ALBERT

Description: Abstract 2891 Poster Board II-867 Thalidomide maintenance therapy after completion of induction therapy plus ASCT and also after conventional therapy yielded conflicting results with some trials showing improvement in overall survival and others not. This study evaluates the efficacy of Thalidomide plus Interferon a2b (Thal-IFN) in comparison to interferon a2b (IFN) as maintenance therapy in elderly pts with multiple myeloma. For induction therapy, 289 pts had been randomized to either Thalidomide-Dexamethasone or to Melphalan-Prednisolone; results of this part of the study had been reported previously (BLOOD, 113, 3435-3442, 2009). 137 pts who had completed 9 cycles of induction therapy and had achieved stable disease or better were eligible for maintenance treatment, and 128 (median age 72 years, range 54 - 86 years) had finally been randomized to either Thal (starting dose: 200mg/day) in combination with IFN-a2b (Schering-Plough, 3 Mega U, TIW) or IFN a2b (IFN) at the same dose/schedule only. All pts were scheduled for zoledronate 4mg, q 4 weeks. Median follow up from randomization to maintenance: 35 mos. Median duration of maintenance therapy: 13.2 mos and 8.3 mos for pts randomized to Thal-IFN or to IFN, respectively (logrank test p=0.20). Maintenance therapy with Thal-IFN resulted in an improvement in the depth of response from PR to VGPR or CR in 5 (8%) and with IFN in 2 (3%) pts, respectively. Progression-free survival (PFS) was significantly longer in the Thal-IFN (27.7 mos) compared to the IFN only maintenance group (13.2 mos), (HR, 0.55; 95% 95% CI, 0.36-0.86; log-rank test, p=0.0068). Analysis of PFS by either Thal-Dex or MP induction therapy showed a significantly shorter PFS in pts started on Thal-Dex and subsequently randomized to IFN maintenance only (7.8 mos, log-rank test, p=0.037). PFS was 27.7 mos in pts started on Thal-Dex followed by Thal-IFN, 20.2 mos in those with MP induction therapy followed by IFN, and 27.6 mos in pts with Thal-IFN maintenance after MP induction therapy. Overall survival (OS) was similar in both groups (Thal-IFN 52.6 mos and IFN 51.4 mos, HR: 0.93, 95% CI: 0.53-1.66, log-rank test; p=0.81). OS by induction therapy did not vary significantly between the four treatment groups (logrank test, p=0.99). No significant difference in OS was seen between pts younger than 75 years and those aged 75 years or older (logrank test, p=0.39). Survival after progression of disease tended to be longer in pts who received IFN maintenance therapy only compared to those started on Thal-IFN (HR: 1.75, 95% CI, 0.97 – 3.14, logrank test: 0.056), while OS was similar between both groups when analyzed from termination of maintenance therapy (HR: 1.20, 95% CI, 0.65 – 2.20, log rang test 0.57). Baseline scores of the EORTC QLQ C30 items general health (Thal-IFN, mean 56; IFN, mean 59) and overall quality of life (Thal-IFN, mean 58; IFN, mean 60) were markedly below the score obtained in an healthy population (mean 75.3 and 73.3 respectively), but did neither differ at baseline between both groups nor did they vary significantly during the course of the maintenance (statistics will be provided). Cytogenetic data were available in 66 pts. PFS tended to be longer in pts with adverse FISH findings [t (4; 14), t (14; 20) Del 17p and abnormalities of 1q21] compared to the standard risk group, but differences were not significant (median: 31.5 vs. 21.6 mos, HR: 1.69, 95% CI, 0.13 – 3.07, log-rank test 0.084). The median of OS was 72.3 mos in those with standard risk and 39.6 mos in those with high risk features (HR: 1.94, 95% CI 0.91-4.13, log rank test: 0.082). In multivariate analysis (Cox model) only Thal-IFN maintenance therapy was shown to correlate significantly with PFS (HR: 0.61, 95% CI: 0.39-0.89, p=0.04) while for poor performance status, low hemoglobin, and low albumin a statistically non-significant correlation with survival was noted. Hematologic toxicity was similar between both groups. Pts on Thal-IFN maintenance experienced significantly more neuropathy (p=0.0024), constipation (p=0.0007) and skin toxicity (p=0.0063) and increase in renal impairment (p=0.037). In addition, there was a tendency for more dyspnea (p=0.40) and more fatigue (p=0.11) in pts on Thal-IFN maintenance therapy. Other non-hematological toxicities were similarly distributed in both therapy arms. In conclusion, Thal-IFN maintenance therapy resulted in increased PFS compared IFN maintenance treatment only, but OS was similar between both groups. Disclosures: Ludwig: Celgene: Honoraria; Mundipharma: Honoraria; AMGEN: Honoraria; Ortho-Biotech : Honoraria; Janssen-Cilag: Research Funding; Roche: Honoraria. Hajek:Janssen-Cilag: Honoraria. Kuhn:Schering-Plough: Employment.

