ALBERT

Description: Abstract 1741 Poster Board I-767 Background In contrast to most normal cells, cancer cells typically produce energy predominantly by glycolysis as demonstrated by O. Warburg more than 50 years ago. Methyljasmonate (MJ), a hormone produced by plants in response to biotic & abiotic stresses such as herbivory and wounding, has been shown to prevent the interaction of hexokinase (Hxk) and voltage dependent anion channels (VDACs), thereby significantly impacting the onset of glycolytic energy production. This may explain promising preclinical results observed with MJ against a variety of cancer cells, including myeloid leukemia and B-cell lymphoma cell lines. Methods and Results We tested the potential of MJ against Multiple Myeloma (MM) cells. We first evaluated the response of 16 different MM cell lines to 24 h of exposure to MJ concentrations of 0.5 – 3.5 mM using MTT assays. 15/16 of the MM cell lines tested displayed an IC50 of 〈 1.5 mM. In contrast, HS-5 stroma cells and peripheral blood mononuclear cells (PBMCs) did not respond to that MJ concentration, and even at a concentration of 2.5 mM MJ showed a maximal reduction of cell viability of 40%. Similarly to MM cell lines, purified CD138+ primary tumor cells of 3 MM patients displayed an IC50 of 〈 1.5 mM, suggesting that the differential sensitivity of MM vs. normal cells to MJ is not restricted to cell lines, but is also observed with primary tumor cells. Importantly, neither co-culture with HS-5 stroma nor IL-6 protected MM cells against MJ. Cell death commitment assays revealed that 1h exposure of 1.5 mM MJ induced cell death. Annexin V/PI FACS analysis of MJ-exposed MM cells showed that the cell death is mainly driven by apoptosis, evidenced by cleavage of caspases 3, 8 and 9 as well as of PARP. However, pre-incubation of MM cells with specific caspase inhibitors such as 10 mM of AC-DEVD-CHO, Z-IETD-fmk, Z-LEHD-fmk or 50 mM of Z-VAD only minimally protects the cancer cells from MJ exposure. Therefore, the impact of the MJ is not solely due to caspase triggered proteolytic cascades. Measurements of cellular ATP content by cell titer glow (CTG; Promega, Madison, WI) assay showed rapid depletion of ATP triggered by MJ action in sensitive MM cell lines. Additionally, we observed that 1 h exposure to 2 mM MJ modulated signaling pathways including IRS1/PI3K/AKT, MEK1/2, as well as Stat3 and JNK. FACS-based cell cycle analysis after propidium iodide staining did not show cell cycle arrest, but rather a rapid transition of cells to G0/G1 No correlation of sensitivity of MM cell lines and the number of mitochondria per cancer cell, as determined by Mitotracker Green (Invitrogen, Carlsbad, CA) -based flow analysis, was observed. We next examined if MJ exhibits either significant antagonism or synergy with established or novel anti-MM agents, including Bortezomib, Lenalidomide, Doxorubicin, Rapamycin or Dexamethasone, but discovered neither. However, MJ displayed synergy when combined with 2-Deoxyglucose. Finally, MJ was tested in vivo in scid/nod mice irradiated with 150 rads, injected with 1× 106 MM1S cells, and then, treated at 500 mg/kg by IP administration on a 5 days on / 2 days off schedule starting two weeks after tumor cell injection, There was an overall survival advantage of MJ-treated animals over the respective controls, with all treated mice (n=10) still alive but 6/10 control mice dead after 27 d. Conclusions Based on its rapidity of anti-MM action, favorable safety profile in preclinical models, distinct pattern of molecular sequelae, and compatibility with established anti-MM agents, MJ represents a promising investigational anti-MM agent. Disclosures Laubach: Novartis: Consultancy, Honoraria. Richardson:Millennium: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Anderson:Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mitsiades:Novartis Pharmaceuticals: Consultancy, Honoraria; Milllennium: Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals : Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: Patents & Royalties; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis Pharmaceuticals: Research Funding.

Description: Abstract 1737 Poster Board I-763 We previously reported the efficacy and B-cell selectivity of the natural product silvestrol in acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL), using both primary cells and B-cell lines. We also showed that silvestrol inhibits translation, resulting in rapid depletion of the short half-life protein Mcl-1 followed by mitochondrial damage and apoptosis. Cencic et al. reported that silvestrol directly blocks translation initiation by aberrantly promoting interaction of eIF4A with capped mRNA (PLoS One 2009; 4(4):e5223). However, the loss of Mcl-1 in breast and prostate cancer cell lines is delayed relative to what we observe in B-leukemias (48 hr vs. 4-6 hr in CLL and ALL cells). Additionally, silvestrol does not reduce Mcl-1 expression in normal T-cells to the same extent that it does in B-cells, potentially explaining in part the relative resistance of T-cells to this agent. We therefore investigated cell-type differences, as well as the importance of Mcl-1, in silvestrol-mediated cytotoxicity. We incubated the ALL cell line 697 with gradually increasing concentrations of silvestrol to generate a cell line (697-R) with resistance to 30 nM silvestrol (IC50 of parental 697 〈 5 nM). No differences between 697-R and the parental line were detected upon detailed immunophenotyping. However, cytogenetic analysis revealed a balanced 7q;9p translocation in 697-R not present in the parental 697 cell line that may be related to the emergence of a resistant clone. We also detected no difference in expression of multi-drug resistance proteins MDR-1 and MRP, which can contribute to resistance to complex amphipathic molecules such as silvestrol. In contrast, we found that baseline Mcl-1 protein expression is strikingly increased in 697-R cells relative to the parental line, although these cells still show similar percent-wise reduction in Mcl-1 upon re-exposure to 80 nM silvestrol. To investigate whether this resistance to silvestrol is reversible, 697-R cells were maintained without silvestrol for 6 weeks (∼18 passages). During this time, viability remained near 99%. Cells were then treated with 30 nM silvestrol. Viability was 94% at 48 hr post-treatment and returned to 99% within a week, while parental 697 cells with the same treatment were completely dead. Baseline Mcl-1 levels remained elevated in 697-R even with prolonged silvestrol-free incubation. These results indicate that the resistance phenotype is not rapidly reversible, as is seen with transient upregulation of multi-drug resistance or stress-response proteins. Additionally, silvestrol moderately induces the transcription of several pro-apoptotic Bcl-2 family members and results in elevated levels of these proteins despite its translation inhibitory activity. Interestingly, no such activity is detected in silvestrol-treated normal T-cells. Together, these results support the hypothesis that in B-cells, silvestrol induces cell death by altering the balance of pro- and anti-apoptotic factors, and that increased Mcl-1 protein can force the balance back toward survival. This work further underscores the importance of Mcl-1 in silvestrol-mediated cytotoxicity. We are now investigating the mechanism of Mcl-1 upregulation in 697-R cells to identify a factor or pathway that can be targeted therapeutically to circumvent resistance. Silvestrol is currently undergoing preclinical pharmacology and toxicology investigation by the U.S. National Cancer Institute Drug Development Group at the Stage IIA level to facilitate its progression to Phase I clinical testing. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1730 Poster Board I-756 The sonic hedgehog (SHH) pathway, associated with proliferative stem cell niches of many organs, is frequently deregulated in diverse cancers. We have previously shown that the SHH pathway augments the survival of tumor cells by inducing antiapoptotic molecules including bcl-2 and provides resistance to a number of conventional cancer therapies. Aberrant activation of the SHH pathway has been associated with activation and maintenance of lymphoid malignancies. Additionally, our recent data indicates that p63, a p53 family member and an important marker of stem cells has multiple interacting nodes with the SHH pathway. Based on these observations, we hypothesized i) that there is crosstalk between the SHH and p53/p63 networks in cells and, ii) inhibition of the SHH pathway with simultaneous activation of the p53 pathway would result in increased cell death in cancer cell lines of lymphoid origin. Using SDS-PAGE followed by immunoblotting and RT-PCR we surveyed a panel of 18 leukemia/lymphoma cell lines for components of the SHH pathway and the p53/p63/p73 network. Robust SHH pathway expression was observed in 15 of the 18 cell lines examined. Interestingly, in p53 deficient cell lines there was an increase in p63/p73 expression as compared to cell lines with wild type (WT) p53. A set of the previously analyzed diffuse large B- cell lymphoma (DLBCL) cell lines were selected and were representative of both p53 deficient and WT cell lines and also the activated B-like DLBCL (ABC) and germinal center B-like DLBCL (GCB) subgroups. These cells were treated with cyclopamine, an inhibitor of the SHH pathway and/or nutlin, an HDM2 antagonist. Cell viability (MTS assay) was measured using both compounds at various drug concentrations and time points. In addition we also investigated the effects of the drugs individually and in combination on components of the p53/p63 and SHH axes and their targets. Our findings suggest that treatment of p53 (WT) cell lines with a combination of nutlin and cyclopamine results in reduced cell survival than treatment with either drug alone and at lower drug concentrations and that the p53 status of the cell line may be more important in determining therapeutic response to the selected compounds. In addition the p53/p63 pathway may have a novel role in regulation of specific components of the SHH pathway in cells of lymphoid origin. In conclusion, these observations provide proof of concept that a combinatorial therapeutic approach, targeting both the p53 and SHH axes would provide a more robust and favorable response in large B cell lymphomas. Acknowledgments This work was supported in part by the The Mehta Family Foundation Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1743 Poster Board I-769 Background Molecular targeting drugs, all-trans retinoic acid (ATRA)and arsenic trioxide (ATO), have major advances in the treatment of acute promyelocytic leukemia (APL). However, resistance to these drugs has been also observed in clinical practice. ATRA acts as a ligand for retinoic acid receptor alpha (RAR) and restores the aberrant transcription repression by PML-RARA fusion protein in APL cells. Previous reports demonstrate that amino-acids substitution, resulting from genetic mutations, in ligand binding domain (LBD) of RARA region of PML-RARA were closely related to drug resistance to ATRA therapy. In contrast, for ATO therapy, the molecular mechanisms of the effectiveness and also the resistance are still unclear. Here we identified a PML-RARA that holds double genetic missense mutations in RARA and PML regions, respectively, from an APL patient, who showed clinically resistance to ATRA and ATO therapy. These mutations were observed as his disease progression, and we are interested in the relationship between these mutations with drug resistance to ATRA and/or ATO. Aims Analyses of the molecular and clinical significance of the double missense mutations of PML-RARA for disease progression and resistance to ATRA and ATO therapy. Results Eight APL patients were treated with ATO in Nagoya University Hospital, Japan, during ∼5 years from Apr. 1, 2000 to Dec. 31, 2004. One out of 8 patients showed clinically ATO resistance. The patient showing ATO resistance firstly diagnosed as APL (M3 variant) from cytogenetic and chromosomal analyses, and complete remission was obtained after combination chemotherapy with ATRA. Molecular CR was confirmed by RT-PCR analysis, but after 3 month from the induction therapy, ATRA-resistant relapse was observed. After treatment with ATO therapy, response was observed, but the effectiveness was gradually decreased, resulting finally into the resistance. The patient died of disease progression. During his 7 years clinical course, leukemia cells were harvested repeatedly from his bone marrow and peripheral blood. RT-PCR using the total RNA from his tumor cells followed by DNA sequencing was performed, with the result of PML-RARA fusion gene with the bcr3 breakpoint in the intron 3 of PML. When using the tumor cells that were harvested at his terminal stage, a missense point mutation in the LBD of the RARA region of PML-RARA was confirmed. Furthermore, missense point mutation in the PML-B2 domain was also confirmed in the same cDNA clones. Interestingly, these mutations were not observed in the leukemia cells obtained at the onset. These mutations were analyzed in each sample that was obtained as his disease progressed, and some correlation between disease progression and/or the drug resistance and the timing of appearance of these two mutations were suggested. These mutated fusion transcripts were cloned into expression vectors, and we are now analyzing the function relating to the drug resistance and disease progression. Conclusions Double genetic missense mutations in the RARA-LBD and PML-B2 of PML-RARA were confirmed in ATRA and ATO resistant patient. These genetic mutations were confirmed in the leukemia cells during his disease progression, and the relationship between those mutations and drug resistances were suggested from the clinical features. Mutations in the PML-B2 domain has not been reported previously, thus, it may be important to show whether this type of mutations are related to the drug resistance, especially to ATO therapy. Disclosures Kiyoi: Novartis Pharma Co. Ltd.: Research Funding; Kyowa Hakko Kirin Co. Ltd.: Consultancy. Naoe:Kyowa Hakko Kirin Co., Ltd. : Research Funding; Chugai Pharmaceutical Co.,Ltd.: Research Funding; Wyeth K.K.: Research Funding.

