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The Kaposi sarcoma–associated herpesvirus (KSHV)–encoded latency-associated nuclear antigen (LANA) modulates viral and cellular gene expression, including interleukin 6 (IL-6), a growth factor for KSHV-associated diseases. LANA-driven IL-6 expression is dependent on the activator protein 1 (AP1) response element (RE) within the IL-6 promoter. We show that LANA activates the AP1 RE in a Jun-dependent fashion and that LANA enhances the transcriptional activity of a GAL4-Jun fusion protein. Coimmunoprecipitation studies documented a physical interaction between LANA and c-Jun in transiently transfected 293 cells as well as the KSHV-infected BCBL-1 primary effusion lymphoma (PEL) cell line. Taken together, these data indicate that LANA is a transcriptional coactivator of c-Jun. In addition, electrophoretic mobility shift assays demonstrated that LANA induces binding of a c-Jun-Fos heterodimer to the AP1 RE, but does not itself bind to the AP1 RE. RNA interference experiments confirmed that LANA activates the AP1 RE, stimulates binding of a c-Jun-Fos heterodimer to the AP1 RE, and induces expression of IL-6. These data indicate that LANA is a transcriptional coactivator of c-Jun, a function that may have implications for the pathogenesis of KSHV-associated diseases.

Abstract 3080 Background: Salvage therapeutic options are limited for multiple myeloma (MM) patients who relapse after autologous hematopoietic stem cell transplantation (ASCT). A second ASCT using a different conditioning regimen may provide long-term disease control. We report efficacy and safety of daily intravenous busulfan (IV Bu) conditioning given with bortezomib for second ASCT. Materials and Methods: In this prospective, multicenter, Phase IIa study, thirty MM patients who relapsed ≥ 1 year after initial ASCT and were candidates for second ASCT were enrolled at eleven centers in the US and Canada. Patients received a test dose of IV Bu (0.8 mg/kg) over 2 hours between Days -12 and -9 prior to ASCT. Pharmacokinetic (PK) analysis from test dose determined Bu exposure as area under the concentration-time curve (AUC). This analysis was used to determine individualized Bu PK-directed dosing for the conditioning regimen in order to achieve a total regimen AUC of 20,000 mM*min. IV Bu was administered over 3 hours once daily from Day -5 to Day -2. Confirmatory PK analysis was conducted in all patients on Day -5. Bu doses were adjusted on Days -3 and -2, if needed. Bortezomib (1.3 mg/m2 QD) was administered as an IV bolus injection on Day -1. Disease response was evaluated prior to the ASCT and at 3 and 6 months post-transplant, based on the International Myeloma Working Group uniform response criteria in 2006. Results: Patient Demographics: Median age at second ASCT was 59 years (range: 48–73). All patients had previously been treated with bortezomib (86.7%), thalidomide (46.7%), and/or lenalidomide (66.7%). All subjects underwent first ASCT with high-dose melphalan. Median time from first ASCT to second ASCT was 28.0 months (range: 12–119). The disease status at second ASCT was seven very good partial response (VGPR; 23.3%), twelve partial response (PR; 40.0%), two stable disease (SD; 6.7%); and nine progressive disease (PD; 30.0%). Safety: The most common grade 3 or 4 adverse event (CTCAE v3.0) was febrile neutropenia in 15 patients (50.0%), followed by stomatitis in 13 patients (43.3%), nausea in four (13.3%) and hypokalemia in three (10.0%). One transplant-related death due to pulmonary complications was reported for a patient with Parkinsonism on post-transplant Day 20. There was no instance of seizure, worsening neuropathy, or hepatic veno-occlusive disease (VOD) meeting the Baltimore criteria. Efficacy: 28 patients had evaluable disease response at least at one time point after second ASCT. Disease response