Description: Abstract 1295 Poster Board I-317 Introduction Recent animal studies suggest that measurable amounts of factor VIIa and antithrombin (AT) complexes are formed and accumulate following rFVIIa administration. The in vivo rate of inhibition has been reported to be faster than the un-stimulated in vitro reaction between AT and free rFVIIa and of the same order of magnitude as the rate determined in the presence of tissue factor. To study the impact of AT inhibition on the elimination of rFVIIa in humans, we measured the pharmacokinetics (PK) of rFVIIa and rFVIIa-AT complex formation in 10 hemophilia A or B patients. Patients and Methods The PK of single-dose rFVIIa 90 μg/kg (Novo Nordisk A/S) was evaluated in 10 severe FVIII- or FIX-deficient patients in a non-bleeding state. The plasma concentrations of FVIIa activity (FVII:C), FVII antigen (FVII:Ag), FVIIa-AT, D-dimer and F1+2 fragment were determined immediately before, and at 0.5, 1, 2, 4 and 6 hours following rFVIIa dosing. Results Significant amounts of FVIIa–AT complex were formed in vivo after rFVIIa administration, and reached a maximum of 5.4 ± 0.8 nmol/L [mean ±SD] at 2 hours following rFVIIa administration and declined to 4.4 ± 0.9 nmol/L at 6 hours, as compared to 0.1 ± 0.05 nmol/L at baseline. While the FVII:C PK data in this study were consistent with previous data, there was greater total body clearance (Cltot), a larger volume of distribution (Vdss) and a shorter plasma half-life (T1/2) of FVII:C relative to FVII:Ag (Table). No change in D-dimer was observed after the administration of rFVIIa, while a slight increase in F1+2 fragment levels to 258 ± 73 pmol/L was measured 4 hours after rFVIIa dosing, as compared to 141 ± 45 pmol/L at baseline. Conclusion A significant divergence between the clearance of rFVIIa, as determined by either FVII:C or FVII:Ag measurements, can be accounted for by AT complex formation. Inhibition by AT appears thus to have a significant impact on the elimination of FVII:C activity from the circulation when rFVIIa is administered at a therapeutic dose. Similar to animal data, the formation of the FVIIa-AT complexes in vivo was faster than anticipated from in vitro studies, indicating that the exposure to the vessel wall stimulates the FVIIa inhibition by AT. Analyses of coagulation parameters did not indicate induction of systemic coagulation. Disclosures Ezban: NovoNordisk A/S: Employment. Pelzer:NovoNordisk: Employment. Agerso:NovoNordisk: Employment. Petersen:NovoNordisk: Employment. Hedner:NovoNordisk: Employment. Carr:NovoNordisk: Employment.