Description: Abstract 1715 Poster Board I-741 Introduction Gene polymorphism coding for drug-metabolizing enzymes may cause individual differences in effectiveness and toxicity of many medications, including cytostatics. Although intensified treatment resulted recently in better prognosis of leukemia, many adverse effects are still observed during therapy resulting from nonspecific activity and narrow therapeutic index of anti-cancer drugs. The objectives of the study: 1. Determining the frequency of selected allele variants: CYP2C9, CYP2C19 in population of Polish children treated for acute leukemia. 2. Analysis of the role of CYP2C9 and CYP2C19 in cyclophosphamide metabolism. 3. Determininig the influence of carrying selected CYP2C9 and C19 polymorphism on cyclophosphamide metabolism in form of increased adverse reaction risk (according to WHO score), relapse and other infringements in treatment protocols and procedures following the use of standard doses of alkylating agents. Patients: 184 patients (106 boys, 78 girls) were examined, age between 1 and 18 (median age 7 years, average age 7.64 years, SD=5.3) treated for acute leukemia (ALL:167, AML:17) at various time points: on diagnosis, during cytostatic treatment and after therapy cessation. All patients were treated with current uniform treatment protocols and procedures. Adverse reactions in each child was recorded according to WHO toxicity scale, during the entire length of observation period and in particular stages (6 periods). Methods Either bone marrow or peripheral blood samples were used for the analyses. DNA isolation procedure was carried out with the use of standard tests. CYP2C9*2,*3 and CYP2C19*2 allelic variant determination was carried out with real-time PCR with double-marked probe hydrolysis with the use of Pre-Developed TaqMan Assay Reagents for Allelic Discrimination sets. PCR products fluorescence was carried out on 7900HT Fast Real-Time PCR device. Results 1. For CYP2C9, IM phenotype was present in 22.3% patients (n=41) and PM phenotype in 7.6% cases (n=14), the most frequently detected polymorphic variant being CYP2C9*2 (75.3%). In case of CYP2C19, IM phenotype occurred in 22.3% patients (n=41) and PM phenotype in 7.1% (n=13). 2. Toxicity risk analysis in patients with acute leukemia did not reveal statistically significant differences depending on CYP2C9 genotype. In patients with CYP2C19*2 allelic variant, statistically lower level of alkaline phosphatase (p=0.013) was observed during the entire treatment and in particular stages (I: p=0.0096; II: p=0.022; III: p=0.038; IV:p=0.0024; V:p=0.044). Lower alkaline phosphatase levels were observed only in patients with ALL (p=0.026). Lower transaminase levels after completion of intensive treatment phase were observed in patients with CYP2C19 mutations (GOT, p=0.044). In patients with CYP2C19 and CYP2C9 polymorphic variants no dependence of risk of adverse reactions with correlation to cyclophosphamide and its dose was observed. 3. In patients with CYP2C9 and CYP2C19 polymorphism no significant relation was discovered. 4. Death risk was not relevant to CYP2C9 or CYP2C19 genotype. Conclusions 1. Occurrence of slow metaboliser phenotype for CYP2C9 and CYP2C19 is more frequent in patients with acute leukemia than in the untreated population. 2. CYP2C9 and CYP2C19 cytochromes are involved in cyclophosphamide metabolism but do not play a major role in drug metabolism. 3. CYP2C9 and CYP2C19 single nucleotide polymorphism is correlated neither with an increased risk of adverse reactions or death during therapy, nor with a relapse of acute leukemia in children. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1720 Poster Board I-746 Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor (1), was found to enhance NK cell mediated antitumor activity by upregulating the surface expression of NKG2D ligands MICA and MICB on hepatoma cells (2). In contrast, VPA was found to impair NK cell antitumor activity by downregulating the surface expression of NKp30 and NKp46 on NK cells by blocking NFkB activation (3). Recently, we also found that VPA efficiently upregulates the surface expression of MICA, MICB, and ULBP family ligands on human osteosarcoma and neuroblastoma cells, but downregulates NKG2D surface expression on human NK cells. Similarly, the HDAC inhibitor MS275 efficiently upregulates expression of MICA, MICB, and ULBP family ligands on human osteosarcoma and neuroblastoma cells, but surprisingly, MS275 significantly upregulates NKG2D expression on NK cells. In addition, we found that VPA inhibited the cell growth of osteosarcoma cell lines more readily than MS275. To investigate the mechanism of these differing effects of VPA and MS275, we compared the effect of these HDAC inhibitors on Signal Transducer and Activator of Transcription (STAT) signaling, which are important signaling pathways in NK cells and many cancers. Here we show that VPA is a potent inhibitor of STAT3 phosphorylation at the tyrosine 705 residue in NK cells, whereas MS275 has no effect on STAT3 phosphorylation (Figure A). Moreover, this inhibition is highly specific, as VPA has no effect on STAT1 and STAT5 phosphorylation (Figure B). To further confirm VPA-mediated STAT3 inhibition, we detected STAT3 expression and the degree of tyrosine phosphorylation affected by VPA in a panel human osteosarcoma cells. We found that constitutive STAT3 phosphorylation is present in osteosarcoma SaOS2 and LM7 cells, but not in OS187 cells, which corresponded to the stronger growth inhibition caused by VPA in SaOS2 and LM7 cells compared to OS187 cells. These results are for the first to show that VPA, a HDAC inhibitor, is also a selective STAT3 inhibitor. VPA can upregulate NKG2D ligand expression on the tumor cell surface by HDAC inhibition, but may downregulate NKG2D expression on the NK cell surface by STAT3 inactivation. The antitumor activity of VPA may be via a mechanism of both HDAC inhibition and STAT3 inactivation. The knowledge of potent STAT3 inhibition by VPA is important for identifying potential new clinical applications for this drug. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1719 Poster Board I-745 Acute myeloid leukemia (AML) is an aggressive disease with heterogeneous genetics and variable prognosis. The presence of an internal tandem duplication within the FLT3 gene (Flt3 ITD) is a marker for poor prognosis and has been linked to anthracycline resistance in cell lines and primary patient samples in vitro. The effect of this mutation on response to chemotherapy in vivo has not been examined and its effect on response to cytarabine is not known. In this study we use a genetically defined mouse model of AML to examine the effects of the Flt3 ITD on response to cytarabine and the anthracycline doxorubicin in vitro and in vivo. In vitro the Flt3 ITD conferred resistance to doxorubicin and the combination of doxorubicin and cytarabine but sensitivity to cytarabine alone in comparison to the identical leukemia without the Flt3 ITD. In vivo the presence of the Flt3 ITD provided an advantage in leukemic engraftment and accelerated disease onset. This advantage could be partially reversed by treatment of the animals with cytarabine but not by treatment with doxorubicin. Surprisingly, in vivo the Flt3 ITD conferred a marked increase in sensitivity to cytarabine when compared to the parental leukemia without this mutation. In contrast to the parental leukemia, the addition of doxorubicin to cytarabine provided no advantage over cytarabine alone. When the DNA damage response was assessed the presence of the Flt3 ITD resulted in an increase in the levels of p53 following treatment with either doxorubicin or cytarabine. Induction of the p53 target genes p21 and MDM2 was also increased. Surprisingly, the Flt3 ITD had no effect on disease onset or chemotherapy response in vitro or in vivo in the setting of p53 null AML. These data when taken together demonstrate that the Flt3 ITD confers a mixed sensitivity and resistance to standard chemotherapy and provides an engraftment advantage in a manner that depends on an intact p53 allele. This may at least in part explain the rarity of dual p53 null and Flt3 ITD positive AML. Furthermore, these data suggest that patients with Fl3 ITD positive AML may benefit more from treatment with high dose Ara-C then with combinations containing an anthracycline. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1705 Poster Board I-731 The CD16 (FcgammaRIIIa) has a functional polymorphism at position 158. CD16 with valine at position 158 (V) has higher affinity for human IgG1 than does CD16 with phenylalanine at position 158 (F). Follicular lymphoma patients with V have a higher response rate to single agent rituximab. In vitro, NK cells from subjects with V are activated at lower rituximab concentrations than NK cells from subjects with F. Little is known about the in vivo effects of rituximab on NK activation, and the influence CD16 polymorphisms have on that activation. The current studies were designed to explore the relationship between CD16 polymorphisms and NK cell activation in patients receiving rituximab for lymphoma. Subjects included 17 lymphoma patients with a variety of histologies scheduled to receive rituximab at the standard dose (375 mg/M2). Some had received prior treatment with rituximab, but not within the prior 6 months. Subjects did not receive recent or concurrent prednisone or immunosuppressive drugs. Blood was obtained immediately prior to initiation of the rituximab infusion, and 4 hours after the initiation of the infusion. CBC, NK cell number, NK cell CD16 and NK cell CD54 were determined at time 0 and time 4hrs, and changes in each parameter determined. All subjects were genotyped related to the CD16 position 158 polymorphism. Genotype testing revealed 9 subjects were FF, 7 were VF, and 1 was VV. NK cell numbers for the group as a whole were lower 4 hours after initiation of therapy when compared to pre-therapy. Phenotypic changes consistent with NK cell activation at 4 hours included downmodulation of CD16 and upregulation of the adhesion molecule CD54 (ICAM-1). Subset analysis demonstrated these changes were largely limited to subjects with VF/VV. No significant change in NK cell number or activation was seen in those with FF. There was no clear correlation between infusion reactions and genotype or NK activation. Ongoing analyses include evaluation of complement, rituximab and cytokine levels. All (n=17) p value VF/VV (n=8) p value FF (n=9) p value p value FF vs VF/VV D NK Cell # Post Rx/PreRx 0.72 +/- 0.81 0.05 0.41 +/- 0.31 0.008 1.00 +/- 1.03 0.62 0.31 D NK CD54 Post Rx/PreRx 2.14 +/- 1.69 0.0006 3.05 +/- 2.1 0.008 1.33 +/- 0.53 0.11 0.01 D NK CD16 Post Rx/PreRx 0.9 +/- 0.17 0.02 0.86 +/- 0.22 0.11 0.94 +/- 0.10 0.15 0.56 We conclude that NK activation occurs within 4 hours of infusion of rituximab. A decrease in the number of circulating NK cells occurs within 4 hours and is consistent with trafficking of NK cells out of the circulation. Subset analysis demonstrates changes in NK cell number and phenotype are statistically significant in subjects with the high affinity CD16 phenotype, but not the low affinity phenotype. This finding may help explain the enhanced efficacy of rituximab that has been observed for patients with high affinity CD16. Disclosures Link: Genentech: Consultancy.

