – 80) years received a total of 506 cycles of high-dose MTX. 39 of 119 evaluable pts (33%) experienced any severe (°3/4) toxicity. Incidence of severe toxicity was significantly different for pts with MTHFR 677 TT (95%) when compared to CC/CT genotype (48%; p=.0024). Not considering hematologic parameters, severe toxicity was even more strongly associated with homozygosity for 677TT (p=.0006). A moderate association with lower ANC was found for MTHFR C677T (p=.049) while °3/4 neutropenia was strongly associated with RFC 80 GG vs. AA/GA (p=.0057). In hand, lower WBC nadirs occurred in pts with RFC 80 GG vs. AA/GA (p=.035). Conclusion: We demonstrate for the first time that pharmacogenetic studies might identify PCNSL pts who are at risk for severe acute hematologic and non-hematologic toxicity when treated with HD MTX. Correlation of (late) neurotoxicity with polymorphisms of folate metabolizing genes, however, will require longer follow-up.

Description: The decay-accelerating factor (DAF), an integral membrane protein of approximately 75,000 mol wt that regulates the stability of the C3 convertases of the classical and alternative complement pathways, was initially isolated from normal erythrocyte stroma and used to prepare a polyclonal antiserum. Previously, anti-DAF antiserum has been used to immunoprecipitate DAF from surface-labeled normal erythrocytes and to document the deficiency of DAF on the surface of erythrocytes from patients with paroxysmal nocturnal hemoglobinuria, a condition in which erythrocytes express abnormal sensitivity to complement-mediated lysis. DAF has now been demonstrated by cytofluorography with anti-DAF F(ab')2 and fluoresceinated second antibody to be present on the surface of resting polymorphonuclear leukocytes (PMN), monocytes, lymphocytes, and platelets. Populations of PMN, monocytes, and platelets each exhibited a unimodal distribution of fluorescent staining, reflecting uniform cellular expression of DAF antigen, while the lymphocyte population had a skewed pattern of staining, indicating the heterogeneous expression of DAF antigen. For platelets, the shift in mean fluorescence channel observed with cytofluorographic analysis was minimal, but the presence of surface DAF on platelets was demonstrated by specific and saturable anti-DAF F(ab')2 binding. The DAF antigen, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of dithiothreitol- reduced anti-DAF immunoprecipitates prepared from surface-labeled, isolated populations of cells, presented a single polypeptide chain of approximately 84,000 mol wt for PMN and 75,000 to 80,000 mol wt for monocytes, T and B lymphocytes, and platelets. Thus, the complement regulatory protein, DAF, is expressed on the surface of all major types of circulating blood cells from normal donors.

Description: The presence of cytoplasmic lipid bodies in human eosinophils and the participation of these lipid bodies in the metabolism of arachidonic acid by human eosinophils have been studied. Lipid bodies, structures of roughly spherical shape and variable size within the cytoplasm, were identified with transmission electron microscopy by their shape and variable osmiophilia and by their lack of a limiting membrane. While generally absent from eosinophils of normal peripheral blood, lipid bodies were found in tissue eosinophils and in blood eosinophils from patients with eosinophilia. A role for lipid bodies in arachidonic acid metabolism was determined with eosinophils obtained from two eosinophilic patients. After incubation for 30 to 60 minutes with 3H- arachidonic acid, purified eosinophils took up 50% to 98% of the tritium label. By electron microscopic autoradiography, almost all tritium label was localized to lipid bodies. Only 3.6% of the cell- incorporated tritium label was free arachidonic acid, while 5.8% was neutral lipids and 66% was phospholipid. Thus, most of the tritiated arachidonic acid incorporated by human eosinophils was present in esterified form, predominantly as phospholipids, and almost all of the tritiated lipids were localized ultrastructurally to cytoplasmic lipid bodies. These results provide evidence that lipid bodies of human eosinophils have a role in the cellular metabolism of arachidonic acid.

Description: Abstract 2727 Poster Board II-703 INTRODUCTION: This study aimed to determine activity and safety of weekly bortezomib and rituximab in patients with relapsed/refractory Waldenstrom's Macroglobulinemia (WM). METHODS: Patients who had at least one previous therapy were eligible. All patients received bortezomib IV weekly at 1.6 mg/m2 on days 1, 8, 15, q 28 days x 6 cycles, and rituximab 375 mg/m2 weekly on cycles 1 and 4. Primary endpoint was the percent of patients with at least a minor response. RESULTS: Thirty-seven patients were treated. Majority of patients (78%) completed treatment per protocol. At least minimal response (MR) or better was observed in 81% (95% CI: [65,92]) with 2 patients (5%) in complete remission (CR)/near CR, 17 (46%) in partial response (PR), and 11(30%) in MR. The median time to progression was 16.4 months (95% CI, 11.4–21.1). Death occurred in 1 patient due to viral pneumonia. The most common grade 3 and 4 therapy related adverse events included reversible neutropenia in 16%, anemia in 11%, and thrombocytopenia in 14%. Grade-3 peripheral neuropathy occurred in only 2 patients (5%). The median event-free survival (EFS) is 12 months (95% CI, 11–20) with estimated 12 month and 18 month EFS of 49% (95% CI: [31, 67%]) and 38% (95% CI: [20, 56%]). The median overall survival has not been reached. CONCLUSIONS: The combination of weekly bortezomib and rituximab showed significant activity and minimal neurological toxicity in patients with relapsed WM. Disclosures: Ghobrial: Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Anderson:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Richardson:Millennium Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Johnson and Johnson: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Keryx: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Treon:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding, Speakers Bureau; Genentech: Honoraria, Research Funding, Speakers Bureau. Matous:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Cephalon: Membership on an entity's Board of Directors or advisory committees.