Description: The proteasome is a multi-catalytic proteinase complex that is integral to intracellular proteolysis, and plays a key role in many cell functions. Targeting the proteasome with small molecule inhibitors has been validated as a rational therapeutic strategy for patients with relapsed/refractory myeloma with the approval of the first proteasome inhibitor, bortezomib (VELCADE®), for this indication. Additional studies are ongoing to better define the role of this agent in myeloma and other diseases, including non-Hodgkin’s lymphoma. Since bortezomib is a reversible proteasome inhibitor, we considered the possibility that an irreversible agent might have novel, potentially attractive properties. To begin to evaluate this hypothesis, we have studied the efficacy of a novel epoxomicin-related proteasome inhibitor, PR-171, which binds irreversibly and with a high degree of specificity in vitro to the chymotrypsin-like subunit of the proteasome. PR-171 was able to inhibit proliferation of both interleukin (IL)-6-dependent ANBL-6 and KAS-6 cell lines, as well as IL-6-independent models, including RPMI 8226 and U266 cells, in a concentration- and time-dependent fashion. IL-6-dependent cells generally displayed a greater sensitivity to PR-171-mediated effects than IL-6-independent cells. Experiments modeling the in vivo pharmacokinetics of proteasome inhibitors, with a one-hour pulse of drug followed by a washout, showed that PR-171 indeed inhibited the chymotrypsin-like activity of the proteasome without effects on other proteasome proteases. Inhibition of cell proliferation was associated with an induction of programmed cell death, as judged by the appearance of apoptotic oligonucleosome DNA fragments, as well as by the activation of caspase-3. This common effector caspase was activated by both the extrinsic and intrinsic pathways, in that both caspase-8 and caspase-9 were potently induced. Additionally, pulse treatment of PR-171 induced activation of c-Jun-N-terminal kinase, a key signaling molecule in stress-induced and proteasome inhibitor-induced apoptosis. Other members of the stress response-signaling pathway, including heat shock protein-70 and mitogen activated protein kinase phosphatase-1, were induced as well. Finally, both continuous and pulse treatment with PR-171 was also able to inhibit proliferation in freshly purified patient-derived multiple myeloma plasma cells, including isolates from patients with both newly diagnosed, previously untreated disease, as well as isolates from patients who had progressed on other standard therapies, including bortezomib. Importantly, PR-171 was active in both myeloma cell line models and patient-derived samples with chromosome 13 abnormalities. Taken together, these data indicate that PR-171 is a promising, novel proteasome inhibitor with activity against models of multiple myeloma, providing a rational basis for its translation into the clinic.

Description: Telomeres consist of repetitive DNA sequences and specific proteins, creating a specialized structure called the telosome. Unraveling the specific telomerase and telosome changes in leukemia is thought for providing new knowledge about oncogenesis, together with useful clinical markers and specific therapeutic targets. Here we measured telomerase activity (TA) by a quantitative TRAP assay in 57 human acute leukemia samples including 20 acute lymphoblastic leukemias (ALL) and 37 acute myeloid leukemias (AML). AML, TALL (n=7) and BALL (n=13) displayed significantly higher levels of TA than their normal counterparts [normal bone marrow CD34+ cells deriving from donors (BMCs, 17 samples) for AML, B-lymphocytes (n=20) for BALL and T-lymphocytes (n=24) for TALL; p

Description: Recently, the identification of the gain of function mutation JAK2V617F delivered important insights into the pathogenesis of BCR/ABL negative myeloproliferative disorders (MPD). JAK2V617F is detectable in more than 90% of polycythemia vera (PV) patients (pts) and in approximately 50% of pts with essential thrombocythemia (ET) or primary myelofibrosis (PMF), representing the genetic hallmark of BCR/ABL negative disease. However, about 30% of MPD pts lack the JAK2V617F mutation and previous studies on ET and PV demonstrated that clonality exceeds the percentage of V617F mutated cells. These findings suggest that additional genetic alterations are involved in the pathogenesis of MPD, in both JAK2 mutated and unmutated pts. To identify novel genetic aberrations and to determine whether specific lesions are associated with disease phenotype, genomic DNA from granulocytes of 72 MPD pts classified according to the WHO criteria was analyzed using high-resolution, genome-wide microarray techniques [disease, number analyzed, JAK2 mutation status: PMF, n=14, 9/14; post-ET MF, n=5, 3/5; post-PV MF, n=5, 5/5; PV, n=37, 37/37; ET, n=11, 11/11]. In a first approach, all cases were investigated by comparative genomic hybridization to 8k arrays (array CGH) with an average probe spacing of less than 1 Mb. While no genomic imbalances were found in ET, 11% of PV pts (n=4) exhibited large (〉10 Mb) deletions on 20q (n=2) or gains on 9p and 1q (n=1, each). In addition, small (