Description: Abstract 1701 Poster Board I-727 Introduction There are still controversies about the impact of erythropoiesis stimulating agents (ESA) on survival of patients with malignancies. The Groupe d'Etude des Lymphomes de l'Adulte (GELA) has conducted a multicentric prospective randomized phase III study to evaluate efficacy and safety of Darbepoetin alfa (DA) in elderly patients with DLBCL treated by immunochemotherapy. Here, we report the results of the planned interim analysis, held after the inclusion of the first 202 patients, with a median follow-up of 24 months. Patients and methods: Patients between 60 and 80 years old with DLBCL and aaIPI ≥ 1 were eligible. They were firstly randomized between two immunochemotherapy regimens combining Rituximab and CHOP given every 2 (R-CHOP14) or 3 weeks (R-CHOP21) for 8 cycles. They were subsequently randomized between an investigational arm with DA (Arm 1) given in order to maintain hemoglobin level between 13 and 15 g/dL and a conventional arm (Arm 2) with usual management of chemotherapy-induced anemia, including red blood cell transfusions and ESA according to usual practices. The objective was to evaluate the efficacy of DA in association with chemotherapy (R-CHOP) as measured by the EFS at 2 years, events being defined as death from any cause, relapse for complete responders and unconfirmed complete responders, progression during or after treatment and changes of therapy during allocated treatment. Secondary objectives were OS, PFS, DFS, response rate and analysis of toxicity. Results 202 patients were included and 201 received study treatment ; 90 patients were randomized in DA group and 111 in conventional treatment group. Median age was 72 years. Patients' characteristics were similar in both arms with a slightly higher proportion of patients with aaIPI 2-3 in conventional arm : 68% compared to 56% (p=NS). Median hemoglobin (Hb) level at randomization was 12.2 g/dL. The median Hb level during treatment in DA arm was 11.9 g/dL and 10.7 g/dL in conventional arm respectively. Overall, 41 patients in conventional group received at least one dose of ESA during treatment. Eighteen percent of patients in DA group had at least one episode of Hb 〉 15 g/dL during treatment compared to 6% in conventional treatment group. Response rate (CR+CRu) after treatment was similar in both groups (69% in DA arm, 72% in conventional arm). Two-year EFS was 59% in DA arm compared with 49% in conventional arm (p=NS). A similar trend was observed for 2-year PFS (61% vs 51%), 2-year DFS (71% vs 56%) and 2-year OS (74% vs 63%) (p=NS for all). Grade 3-4 hematological toxicity was similar in the two groups. In contrast, proportion of patients receiving at least 1 red cell transfusion was higher in conventional arm (49% versus 36%). A total of 288 AEs were reported as serious (130 in DA group and 158 in symptomatic treatment group), concerning 122 patients (61%). Most SAEs were related to infectious complications. The rate of thromboembolic SAEs (composite criteria with pulmonary embolism, venous thrombosis from any site, coronary disease including myocardial infarction and cerebrovascular event) is slightly higher in DA group (13 SAEs) than in conventional arm (9 SAEs). Finally, the number of patients who died because of treatment toxicity was comparable in DA group (6 patients, 7%) and in conventional treatment group (7 patients, 6%). Conclusions The results of interim analysis of the LNH 03-6B provide encouraging results about efficacy and safety of a prophylactic use of DA in this population. It should be confirmed by the final analysis of the LNH03-6B trial which is planned in 2010. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1712 Poster Board I-738 [Background and Purpose] Everolimus (RAD001), an oral derivative of rapamycin, inhibits the mammalian target of rapamycin (mTOR) serine-threonine kinase, which plays a key role in regulating cell growth and proliferation. Although anti-lymphoma effects of RAD001 have been shown in preclinical studies and one phase II clinical study in NHL, there is no PK data in NHL patients (pts). A phase I (P1) study was conducted to evaluate safety, PK profile, and efficacy of RAD001 in Japanese pts with relapsed and/or refractory NHL. [Patients and Methods] Pts with relapsed or refractory NHL, age ≥20, and PS 0-2 (ECOG) were enrolled and treated with RAD001, administered orally once daily at either 5 or 10 mg. Dose escalation was based on the clinical assessment of safety, incidence of dose-limiting toxicity (DLT), and probability of incidence of DLT. The latter probability distribution was estimated by a Bayesian logistic model, using prior information from other P1 trials with this agent in solid tumors. DLTs were evaluated in 6 pts at each dose level during the initial 28 days of study treatment (cycle 1). PK parameters were evaluated on days 1 and 15 of cycle 1. Response was assessed according to the International Workshop Criteria for NHL (1999). [Results] A total of 13 pts were enrolled into 2 cohorts as follows; 5 mg (7 pts, DLBCL: 2; MCL: 1; FL: 1; PTCL: 1; CTCL: 1; ALCL: 1) and 10 mg (6 pts, FL: 4; MCL: 1; ALCL: 1). 12 pts were evaluable for DLTs. 1 pt in the 5 mg cohort was inevaluable for DLTs due to early disease progression. No DLTs were observed at either dose level. Frequently encountered potentially drug-related adverse events (AEs) (≥40%) include leukopenia (8/13, 62%), thrombocytopenia (8/13, 62%), elevated AST (9/13, 69%), stomatitis (7/13, 54%), anemia (6/13, 46%), nasopharyngitis (6/13, 46%), and elevated ALT (6/13, 46%). Potentially drug-related Gr 3 or 4 toxicities include anemia (Gr 4 : 1), thrombocytopenia (Gr 4 : 1; Gr 3: 1), lymphopenia (Gr 4 : 1; Gr 3: 3), elevated LDH (Gr 3: 1), anorexia (Gr 3: 1), hyperglycemia (Gr 3: 1), fatigue (Gr 3: 1), hepatic function abnormal (Gr 3: 1), P. jiroveci pneumonia (Gr 3: 1), hyperkalemia (Gr 3: 1), and hypokalemia (Gr 3: 1). All toxicities cited above were transient and reversible. Non-infectious pneumonitis (Gr 1) was observed in 1 pt but resolved following discontinuation of RAD001 without additional therapy. Tmax was achieved within 4 hours, and Cmax and AUCtau both showed dose-proportional increases. The accumulation ratio after daily administration was approximately 2-fold. These data are consistent with those from a previous P1 study in solid tumors. A total of 4 objective responses (OR) were observed (summarized below): [Conclusions] RAD001 is well tolerated in this pt population at doses up to 10 mg/day and shows preliminary evidence of activity in relapsed or refractory NHL. The PK profile in pts with NHL is similar to that in pts with solid tumors. A global randomized phase III study to determine efficacy of RAD001 as adjuvant therapy following front-line R-CHOP induction in poor-risk DLBCL is currently ongoing. The data from this P1 study led directly to Japan's decision to participate in the global phase III study. Disclosures Shimada: Novartis Pharma K.K. Japan: Employment. Kobayashi:Novartis Pharma K.K. Japan: Employment.