at 3 months were two complete responses (CR; 6.7%), five VGPR (16.7%), four PR (13.3%), eight SD (26.7%), nine PD (30.0%), and two cases without evaluable assessment (6.7%). Two patients who achieved CR at 3 months had PD and VGPR prior to ASCT, respectively. Disease response at 6 months were one stringent CR (sCR; 3.3%), one CR (3.3%), four VGPR (13.3%), seven SD (23.3%), fourteen PD (46.7%), and three cases without evaluable assessment (10.0%). Median progression-free survival was 191 days, while median overall survival has not been reached yet. PK: 40.0% (n=12/30) of patients had AUC outside the expected range from pre-transplant test dose, 0.8 mg/kg of IV Bu: eleven cases with AUC 1,500 μM*min. If only weight was used (e.g. 3.2 mg/kg daily) to determine the dose without considering difference in individual busulfan metabolism, this 40% would have been dosed outside the total target AUC range. Based on test PK, IV Bu dosing for conditioning was individualized ranging between 1.99 and 4.73 mg/kg, which resulted in 93.3% of patients (n=28/30) falling between 16,000 and 24,000 μM*min as a total target AUC without any further dose alteration during conditioning. Only 2 patients (6.7%) needed dose reduction on Days -3 and -2. Mean Bu clearance for test dose and on Day -5 were comparable, 3.00 and 2.92 ml/min/kg, respectively. Conclusions: Disclosures: Stadtmauer: Millenium: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Off Label Use: IV busulfan and bortezomib-based conditioning regimen prior to transplant for myeloma. Freytes:Otsuka Pharmaceuticals: Research Funding. Shaughnessy:Otsuka: Honoraria, Speakers Bureau. White:Otsuka: Honoraria, Research Funding. Rodriguez:Otsuka: Consultancy, Research Funding, Speakers Bureau; Millennium: Research Funding, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; SOBI: Consultancy, Speakers Bureau. Sun:Otsuka Pharmaceutical Development & Commercialization, Inc.: Employment. Armstrong:Otsuka: Employment. Smith:Otsuka Pharmaceutical Development & Commercialization, Inc: Consultancy. Elekes:Otsuka Pharmaceutical Development & Commercialisation., Inc.: Employment. Kato:Otsuka Pharmaceutical Development & Commercialization, Inc.: Employment. Reece:Otsuka: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Millinneum Pharmaceuticals: Research Funding; Merck: Consultancy, Honoraria, Research Funding.

Abstract 813 Background: Published reports indicate that oral or intravenous (IV) busulfan in combination with cyclophosphamide and VP-16 (BuCyVP-16) is an effective conditioning regimen with acceptable safety profile for lymphoma patients prior to autologous hematopoietic stem cell transplantation (ASCT). Since the therapeutic window of Bu is narrow, it is important to standardize the systemic exposure during conditioning. Although the IV formulation of Bu eliminates the problem of variable drug absorption, unpredictable systemic exposure can still occur due to interpatient differences in Bu clearance. Therefore, tighter control of systemic Bu exposure using pharmacokinetics (PK) may lead to improved efficacy and further decrease in toxicity. The aim of this multi-center, single-arm, Phase II study was to prospectively evaluate the safety and efficacy of IV BuCyVP-16 regimen in lymphoma patients undergoing ASCT, after optimizing Bu exposure using PK-directed dosing. Methods: Patients with chemosensitive, relapsed or primary-refractory Hodgkin and B-cell non-Hodgkin lymphoma undergoing the first ASCT received a test dose of IV Bu (0.8 mg/kg) given as a 2-hour infusion 11 to 14 days before transplant. Bu exposure was determined as area under the concentration-time curve (AUC) using six serial blood samples after the test dose administration. Doses for the conditioning regimen were then calculated to result in total AUC (conditioning + test) of 20,000 mM*min. One-fourth of the resulting calculated Bu dose was given as a 3-hour infusion on Day -8, followed by a second, confirmatory PK analysis. The same daily Bu dose was administered on the next 3 days, unless the confirmatory PK analysis showed that this would result in total AUC outside the target range (>24,000 mM*min or Results: A total of 202 subjects with Hodgkin (n=65) and non-Hodgkin lymphoma (n=137) were enrolled from 32 centers in the US and Canada. 