Description: Thalidomide-Dexamethasone (TD) is an active regimen both in patients (pts) with relapsing/refractory and in untreated pts with multiple myeloma (MM). Here we compare TD with standard Melphalan-Prednisone (MP) in previously untreated elderly pts with MM. 274 pts have been enrolled (median age: 72 yrs, stage I: 9 (3%), stage II: 84 (31%), stage III: 179 (65%). Pts were randomized to T 200mg/day and D 40mg, days 1–4 and 15–18 (on odd cycles) and days 1–4 (on even cycles) or M 0.25mg/kg days 1–4 and P2mg/kg days 1–4, q 4–6 weeks. T should be dosed up to 400mg/day, if feasible. Pts achieving response or SD were randomized to maintenance therapy either with T (≤200mg/day)-Interferon-a2b (IFN, 3Mega U, TIW) or IFN (3Mega U/TIW). Zoledronic acid (4mg) was administered monthly to all pts during the entire treatment period. Response is defined according Blade’s criteria, plus nCR defined as IF positive or 〉90%↓ in PP and VGPR defined as 〉75% ↓reduction in PP. 231 pts are evaluable per protocol. Best response to TD: CR (14%) nCR 17%, VGPR 17%, PR 21%, yielding an ORR (CR-PR) of 68%. Best response to MP: CR 7%, nCR 8%, VGPR 14%, PR 22%; ORR 51% (ORR in TD vs. MP p=.0044). Time to response and time to best response were shorter in the TD (6, 16 weeks, respectively) compared to the MP group (16, 25 weeks, respectively; p

Description: Abstract 3485 Poster Board III-422 Introduction The clinical response of hemophilia patients with inhibitors to bypassing agent therapy can be unexpectedly poor. Indeed, there are reports of “poor responders” who either require alternative treatment or dose escalation. The lack of correlation between routine coagulation assays or factor antigen/activity levels with clinical efficacy contributes to the problem in these patients. Analysis of the clotting characteristics of “poor responders” is limited. In a recent study of rFVIIa PK in 10 non-bleeding hemophilia A and B patients, we noted that four of the ten had a remarkable attenuated response as seen in whole blood assays. We report here a comparison of clotting parameters in the “poor responders” versus robust responders to rFVIIa infusion, and attempt to define what might be the source of the altered response. Patients and Methods Ten severe FVIII or FIX deficient patients in a non-bleeding state were infused with a single-dose of rFVIIa (90 μg/kg). Platelet contractile force (PCF), clot structure (CEM,MA,MCF), and clot formation time (FOT,R,CT) were analyzed in whole blood by Hemodyne HAS, Thromboelastography, and Rotation Thromboelastography before, and at 0.5,1,2,4 and 6 hours following rFVIIa dosing. Thrombin generation parameters (Tlag, Cmax) were measured in PRP by the Calibrated Automated Thrombogram. Plasma concentrations of FVII:C and FVII:Ag were measured at each time point. Patients with a clot formation time (FOT, R, CT) ≥ 15 minutes following rFVIIa dosing were termed “Poor Responders”. Results There were few inter-group differences in baseline clotting characteristics. The values for all parameters are provided in Table 1. FVII PK were not different between the Responders and Poor-Responders. This can be appreciated from the PK parameters listed in the table as well as from relative lack of variability for the composite rFVIIa levels seen for the entire group of 10 patients (Figure 1, panels 1 and 2). Both groups had similar FVIIa Cmax and total body clearance values. However, the responders made significantly stronger clots (PCF, CEM) as can be appreciated in table 1 and even more dramatically in panels 3 and 4 of figure 1. In these panels, the responders and poor responders are plotted as separate groups. Even though the groups are small (n=6 vs 4) the minimal response (both in magnitude and duration) to rFVIIa in terms of platelet function (PCF) and clot structure (CEM) is grossly apparent. All three whole blood assays revealed significantly shorter time to clot formation (R, FOT, CT) in the responders. However, the MA (TEG) and MCF (ROTEG), and thrombin generation parameters (Tlag, max) failed to show significant inter-group differences following rFVIIa dosing. Conclusions These data suggest that the differences observed between Responders and Poor Responders are not due to PK influences, but may be related to differences in the effects of thrombin on platelet function. It is possible that whole blood assays may serve as a tool to monitor the clinical effects of rFVIIa. Changes in clot stiffness were better characterized by CEM compared to MA and MCF. There was good correlation between FOT, R and CT parameters to detect onset of clot formation. The thrombin parameters were highly dependent on sample type and triggering agent and did not significantly vary between the two groups. Further studies are needed to clarify the clinical significance of these findings. Disclosures: Ezban: NovoNordisk A/S: Employment. Hedner:NovoNordisk A/S: Employment.