Description: Abstract 171 Introduction: SVT is a common disease, associated with risks of clinically relevant venous thromboembolic complications. Currently, treatment of isolated SVT (i.e. without concomitant deep-vein thrombosis, DVT, or pulmonary embolism, PE) is based on analgesic agents, topical or oral nonsteroidal anti-inflammatory drugs, elastic stockings and occasionally surgery. Previous clinical trials have suggested the benefit of an extended antithrombotic therapy. Yet the optimal antithrombotic treatment remains unknown. Fondaparinux (Arixtra®) is a synthetic selective inhibitor of factor Xa with a favorable benefit-risk profile in the prevention and treatment of venous and arterial thrombosis in various medical and surgical settings. The large-scale, double-blind, randomized, placebo-controlled, CALISTO trial was designed to evaluate the benefit-risk ratio of fondaparinux 2.5 mg subcutaneously once daily during 45 days in 3000 patients with symptomatic, isolated SVT of the lower limbs documented by compression ultrasound (CUS). Methods: The study concerned male or female patients 18 years of age or greater with acute symptomatic isolated SVT of the lower limbs at least 5 cm long documented by standard CUS. After randomization (Day 1), subjects received fondaparinux 2.5 mg or placebo subcutaneously once daily up to Day 45. Study treatments were administered on an outpatient basis. Follow-up continued up to Day 75. Permitted medications included analgesic agents, topical non-steroidal anti-inflammatory drugs, graduated compression (elastic) stockings, aspirin at low dose (up to 325 mg per day) and other oral antiplatelet agents if the subject was receiving these drugs at the time of screening for a chronic medical condition. The primary efficacy outcome was confirmed symptomatic thromboembolic complications (a composite of PE, DVT, extension of SVT with thrombus head ≤3 cm from the saphenofemoral junction or recurrence of SVT) or all-cause death up to Day 45. Safety outcomes included major bleeding, clinically relevant non-major bleeding and death. All efficacy and safety outcomes were adjudicated by a central adjudication committee, unaware of treatment assignment. Results: The study started in March 2007 and inclusions were completed on May 15th 2009, with the recruitment of 3002 patients. The follow-up of the last patient was completed on July 31st 2009. Overall, on blinded preliminary data available on June 23rd 2009 (n=2994), the median age of patients was 58 [range: 19-92] years; 63.9% were women; 37.0% were obese (BMI ≥30 kg/m2). The main predisposing risk factors for SVT were varicose veins (88.6%) and a history of SVT (11.9%). More than one SVT was observed on baseline CUS in 19.2% of patients. The qualifying SVT predominantly involved the great saphenous vein alone (64.6% of patients). At Day 45, the overall incidence of adjudicated and adjudicated-pending symptomatic thromboembolic complications or death was 4.4% (Table). There was one adjudicated major bleeding. The final results according to treatment groups will be presented during the congress. Conclusions: There is an unmet medical need for an evidence-based anticoagulant treatment for isolated SVT. CALISTO is the first large randomized, controlled trial designed to establish standard anticoagulant therapy in patients with isolated SVT. It also provides a large database on the clinical characteristics of this disease. Disclosures: Décousus: GSK: Consultancy, Research Funding. Leizorovicz:GSK: Consultancy.

Description: Abstract 1702 Poster Board I-728 Background ASCT consolidation in the first remission is the treatment of choice in MCL patients. However lack of adequate response to the first line therapy, elderly age and co-existing co-morbidities makes it feasible for less than a third of patients. Methods Retrospective analysis of 279 MCL cases treated at 10 PLRG centers was performed: 52% of them (144 pts) received Rituximab in induction therapy; 35% (97 pts) were subjected to post-induction therapy (ASTC – 16%, radioimmunotherapy consolidation – 13% and Rituximab maintenance – 6%). There were no significant differences in risk factor distribution among analyzed subgroups. Results 1) At 5 years OS was 40% in the whole group: 77% for those subjected to post-induction therapy vs 25% for those who were not; 5 year PFS is 20%, 48% and 5% respectively (p

Description: Abstract 1693 Poster Board I-719 A central goal of cancer immunotherapy is an adaptive immune response against tumors. The memory T cell is a critical mediator of this response as it can give rise to effector cells as well as self-renew. Regulatory T cells (Tregs) present a barrier to effective cancer immunotherapy. Indeed, cancer patients have increased numbers of CD4+CD25+ Tregs and cancer vaccination strategies in some cases expand this cell population. Here, we demonstrate that (1) CD4+CD44hi memory T cells are effective in mediating anti-tumor immunity and (2) that expression of CD137 can be used to exclude tumor-reactive Tregs from the CD4+CD44hi population. We have established a model for adoptive therapy of mantle cell lymphoma in which CD4 T cells mediate anti-tumor immunity. Specifically, we use a whole tumor-cell vaccine to induce anti-tumor immune cells in vivo. These cells are isolated and adoptively transferred into lethally irradiated, syngeneic, recipient mice. We show that CD4 but not CD8 T cells from vaccinated donor mice can prevent tumor growth when adoptively transferred into irradiated recipient mice. We observed that a majority of anti-tumor T cells display a memory phenotype. 10 days after transfer, T cells from recipient mice were co-cultured with irradiated lymphoma cells for 24 hours. Only CD4 T cells produced IFN-γ in response to co-culture and greater than 95% of IFN-γ+ CD4 T cells expressed the memory marker CD44. Finally, we observed that transferred CD4+CD44hi T cells persisted for over 100 days suggesting that this CD4 subset is important for lasting anti-tumor immunity. Contaminating Tregs may limit the effectiveness of our CD4 T cell adoptive therapy. In order to functionally deplete these Tregs from the CD4 population, we sought to identify a surface marker that could uniquely distinguish tumor-reactive Tregs from other CD4 T cells. T cells from vaccinated donor mice were co-cultured with irradiated lymphoma cells for 24 hours. We evaluated a panel of activation markers and observed that CD137 expression defined a distinct population of Tregs. Based on these results, we used flow cytometry to isolate a sub-population of CD4+CD44hiCD137- T cells from vaccinated donor mice. Adoptive transfer of less than 100,000 CD44hiCD137- but not other sub-populations of CD4 T cells provided significant protection from tumor challenge. These results suggest that CD137 defines a novel population of tumor-reactive Tregs. This marker can facilitate the enrichment of anti-tumor CD4 memory T cells within the CD44hiCD137- population. In conclusion, these findings highlight the potential use of CD4 T cells in adoptive therapy for mantle cell lymphoma. Disclosures No relevant conflicts of interest to declare.

Description: 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.