196 subjects had both test PK and confirmatory PK results. Test PK demonstrated that 36.2% (n=71) of the patients had exposure outside of expected range (1,250 μM*min ± 20%): higher AUC (>1,500 μM*min) in five patients (2.6 %) and lower AUC ( An early subset-analysis by age in June 2011 revealed that 4 of 18 subjects older than 65 years suffered TRM. Consequently, the protocol was amended to lower the inclusion age limit to 65. The final TRM at day 100 was 4/18 (22.2% [95%CI; 6.4–47.6%]) for patients older than 65 years and 5/184 (2.7% [95%CI; 0.9–6.2%]) for those 65 years old or younger. PK results indicated no extraordinary Bu exposure in TRM cases. The most frequently observed grade ≥ 3 adverse events (CTCAE ver. 3.0) were febrile neutropenia 55.1 % (Grade 3: 52.2%; Grade 4: 2.9%; no Grade 5), stomatitis 37.7% (all Grade 3 events) and nausea 9.7% (all Grade 3 events). No case of hepatic veno-occlusive disease (HVOD) meeting Baltimore criteria was reported. Conclusions: A pre-conditioning test dose estimated individual PK parameters and accurately predicted Day-8 PK in 95% of the subjects. This strategy personalized the dosing for optimal Bu exposure preventing the Bu overexposure or underexposure that would have occurred in over a third of patients. The toxicity of PK-guided IV BuCyVP-16 preparative regimen was low for patients younger than 66 years of age. This approach resulted in no incidence of HVOD. Disclosures: Costa: Otsuka: Research Funding. Off Label Use: Off label use of busulfan in non hodgkin and hodgkin lymphoma. Waller:Outsuka: Research Funding. Freytes:Otsuka Pharmaceuticals: Research Funding. Shea:Otsuka : Research Funding. Rodriguez:Otsuka: Consultancy, Research Funding, Speakers Bureau; Millennium: Research Funding, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; SOBI: Consultancy, Speakers Bureau. Sun:Otsuka Pharmaceutical Development & Commercialization, Inc.: Employment. Armstrong:Otsuka: Employment. Smith:Otsuka America Pharmaceuticals Inc: Consultancy. Elekes:Otsuka Pharmaceutical Development & Commercialization, Inc.: Employment. Kato:Otsuka Pharmaceutical Development & Commercialization, Inc.: Employment. Vaughan:Pierre Fabre: Honoraria.

Abstract 4509 BACKGROUND: Once multiple myeloma (MM) relapses after a first autologous hematopoietic stem cell transplantation (HSCT), long-term disease control is challenging because of limited therapeutic options. A second autologous HSCT is one of the options although the choice of conditioning regimens has not been thoroughly investigated. Past studies demonstrated that adding oral busulfan (Bu) to melphalan for conditioning regimen prior to a first HSCT resulted in better disease control of MM than melphalan single administration [Lahuerta et al, Haematologica. 2010;95:1913]. Nonetheless, the risk of hepatic veno-occlusive disease (VOD) hinders the use of oral Bu as a part of the conditioning regimen. Intravenous (IV) Bu eliminates the unpredictable bioavailability of the oral drug, whereas pharmacokinetic (PK)-directed dose optimization reduces inter-individual variability in the metabolism of Bu, resulting in decreased incidence of VOD and improvement in the control of hematologic malignancies [Andersson et al, Biol Blood Marrow Transplant 2002;8:477]. In addition, a proteasome inhibitor like bortezomib may have synergy with an alkylating agent for conditioning prior to HSCT in myeloma [Lonial et al, Clin Cancer Res 2010; 16: 5079], but