Description: Introduction: Kinesin spindle protein is a mitotic kinesin that is expressed only in proliferating cells and plays a key role in spindle pole separation, formation of a bipolar mitotic spindle, and thus in satisfaction of the mitotic checkpoint. Ispinesib (SB-715992) is a potent and selective inhibitor of kinesin spindle protein with a Ki of 0.6 nM, has cytotoxic activity at less than 10 nM in a spectrum of tumor cell lines, and disrupts the assembly of functional bipolar mitotic spindles. Methods: This study sought to examine whether spindle disruption by inhibition of kinesin spindle protein with ispinesib may have therapeutic potential in the treatment of multiple myeloma. Results: Ispinesib reduced cell viability in both interleukin-6-independent (RPMI 8226 and U266) and interleukin-6-dependent (ANBL-6 and KAS-6/1) models of multiple myeloma in a time- and concentration-dependent fashion. The average IC50 for ispinesib against these cell lines was 3.0 nM, 1.7 nM, 1.8 nM, and 1.8 nM, respectively. Cell cycle analysis showed that ispinesib induced growth arrest of myeloma cells with 4N DNA content (in M phase) within 24-hours. Two days after treatment at a 1 nM concentration, cells were able to recover from M phase arrest and resume normal cycling but, after exposure to 10 nM, treated cells could not escape M phase arrest, and instead entered apoptosis as determined by an increased sub-G1 population. Ispinesib was able to overcome resistance to melphalan in that the IC50 in melphalan-resistant RPMI 8226/LR5 cells (1.6 ± 0.2 nM) was comparable to that in parental RPMI 8226 controls (3.0 ± 0.9 nM). Similarly, ispinesib was also able to overcome dexamethasone resistance and bortezomib resistance. In regard to the latter, KAS-6/VR5 bortezomib-resistant cells (IC50 of 12.5 nM for bortezomib) retained sensitivity to ispinesib (IC50 1.6 ± 0.2 nM). Combination therapy with ispinesib and bortezomib in these cells resulted in enhanced levels of specific apoptosis (24%) that were greater than the sum of either agents alone (7% for ispinesib and 1% for bortezomib), suggesting synergy. Importantly, ispinesib was also active against freshly isolated CD138+ patient-derived multiple myeloma cells, while relatively sparing CD138− cells. Conclusions: Taken together, these studies demonstrate that kinesin spindle protein inhibition with ispinesib was able to induce growth arrest and apoptosis in myeloma cells, and overcome resistance to both conventional drugs and novel agents such as bortezomib. Moreover, the preferential activity against transformed plasma cells with sparing of normal bone marrow cells provides a strong rationale for translation of this agent into the clinic to combat relapsed/refractory multiple myeloma.

Description: Introduction: The proteasome is a large (~2.5 MDa), ATP-dependent, intracellular protease responsible for degrading ubiquitinated proteins as part of the ubiquitin-proteasome pathway. The immunoproteasome is a unique proteasomal variant with distinct catalytic subunits termed low molecular mass proteins that functions predominately in cells derived from hematopoietic precursors, and differs from the constitutive proteasome found in most other cells. Bortezomib (VELCADE®; Millennium Pharmaceuticals, Inc.) is a first-in-class proteasome inhibitor, which is approved for treatment of multiple myeloma patients who have received at least one prior therapy. While the overall safety profile of bortezomib is manageable and predictable, some toxicities, such as peripheral neuropathy, associated with bortezomib treatment can be dose-limiting. These toxicities may be due to the inhibition of all isoforms of the proteasome. Development of immunoproteasome-specific inhibitors (IPSIs) would allow for targeted therapy against cancers arising from hematologic origins, thereby sparing normal tissues, such as gastrointestinal and neurological tissues. Methods: We have identified several novel IPSIs, most notably IPSI-001, with selective activity against the immunoproteasome, which we therefore sought to characterize. Results: Expression of proteins associated with the immunoproteasome (low molecular mass protein-2; 11S Reg-α) was found primarily in cell lines of hematopoietic origin, while solid tumor cell lines exhibited expression of constitutive proteasome proteins (β5; 19S S6′). IPSI-001 exposure induced preferential inhibition of the chymotrypsin-like activity, the