Description: Abstract 1672 Poster Board I-698 Introduction PCNSL is a rare form of non-Hodgkin lymphoma. High-dose methotrexate (HD-MTX) is the backbone of therapy but uncertainty remains about what additional chemotherapies should be added to HD-MTX to improve response rates. Methods After receiving IRB consent, we retrospectively evaluated patients with PCNSL treated at our hospital with combination M/R/T at initial diagnosis or at relapse. Patients were treated in 28-day induction cycles as follows: HD-MTX (8g/m2- dose adjusted based on creatinine clearance) on days 1 and 15; rituximab (375 mg/m2) on days 3, 10, 17, and 24; and temozolomide (150-200 mg/m2) on days 7-11. HD-MTX was given every 2 weeks until complete response (CR) and for 2 additional treatments followed by monthly maintenance treatments for 11 months. Rituximab was given weekly for a total of 8 weeks. Temozolomide was continued for 6 months after CR. Brain MRI was done after every other methotrexate treatment to assess response. Results Sixteen patients were treated between February 2006 and August 2009. Ten patients received MRT as first-line therapy at the time of initial diagnosis and 6 received MRT as salvage therapy at first or second recurrence. The median age of newly diagnosed patients was 58 (range 47-76) and of relapsed patients was 60 (range 46-76). CSF cytology was atypical in 5/15 patients who underwent lumbar puncture (4 first-line, 1 relapse). After first-line therapy, there were 9 CRs (median cycles to CR = 3) and 1 PR (patient still receiving induction treatment). After salvage therapy, there were 4 CRs (median cycles to CR = 4) and 2 PRs (both patients died while receiving treatment- 1 from an unrelated myocardial infarction). With a median follow-up of 10.3 months in the first-line group and 7.2 months in the relapse group, only 3 patients have progressed; one of whom had clinical progression after a PR and a second who relapsed in the skin. Treatment was well tolerated with reversible grade 4 transammonitis in 1 patient and grade 3 hematological toxicities in 8 patients. One patient experienced grade 4 thrombocytopenia related to temozolomide requiring dose reduction for subsequent cycles. Conclusions Combination MRT is well tolerated and resulted in a promising early response rate. The median number of cycles needed to achieve a CR for patients with newly diagnosed PCNSL was less than the 6 cycles we have previously reported for HD-MTX monotherapy suggesting that adding rituximab and temozolomide is beneficial to patients. This combination warrants further evaluation in larger scale, prospective studies. Disclosures Off Label Use: temozolomide and rituximab for PCNSL..

Description: Abstract 1666 Poster Board I-692 Diffuse large B-cell lymphoma (DLBCL) is primarily treated with Rituximab – CHOP immunochemotherapy with varying results. Improved prediction at diagnosis of outcome for an individual patient remains a key goal in DLBCL management. This would allow the selection of patients who could benefit from new therapies. A large amount of data on new immunohistochemical and molecular factors has been collected in the last decade, but the International Prognostic Index (IPI) and Revised IPI are still mostly used for identification of prognostic groups. Both of these indices have important limitations. They rely on a small number of dichotomised predictive variables and patients are assigned to one of a small number of risk groups rather than giving a direct prediction, such as a predictive survival probability or a predictive median lifetime, on a continuous scale for each patient. We have developed a new prognostic index based on Bayesian survival modelling. This has allowed an increase in the number of predictive variables and removal of the need to dichotomise them. Importantly, the new index can give predictions for patients where not all of the predictive variables are observed. It can easily be modified in the future by the addition of new variables, e.g. molecular factors or new treatment regimens. The index was developed using data on a population-based cohort of DLBCL patients prospectively registered with the Scotland and Newcastle Lymphoma Group (SNLG) between 1990 and 2003. The complete data on patients' outcomes were available for 1863 from 2025 registered patients and of this cohort 1391 patients were treated with anthracycline-based chemotherapy and the new index is based on this group. Bayesian statistical inference was used with a three-component Weibull mixture model in which both the hazard multiplier and the component membership probabilities depend on the predictive covariates. The three mixture components correspond to the following three groups: patients who will suffer from primary progressive disease or relapse within one year from diagnosis; patients who will relapse at a later point; those who will have no relapse. A novel method of modelling to deal with cases with missing covariates was used. Both time to first relapse (TFR) and overall survival (OS) were modelled. The following factors were included in the current version of the model: age, sex, performance status by ECOG, clinical stage, B-symptoms, extranodal and bone marrow involvement, Hb, WBC, lactate dehydrogenase, albumin, alkaline phosphatase and urea. The new index is available in the form of a simple computer program and can predict the outcome of patients as: a predicted TFR and OS or a probability of belonging to one of the three groups. In order to check the index, the whole cohort of patients was separated randomly with stratification by IPI into a training-prediction set (2/3 of patients) and a validation-observed set (1/3 of patients). Fig 1 shows the Kaplan-Meyer plots for TRF and OS for both sets. The index was successfully validated. Our new Bayesian prognostic index for DLBCL gives more reliable predictions at diagnosis for individual patients. In future the model can be modified for new treatment regimens or factors. Presently our group is investigating this issue for patients treated with CHOP-R. Fig1 Kaplan-Meyer plots for TFR and OS in training-prediction (TFRP and OSP) and validation-observed set (TFRO and OSO). Fig1. Kaplan-Meyer plots for TFR and OS in training-prediction (TFRP and OSP) and validation-observed set (TFRO and OSO). Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1722 Poster Board I-748 Conventional anti-cancer drug screening in vitro has traditionally been performed in the absence of accessory cells of the tumor microenvironment. These normal cells of the bone marrow milieu can profoundly alter anti-tumor drug activity. To address this major limitation of traditional in vitro models, we developed the tumor cell-specific in vitro bioluminescence imaging (CS-BLI) assay. In this platform, tumor cells (e.g. myeloma, leukemia and solid tumors) stably expressing bioluminescent reporters are co-cultured with non-malignant accessory cells (e.g. stromal cells) to selectively quantify tumor cell viability to treatments in presence vs. absence of accessory cells. We applied CS-BLI to test various chemical libraries and showed that this platform is high-throughput scalable. We also identified stroma-induced chemoresistance in diverse malignancies, including imatinib-resistance in leukemic cells, as well as MM cell resistance to certain investigational agents. The majority of compounds screened in our studies were less active against tumor cells in the presence of stromal cells compared to their absence. Most interestingly, however, we identified a fraction of compounds which were more active against tumor cells in the presence of stromal cells. For example, we identified reversine, a compound exhibiting this stroma-dependent synthetic lethality in vitro, which we further confirmed in vivo, as it is active in an orthotopic model of diffuse MM bone lesions, but not in conventional subcutaneous xenografts. Mechanistically, in vitro kinase activity assays showed that reversine exhibits a distinct pattern of inhibition against targets such as Auroras, JAK2, and SRC, but not against other important kinases for MM survival, such as AKT1, 2, 3, FGFR3, or GSK3. These observations are compatible with the role of SRC and JAK kinases as downstream regulators of IL-6/IL-6R signaling, a key cascade triggered by tumor-stromal interactions in MM. Further mechanistic evaluation of this interaction at the transcriptional level showed that a stromal-induced gene expression signature in MM tumor cells correlates with inferior overall survival in patients with advanced MM (APEX dataset) and includes enhanced amplitude of signatures for activated Akt, Ras, NF-κB, HIF-1á, myc, hTERT, and IRF4; as well as signatures for biological aggressiveness and stem cell self-renewal. This suggests that selective inhibitors which block the activity of these pathways may exhibit tumor specific stromal-dependent synthetic lethality. Historically, synthetic lethality has focused on how tumor cells harboring specific constitutive oncogenetic lesions are responsive to certain agents, but not in absence of these genetic events. Our study introduces the notion that a synthetic lethal phenotype, rather than being exclusively genotype-dependent, can also be driven by the extrinsic influences of the tumor microenvironment. Importantly, the CS-BLI system can probe both genetically- and microenvironment-related synthetic lethality in a high-throughput scalable manner. This allows the testing of a large number of permutations, including multiple candidate therapeutics, cell lines, and non-malignant accessory cells, thus enabling the previously intractable large-scale evaluation of how genetics and microenvironment play a role in modulating cancer cell response to treatment. Importantly, unlike conventional screening, CS-BLI can also identify agents with increased activity against tumor cells when interacting with stroma. These agents, in the past, may have been discarded from further preclinical or clinical development. We now provide a system with which to evaluate the role of the tumor microenvironment and identify novel agents capable of overcoming its protective effects. Disclosures Munshi: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Richardson:Celgene: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millenium: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Anderson:Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mitsiades:Millennium: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: Patents & Royalties; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis Pharmaceuticals: Research Funding.