the safety of a combination with Bu has not been investigated to date. Here we report results from a multicenter, prospective Phase 2 study to examine the pharmacokinetics of IV Bu and safety with bortezomib as a conditioning regimen for a second autologous HSCT. METHODS: 30 patients with relapsed MM who had a first autologous HSCT ≥1 year prior to the planned HSCT were enrolled from eleven centers in the US and Canada. Patients received a test dose of IV Bu (0.8 mg/kg) over 2 hours between days 12 and 9 prior to HSCT. Serial blood samples were drawn up to 360 min after the start of the infusion. A central laboratory measured Bu concentrations, determined Bu exposure as area under the concentration-time curve (AUC) using WinNonlin software, and recommended individualized PK-directed dosing in order to achieve a total regimen AUC of 20,000 μM·min. Using the patient-specific PK-directed dose, IV Bu was administered over 3 hours once daily from Day -5 to Day -2. In all patients, a second PK analysis was conducted on Day -5 to confirm the PK-directed dose. Bu doses were adjusted on Day -3 and Day -2, if needed. Bortezomib (1.3 mg/m2 QD) was administered as an IV bolus injection on Day -1. RESULTS: All 30 patients who enrolled in this study had been treated with high dose melphalan before a first HSCT. The median time from the first to second HSCT was 31.4 months (range 12.0 – 119.2 months). Disease status prior to the second HSCT was VGPR (n=6), PR (n=12), SD (n=2), and PD (n=10). Test PK revealed that 40.0 % (n=12/30) of the patients had doses outside of target range (1,250 μM·min +/− 20%), based on expected exposure using a fixed dose of 0.8 mg/kg. Specifically, infusion of the test dose resulted in 1,500 μM·min or higher AUC in 3.3% (n=1/30) and 1,000 μM·min or lower AUC in 36.7% (n=11/30) of patients. In order to achieve the target AUC, daily PK-directed dose of IV Bu when conditioning started on Day -5 ranged between 1.99 mg/kg and 4.73 mg/kg: lower than 3.2 mg/kg in 12 subjects and higher than 3.2 mg/kg in the remaining 18. Confirmatory PK on Day -5 demonstrated that mean Bu clearances were comparable to that from test PK: 3.03 ml/min/kg for test dose and 2.93 ml/min/kg for Day - 5. Consequently, using a test dose followed by PK-directed Bu dosing, 93.3% of patients (n=28/30) fell within the target range (AUC, 20,000 μM·min +/−20%). Only two patients (6.7%) needed subsequent daily dose adjustment on Day -3 and Day -2. No instances of VOD, seizure, or worsening neuropathy have been reported to date. One death was reported by Day 30 after transplant in a patient with Parkinsonism who died of pulmonary complications. CONCLUSION: This multicenter, prospective study reveals that a pre-transplant test dose PK allows accurate targeting of busulfan dosing. The conditioning regimen of bortezomib and IV busulfan with PK-directed dose optimization is well tolerated in patients with relapsed MM who undergo second autologous HSCT. Disclosures: Freytes: Otsuka Pharmaceuticals: Research Funding. Off Label Use: Busulfex and bortezomib as conditioning regimen for autologous transplant in patients with multiple myeloma. Yeh:Otsuka Pharmaceutical Development & Commercialization, Inc.: Research Funding. Shaughnessy:Otsuka: Consultancy, Honoraria, Speakers Bureau. White:Otsuka: Consultancy, Honoraria. Rodriguez:Millennium: Research Funding, Speakers Bureau; Otsuka: Research Funding. Yu:Otsuka Pharmaceutical Development & Commercialization, Inc.: Research Funding. Sun:Otsuka Pharmaceutical Development & Commercialization, Inc.: Employment. Armstrong:Otsuka Pharmaceutical Development and Commercialization, Inc.: Employment. Elekes:Otsuka Pharmaceutical: Employment. Kato:Otsuka Pharmaceutical Development & Commercialization, Inc.: Employment. Reece:Novartis: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Johnson & Johnson: Research Funding.