rate-limiting step of proteolysis, in hematologic cell lines over solid tumors. This inhibition was associated with an increase in ubiquitinated substrates, activation of c-Jun N-terminal kinase, and accumulation of Bax. IPSI-001 treatment led to preferential induction of apoptosis as evidenced by DNA fragmentation assays and cleavage of β-actin by caspase-3 into an apoptotic marker, fractin. Furthermore, IPSI-001 had potent chymotrypsin-like inhibitory activity in patient samples of chronic lymphocytic leukemia and acute myeloid leukemia. A dose-dependent decrease in proliferation was observed in additional patient samples of acute myeloid leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma treated with IPSI-001. Also, IPSI-001 exposure induced apoptosis in multiple myeloma and chronic lymphocytic leukemia patient samples. In an effort to increase the efficacy of IPSIs, a series of boronic acid analogs were made of several IPSIs. Conversion into the boronate analogs increased the potency by up to 1000-fold against the chymotrypsin-like activity of the immunoproteasome in vitro and in cellulo. Dose-dependent inhibition of proliferation was observed in ANBL-6, KAS-6/1, and Ramos cell lines, which was associated with induction of apoptosis. Conclusions: Studies are ongoing to characterize the specificity and molecular effects associated with IPSI-boronic acid derivatives exposure in immunoproteasome- and constitutive proteasome-containing cell types.

Description: Levels of Mcl-1, an anti-apoptotic Bcl-2 family member that plays an important role in MM cell survival, are tightly regulated by the proteasome, and recent data has demonstrated that proteasome inhibition with bortezomib leads to Mcl-1 accumulation, thereby attenuating its activity. Previously, we reported that inhibition of IL-6 signaling with the chimeric antibody, CNTO328, potentiated the anti-myeloma activity of bortezomib in IL-6-dependent cell line models of MM, and that the enhanced activity was associated with inhibition of downstream IL-6 signaling pathways and repression of bortezomib-mediated induction of the anti-apoptotic heat shock response. Based on our promising results with this combination and the role that IL-6 plays in Mcl-1 regulation, we have extended our preclinical studies and investigated whether CNTO328 could inhibit bortezomib-mediated Mcl-1 induction and how the bone marrow microenvironment affects the activity of the combination. Pre-treatment of the IL-6-dependent MM cell lines KAS-6 and ANBL-6 with CNTO328, but not an isotype control antibody, blunted bortezomib-mediated induction of anti-apoptotic Mcl-1L, and enhanced the cytotoxicity and pro-apoptotic activity of bortezomib. In contrast, CNTO328 did not attenuate bortezomib-mediated Mcl-1L accumulation in the IL-6-independent MM cell line RPMI 8226, nor did it enhance the cytotoxicity of bortezomib in these cells. In the presence of patient-derived bone marrow stromal cells, CNTO328 and bortezomib resulted in a greater reduction of cell viability than with either agent alone in both ANBL-6 and KAS-6 cells. Furthermore, although CNTO328 alone did not lead to an increase in apoptosis of ANBL-6 cells in the presence of bone marrow stroma, it significantly potentiated the pro-apoptotic activity of bortezomib in a synergistic manner at clinically achievable concentrations. As opposed to our cell viability data using the co-culture system, CNTO328 was not able to increase levels of apoptosis in KAS-6 cells either as a single agent or in combination with bortezomib, suggesting that CNTO328 may be inducing cell cycle arrest of KAS-6 cells in the presence of bone marrow stroma but is not able to potentiate the apoptotic activity of bortezomib. Finally, given the important role of IL-6 in glucocorticoid resistance and the additive preclinical and clinical activity of bortezomib and dexamethasone in MM, we evaluated the activity of dexamethasone in combination with CNTO328 and bortezomib in ANBL-6 cells. Whereas the combination of bortezomib (2.5 nM) and CNTO328 reduced viability to 58%, the addition of dexamethasone (10 mM) reduced viability further to 26%. Taken together, the above data demonstrate that CNTO328 enhances the activity of bortezomib in part by attenuating bortezomib-mediated Mcl-1 accumulation, and provide the rationale for clinical evaluation of CNTO328 and bortezomib, with or without dexamethasone, in patients with MM.