Description: Abstract 1678 Poster Board I-704 Background The outcome of patients with PTCL is poor and the majority of patients ultimately have refractory disease to a variety of agents, including standard therapy with CHOP or CHOP-like regimens. Pralatrexate is a rationally designed antifolate that has a high affinity for the reduced folate carrier-1 (RFC-1) and is likely to be retained longer within cancer cells due to efficient polyglutamation by folylpolyglutamyl synthetase. Phase 1 and 2 clinical trials have shown that pralatrexate is safe and active in hematologic malignancies including relapsed or refractory PTCL (O'Connor OA, JCO 2009:10.1200/JCO.2008.20.8470). The PROPEL study, a pivotal, international, Phase 2 single-arm trial, evaluated pralatrexate in patients with relapsed or refractory PTCL who had a median of 3 prior therapies. Pralatrexate treatment was well tolerated and showed an overall response rate of 28% (30/109 patients) by central independent review. The objective of this current analysis was to characterize the treatment response among patients who were considered to have refractory disease, defined as showing 1) no evidence of response to their most recent prior therapy or 2) no evidence of response to all prior therapies. Methods Patients received pralatrexate 30 mg/m2 once weekly for 6 weeks in 7-week cycles, with vitamin B12 and folic acid supplementation. Response to pralatrexate was assessed after each odd-numbered treatment cycle and was based on rigorous centralized review of imaging and clinical data using the International Workshop Criteria (Cheson BD, JCO 1999;17:1244). Results The most commonly used prior therapies were CHOP-based chemotherapy (70%), platinum-based multi-agent chemotherapy (41%), and non-platinum containing combination chemotherapy (39%). Eighteen (16%) patients received autologous stem-cell transplantation (SCT) prior to the PROPEL study. Of the 109 patients who were evaluable for response, 69 (63%) patients had no evidence of response to the most recent therapy and 26 (25%) patients had had no evidence of response to any therapy. The ORR for the 69 patients with no evidence of response to their most recent prior therapy was 25% (n=17) according to central review and 36% (n=25) according to investigator review. The median duration of response was 99 days (range, 41-535) by central review. The median number of prior systemic therapies for these 69 patients was 3 (range, 1-11). Twenty-six patients had no evidence of response to any prior therapy before initiating pralatrexate, having received a median of 2.5 prior therapies (range, 1-7). Five (19%) of these patients responded to pralatrexate by central review and 7 (27%) responded by investigator review. The duration of centrally reviewed response to pralatrexate in the patients with no evidence of response to any prior therapy was 54, 57, 69, 78, and 306 days. Conclusions Pralatrexate has demonstrated activity in patients with PTCL who were refractory to their most recent therapy, including patients who were refractory to all prior therapies. Responses to pralatrexate were observed in patients with disease that was refractory to a range of therapies used in PTCL, including SCT and a variety of single agent and combination chemotherapy regimens, suggesting that pralatrexate can overcome mechanisms of drug resistance in this population. Disclosures Savage: Allos Therapeutics, Inc.: Consultancy, Honoraria. Horwitz:Allos Therapeutics, Inc: Consultancy, Research Funding. Pro:Allos Therapeutics, Inc.: Research Funding. Fruchtman:Allos Therapeutics, Inc.: Employment.

Description: Abstract 1658 Poster Board I-684 Introduction NHL patients with refractory disease or relapsing after autologous or allogeneic stem cell transplant (SCT) have very poor prognosis with currently available salvage chemotherapy. Sorafenib (Nexavar, BAY43-9006, Bayer) is an oral multikinase inhibitor approved by FDA for the treatment of renal cell carcinoma and hepatocellular carcinoma. Sorafenib exerts a broad range of antiproliferative, antiangiogenic, and proapoptotic effects against a variety of nonhematological tumors through the inhibition of the RAF/MEK/ERK pathway, the receptor tyrosine kinases c-kit, Flt3, RET, as well as the proangiogenic vascular endothelial growth factor receptors (VEGFRs), and platelet-derived growth factor receptor-β (PDGFR-β). Several lines of evidence suggest that Sorafenib might have a significant clinical impact in the treatment of malignant lymphomas by overcoming the cytoprotective effects of Bcl-XL, ERK, and Mcl-1 and eventually targeting additional signalling pathways relevant to lymphomagenesis. Our preclinical data demonstrating a marked cytotoxic activity of Sorafenib against NHL cell lines in vitro and in vivo in xenograft models, established the rationale for this currently ongoing phase II study aimed to determine safety and activity of Sorafenib in relapsed/refractory NHL. Methods Between March 2008 and May 2009, 21 patients (16 males and 5 females; median age, 65 years; range, 29-74 years) with relapsed/refractory diffuse large B cell lymphoma (DLBCL, n = 11), follicular lymphoma (FL, n =4), mantle cell lymphoma (MCL, n =2), lymphoplasmacitoid lymphoma (LPL, n =1), chronic lymphocytic leukemia (CLL, n =2), and peripheral T-cell lymphoma (PTCL, n =1) who have failed second- or subsequent-line salvage chemo-radiotherapy were enrolled in this phase II trial. Prior to study entry, patients received a median of 4 (range 2 - 7) lines of treatment, including autologous SCT in 15 (71%) and an additional allogeneic SCT in 5 (24%) cases. At study entry, 7 (33%) patients had relapsed and 14 (67%) refractory disease. Eligibility criteria included absence of any available treatment options of proven efficacy, at least one target lesion ≥2 cm, ECOG performance status of 0-1, and adequate bone marrow, liver and renal functions. Sorafenib (400 mg BID, per os) was administered continuously until disease progression or appearance of clinical significant toxicity probably related to study drug. Tumor responses were assessed according to the revised response criteria for malignant lymphoma of the International Working Group. NCI CTCAE v3.0 was used for toxicity assessment. Results To date, 21 patients received a median of 3 months (range, 1 – 11) of Sorafenib therapy. All patients are evaluable for toxicity and response, and 1 patient is still on therapy. Overall, therapy was well tolerated without significant adverse events. The most common drug-related non-hematological toxicities were grade 1-2 mucositis (14%), diarrhea (24%), hand-foot syndrome (24%), anorexia (29%), and fatigue (29%). Grade 3-4 hand-foot syndrome occurred in 19% of patients. Hematological toxicities included grade 1-2 neutropenia (10%) and thrombocytopenia (24%). Grade 3-4 neutropenia and thrombocytopenia were observed in 14%, and 24% of patients, respectively. Best response to Sorafenib included 1 (5%) complete remission (CR) occurring in the patient with LPL, and 1 (5%) partial remission (PR) in a patient with cutaneous DLBCL, for an overall response rate (ORR) of 10%. In both patients, response duration was 6 months. In addition, 9 (42%) patients achieved stable disease (SD) for a median of 3 months (range, 2 – 10), with 3 (14%) patients achieving SD for ≥6 months, while 10 (48%) patients progressed. Upon Sorafenib treatment, an extensive necrosis involving the central area of the tumor associated with a nearly complete disappearance of tumor vascularization was documented by computed tomography and contrast-enhanced ultrasound in two DLBCL and one FL patients bearing latero-cervical or abdominal lymphoid masses. Conclusions Sorafenib as a single agent was well tolerated. Despite limited clinical activity (10% ORR), disease stabilization was experienced by 42% of patients. The potent antiangiogenic activity of Sorafenib in NHL patients bearing highly vascularized lymphoid masses suggests that further research should focus on combinations of Sorafenib with molecularly targeted agents eventually exerting antivascular activities. Disclosures Off Label Use: The multikinase inhibitor Sorafenib has been used in a phase II trial in patients with relapsed/refractory NHL.

Description: Abstract 1685 Poster Board I-711 Adult T-cell lymphoma/leukemia (ATLL) is a T-cell malignancy caused by human T-lymphotrophic virus type I (HTLV-I), and its therapeutic outcome is still remains very poor. Therefore, novel therapeutic strategies are needed to improve patient outcome. In this study, we elucidated the therapeutic potential to target anti-apoptotic Bcl-2 family proteins for the treatment of ATLL by using ABT-737 (Abbott Laboratories, Abbott Park, IL, USA), a small molecule inhibitor of Bcl-2, Bcl-XL and Bcl-w. We first validated the rationale of this study by assessing the expression of Bcl-2 family proteins among 25 lymph-node specimens derived from ATLL patients by using immunohistochemistory. Both or either of Bcl-2 and Bcl-XL proteins was highly expressed in 80% of specimens. We next examined the cytotoxicity of ABT-737 against ATLL cell lines. ABT-737 significantly inhibited growth of MT-1, MT-2 and HuT 102 cells with a concentration of 50 percent inhibition (IC50) at 72 h of 2.4, 0.23 and 0.008μM, respectively. We then elucidated the mechanism of growth inhibition induced by ABT-737 using MT-1 and MT-2 cells. ABT-737 induced apoptosis in MT-1, MT-2 cells with cleavage of caspase 9, 3 and PARP. ABT-737 also induced apoptosis in fresh tumor cells derived from patients with ATLL. We next elucidated the potential of ABT-737 to enhance the cytotoxicity induced by conventional chemotherapeutic agents. The interaction between them was evaluated using the Chou-Talalay method by determining the combination index. ABT-737 synergistically enhanced the cytotoxicity and apoptosis induced by either of doxorubicin, vincristine or etoposide, which is a current key drug to treat ATLL. Most importantly, ABT-737 significantly inhibited tumor growth of in vivo ATLL model using SCID mice inoculated by HuT 102 cells subcutaneously. The mean tumor volume, weight and serum level of soluble interleukin-2 receptor á of ABT-737 (100mg/kg/day)-treated mice were significantly lower than those of vehicle-treated mice after treatment for 21 days. Moreover, massive induction of apoptosis in tumors treated by ABT-737 was observed by immunofluorescent TUNEL assay. These results suggest that ABT-737 used either alone or in combination with conventional cytotoxic drugs, represents a promising novel targeted approach to overcome drug resistance and improve patient outcome in ATLL. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1679 Poster Board I-705 Background Lenalidomide (Revlimid®) is a potent immunomodulatory agent that has recently demonstrated clinical activity in the treatment of relapsed/refractory Multiple Myeloma and low- and int-1 risk Myelodysplastic syndromes with the del (5q) mutation. Laboratory data has shown that lenalidomide enhances T- and NK-cell mediated immune synapse formation and ADCC, and it exerts antiproliferative activity. In relapsed/refractory (R/R) NHL lenalidomide treatment achieved modest responses, although duration of response was 〉16.5 months (Witzig et al JCO, in press). Synergistic activity between lenalidomide and rituximab has been reported in lymphoma cell and animal models. Here we assess safety and efficacy of the lenalidomide and rituximab combination (R2) in patients with R/R indolent NHL. Methods Eligible patients had relapsed/refractory indolent NHL with measurable disease, ≥1 prior therapy, and ECOG PS 0-2. Oral lenalidomide was administered once daily on days 1-21 of a 28-day cycle, and was continued until disease progression. Rituximab 375mg/m2 IV was initiated on day 15 of cycle 1, and repeated weekly for a total of 4 doses; if after cycle 2 patient had less than a complete response (CR), 4 additional doses could be administered at discretion of the treating physician. After Gr. 3 tumor lysis syndrome (TLS) developed in 2 of the first 4 patients, the protocol was amended to reduce lenalidomide starting dose from 25 mg to 20mg daily, and TLS prophylaxis was provided with allopurinol in subsequently enrolled patients. Response and progression were evaluated according to IWG criteria (Cheson et al 1999). Results Of 15 patients currently enrolled on study, 14 received 〉2 cycles of R2, and 12 are evaluable for response. Median age for evaluable patients is 60 yrs (range: 50-91), 5 pts are female, and histologies include FL (n=9), SLL (n=1), and marginal zone lymphoma (n=2). Median time from diagnosis to treatment on study was 7.5 years (3.3-19), median number of prior therapies was 4 (range:1-11), and 9 patients were considered heavily pretreated (defined as ≥ 3 prior courses of therapy). All patients received prior rituximab, 6 patients had rituximab-resistant disease (defined as no response or relapse ' 6 months after initiating rituximab), and 5 patients had received prior radioimmunotherapy. Of 12 evaluable patients, 10 (83.3%) responded to R2, including 5 patients (41%) with a CR and 4 patients (33%) with a PR. Responses occurred in 4 of 6 patients (66%) with rituximab refractory disease, and in 7 of 9 (77%) heavily pretreated patients. Of 9 patients with R/R FL, 5 (55%) achieved a CR and 3 (33%) had a PR in response to R2, for an 88% ORR. At a median follow-up of 12 months, the median progression free survival has not been reached. Most common grade 3/4 adverse events were fatigue (2/12, 16%), neutropenia (3/12, 25%), lymphopenia (4/12, 33%), and hyponatremia (2/12, 16%). After prophylaxis was initiated, TLS was not observed at the 20 mg dose level. Conclusion This is the first study to demonstrate that the R2 combination is generally well tolerated and active in patients with R/R indolent NHL. Clinical activity was particularly noted among patients with R/R FL, who achieved 88% OR and 55% CR rate. The tolerability profile of R2 was consistent with other studies of each agent individually in the R/R setting. TLS prophylaxis and monitoring is recommended, particularly during initial cycles of treatment. These data support further evaluation of the potential clinical synergy between rituximab and lenalidomide in larger studies of FL and in earlier lines of therapy. Disclosures Off Label Use: Lenalidomide for treatment of non-hodgkin's lymphoma. Tuscano:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 1671 Poster Board I-697 Background Bevacizumab (Avastin), a humanized monoclonal antibody directed against vascular endothelial growth factor (VEGF-A), is approved in combination with chemotherapy in lung, colorectal, breast and brain cancers. VEGF-A expression is reported in all lymphoma subtypes, with strongest expression in PTCL. Given the relatively poor prognosis of PTCL with standard therapy, we combined bevacizumab (A) with CHOP in the ECOG 2404 phase II trial. Methods Untreated PTCL pts with a normal baseline cardiac ejection fraction received bevacizumab 15 mg/ kg IV and CHOP chemotherapy on day 1 for 6-8 cycles. Patients (pts) with CR/PR/SD received maintenance therapy with bevacizumab 15 mg/ kg IV q 3 weeks for 4 cycles. Due to the reported 3.7% incidence of cardiac toxicity associated with bevacizumab in metastatic breast cancer pt previously exposed to anthracyclines (Avastin package insert), cardiac function was monitored prospectively with ejection fraction (EF) determination at the completion of A-CHOP prior to the start of bevacizumab maintenance. Results Among 33 pts who came off study, toxicity data are available for all and coded using CTCAE Version 3.0. 22 received at least 4 cycles of A-CHOP (median treatment duration 3.6 months (〈 1-10.6 months). One or more designated cardiac toxicities were recorded for 5 of these 22 pts. Congestive heart failure (CHF), defined as grade 2-4 left ventricular dysfunction was recorded in 4 pt (18%, 90% CI 5.2%-40.3%): grade 4 (n=1), grade 3 (n=3). In 3 pts clinical CHF occurred after A-CHOP x 6 and in one during bevacizumab maintenance after A-CHOP x 8. The median time to development of a cardiac adverse event was 5 months (3.5 – 8.8 months). One pt required a ventricular assist device in addition to medical management. In 3 of the 4 pts, ongoing medical management for CHF has been required. Other cardiac toxicities included ventricular arrhthymia (one grade 4, one grade 2), and one grade 2 cardiac troponin elevation. In addition, one patient experienced sudden death of uncertain cause. Three pts developed thromboses or embolic toxcities (two grade 4 and one grade 3). Conclusions We observed four cases of clinical CHF early after cumulative doxorubicin doses of 300-400 mg/m2 in our study, The incidence of CHF among study patients who received 4 or more A-CHOP cycles is concerning and suggests that bevacizumab may potentiate the adverse cardiac effects of anthracyclines, resulting in clinically significant toxicity. Careful monitoring of cardiac function is warranted in ongoing trials combining bevacizumab and doxorubicin in lymphomas and solid tumors to assess long-term safety and better understand underlying mechanisms and risks. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1649 Poster Board I-675 Non-conventional γσ T lymphocytes are innate immunity effectors bearing potent anti-tumoral activity, particularly against malignant B cells. IPH1101 is an agonist of γσ T cells, which in the presence of low doses of IL-2 potentiates their direct cytotoxic activity. ADCC is a major molecular mechanism underlying rituximab's efficacy. Increasing the number and the activation state of killer lymphocytes mediating ADCC is therefore believed to be beneficial for therapeutic potency. Since γσ T cells have been found to be capable of mediating ADCC, modulating γσ T cells in the context of rituximab is worth being tested in a clinical trial. The main purpose is to assess the clinical efficacy of IPH1101 with low doses of IL-2, combined with a standard rituximab treatment, in patients (pts) with follicular lymphoma. This is an open label, multinational study consisting of a dose escalation Phase (ph) I-like part followed by a ph II part. The ph I part has shown a good safety and immuno-biological efficacy profile for the highest dose of IL-2. Consequently, the pts of the phase II part were treated with the combination of rituximab (375 mg/m2) administered 4 times weekly, IPH1101 (750 mg/m2) administered i.v. 3 times (every 3 weeks) and IL-2 (8 MIU) administered daily s.c. for 5 days starting on the day of each IPH1101 administration. All pts presented low grade FL which had relapsed after 1 to 4 lines of previous therapy including at least one rituximab-containing line. Inclusion criteria set forth that pts should have no lesion 〉 7 cm. Results We report here the end of recruitment and updated data on 45 patients and more than 100 cycles of IPH1101. Overall safety was good, and most of the drug-related adverse events were, as expected, flu like symptoms of grade 1 or 2. The SAEs reported were 2 hypotensions, 1 bronchospasm, 1 allergic reaction (back pain), 1 glomerular filtration decrease, 1 ALAT elevation, 1 hypertension and 1 asthenia. The immuno-biological follow up demonstrated the very good pharmacodynamic profile of IPH1101 in these patients and these data are presented in details in another abstract from Lucas et al. To date, in the first set of evaluable patients (12 pts) and after at least 3 months post treatment, investigators reported about 75% of response and 50% of CR. Most of the responses are already confirmed by an independent panel. Conclusion These observations confirm the good safety and the biological rationale of this approach. Furthermore, the response rate in this first set of pts is encouraging in the context of previously treated patients. Updated results will be presented at the meeting. Disclosures Laurent: Innate pharma: Honoraria. Lafaye de Micheaux:Innate Pharma: Employment. Solal-Celigny:Innate Pharma: Research Funding. Soubeyran:Innate Pharma: Research Funding. Delwail:Innate Pharma: Honoraria. Ghesquières:innate pharma: Honoraria. Thieblemont:Innate Pharma: Honoraria. Jourdan:Innate Pharma: Honoraria. Beautier:Innate Pharma: Employment. Squiban:Innate pharma: Employment. Rossi:Innate Pharma: Honoraria.

Description: Abstract 1653 Poster Board I-679 Background The immune system is involved in AML control and Natural Killer (NK) cells are among the most promising effectors. The therapeutic potential of NK cells has been revealed by the Killer Immunoglobulin Receptor (KIR) mismatch allogeneic transplant model where the anti-leukemic effect of the graft, is due to unleashed NK cells towards AML blasts, as suggested by enhanced in vitro lytic activity of KIR-HLA mismatched donor NK cells against recipient blasts (Miller et al. 2005; Ruggeri et al. 2002). Receptors involved in the function of NK cells against AML blasts have been identified (Pende et al., 2005). Some of these receptors are altered in AML patients at diagnosis and might be involved in the immune escape of AML blasts (Costello et al., 2002). However, the status of NK cells during early stages of patient's chemotherapy (CT) treatment is unknown. The present study monitored status of NK cells during early stages following patient's remission after CT that may be critical for their long lasting clinical response, and results might provide new targets for immunotherapy. Methods We enrolled 20 elderly patients (60 to 80 years old) with non promyelocytic AML in first CR following induction and pre-consolidation CT with normal renal and hepatic functions. Patient peripheral NK, gd T and CD8 T cells were analyzed before consolidation CT and every other week after treatment for 8 weeks. 6-colors flow cytometry was performed to investigate the expression of MHC receptors (CD158a, b, e, i, CD85j and NKG2A), activating receptors (NKp30, NKp46, NKG2D, CD16, DNAM-1, 2B4) as well as their differentiation status (perforin and granzyme expression). Their function, as determined by cytotoxicity (51Cr release and CD107 expression) and cytokine production (intracellular staining of IFN-g), was analyzed using purified NK cells stimulated by K562, or in redirected assays using NKp30, NKp46 and CD16 mAbs. Results NK cell counts were depressed away from the induction and pre-consolidation CT as compared to NK cell counts of age-matched controls (ctl) (95±107 NK/μL vs 229±91 NK/μL respectively); they were further depressed during the first 2 weeks post-consolidation CT (55±57 NK/μL), but were back to pre-consolidation CT level at 4 weeks (105±102 NK/μL). In contrast, CD8 T cells and gd T cells counts were normal even at early times post-CT. Expression of 2B4 was depressed at all time points. In contrast, NKp30 expression was lower at diagnosis and close to ctl level post-consolidation CT (p=0.0003) and NKp46 expression increased after CT (p

Description: Abstract 1586 Poster Board I-612 Background Mutations in the nucleophosmin 1 (NPM1) gene represent the most frequent gene mutations in acute myeloid leukemia (AML), with highest frequency (50-60%) in cytogenetically normal (CN)-AML. Several studies have shown the applicability and prognostic value of an NPM1 mutation (NPM1mut)-based assay for detection of minimal residual disease (MRD). So far, there are no studies evaluating the prognostic value of NPM1mut MRD levels in a large controlled cohort of AML patients (pts) enrolled on prospective clinical trials. Aims To evaluate the prognostic value of NPM1mut MRD levels in younger (16 to 60 years) AML pts harbouring NPM1 mutations type A, B or D, and to assess the influence of concurrent FLT3 internal tandem duplications (ITD). Methods All pts were enrolled in the prospective AMLSG 07-04 and AML HD98A treatment trials. Treatment comprised double induction therapy with ICE (idarubicin, cytarabine, etoposide) followed by high-dose cytarabine-based consolidation, autologous or allogeneic stem cell transplantation. Levels of NPM1mut expression ratios, defined as NPM1mut copies per 104ABL copies, were determined by RQ-PCR using TaqMan technology. Dilution series showed a maximum sensitivity of 10-6 and high specificity as no wildtype NPM1 could be detected. Results A total of 1079 samples, [bone marrow (BM), n=1062; peripheral blood, n= 17) from 212 pts were analyzed at diagnosis, after each treatment cycle, during follow-up and at relapse (median number of samples per pt, n=4; range, 1-16). NPM1mut expression ratios at diagnosis varied between 1.1×104 and 10.4×106 (median, 6.9×105). Pretreatment transcript levels were not associated with clinical characteristics (e.g., age, white cell counts, BM blasts) and did not impact on relapse-free (RFS) and overall survival (OS). Following the first induction cycle, the median decrease of the MRD level ratio normalized to pretreatment levels was 4.21×10-3, independent of the presence of concurrent FLT3-ITD (p=0.39). After the 2nd induction cycle, the median reduction of MRD levels was significantly stronger in the FLT3-ITDneg group (6.75×10-5) compared with the FLT3-ITDpos group (4.19×10-4) (p=0.003) and this differential effect was observed throughout consolidation therapy. For evaluation of the prognostic impact of NPM1mut MRD levels, we compared patients achieving PCR-negativity with those with positive values at different checkpoints. The first reliable checkpoint was after double-induction therapy: the cumulative incidence of relapse (CIR) at 4 years of PCR-negative patients (n=27) was 0% compared with 48% (SE, 4.4%, p

Description: Abstract 1626 Poster Board I-652 Introduction Familial Platelet Disorder with Propensity to Acute Myelogenous Leukemia (FPD/AML; OMIM 601399) is a rare disorder with an autosomal dominant inheritance pattern characterized by varying degrees of thrombocytopenia, clinical bleeding due to platelet dysfunction, and an increased risk of developing myeloid malignancy. To date, twenty-six families with this disorder have been described and all carry germline RUNX1 mutations as the causative genetic abnormality. The spectrum of RUNX1 mutations includes point mutations within the RUNT domain and frameshift and termination mutations throughout the body of the gene. Here we report identification of a new family with FPD with a novel nonsense mutation resulting in premature protein termination at amino acid 388. Patients and Methods Our four generation pedigree includes a mother (II:4) diagnosed with dysplasia and normal karyotype acute myeloid leukemia now in remission after a matched sibling allogeneic stem cell transplant, and her daughter (III:2) with thrombocytopenia since childhood, excessive bleeding with childbirth, and 5q- syndrome diagnosed at 37 years old. Genomic DNA was obtained from all available family members, and RUNX1 cDNA (transcription variants a through c) was sequenced. In addition, RUNX1 cDNA was analyzed for second mutations in bone marrow samples from both patients at the time of diagnosis of their initial bone marrow malignancy. Results RUNX1 sequencing of germline DNA revealed heterozygosity for a novel nonsense mutation in exon 8 (c.1163C〉A), which is predicted to result in premature protein truncation (p.Ser388X). Full sequencing of RUNX1 cDNA from II:4's AML does not show any secondary mutations. Our current efforts include full sequencing of RUNX1 cDNA from III:2's bone marrow malignancy as well as functional studies of the truncated protein. Conclusions We have identified a novel 3' RUNX1 mutation within exon 8, which is predicted to result in premature protein truncation at amino acid 388. To date, this is the most distal mutation identified in an FPD/AML pedigree. The identification of this mutation suggests that the last 100 amino acids, which are known to contain the RUNX1 inhibition domain, contribute an essential function. Further characterization of this RUNX1 mutation and its encoded truncated protein may yield insight into RUNX1's role in leukemogenesis in FPD and de novo AML. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 159 The anemias of chronic disease (ACD) are a common complication of malignancy, inflammation and kidney disorders. In ACD, there is dysregulation of iron homeostasis, decreased proliferation of erythroid progenitors, diminished production of erythropoietin (EPO), and shortened lifespan of RBC. Multiple pathophysiologic mechanisms have been implicated in the development of ACD, including elevated production of hepcidin and inflammatory cytokines, IFNγ, TRAIL, Interleukins-1β, 6, 10, 15, & TNFα. These cytokines are thought to directly inhibit erythroid differentiation through unknown mechanisms. The current study addressed the hypothesis that inhibition of erythropoiesis in ACD may arise through synergistic effects of iron deprivation and specific inflammatory cytokines. To identify relevant cytokines, candidate factors were applied to primary human erythroid progenitors in iron replete and restricted cultures. Peripheral blood human CD34+ progenitors from healthy donors underwent standard prestimulation for 72 hours, followed by culture in unilineage erythroid medium (4.5 U/ml EPO + 25ng/ml SCF) for 4-5 days under iron replete (100% transferrin saturation) or iron restricted (15% transferrin saturation) conditions. Candidate cytokines were screened for effects on viability, proliferation, and differentiation using cell counting and flow cytometric analysis of the erythroid cell surface marker GPA and the megakaryocytic antigen CD41a. Contrary to previous reports, the majority of cytokines (TRAIL & Interleukins-1β, 6, 10, 15) showed no effects on erythroid proliferation or differentiation under iron replete or restricted conditions. By contrast, both IFNγ and TNFα displayed potent inhibitory effects under iron restricted conditions but only weak effects in iron replete cultures. Typically, iron restriction alone reduced the proportion of GPA+ cells by 50%, whereas IFNγ or TNFα combined with iron restriction caused a 90% reduction. While both cytokines cooperated with iron restriction in blocking upregulation of GPA and promoting cell death, each cytokine also had distinctive effects on morphology and differentiation. IFNγ enhanced megakaryocytic development, while TNFα retained cells as immature, CD34+ progenitors. The synergistic inhibition of erythroid differentiation with iron restriction and TNFα was confirmed in vivo using a murine model of dietary iron deprivation coupled with continuous infusion of low-dose TNFα. Regarding the mechanism for this synergy, we have previously shown that erythroid iron deprivation leads to inactivation of the aconitase enzymes, which normally convert citrate to isocitrate, and that provision of exogenous isocitrate abrogates the erythroid inhibition associated with iron deprivation. Accordingly, participation of this pathway was assessed in the more potent erythroid inhibition associated with IFNγ or TNFα plus iron deprivation. Strikingly, isocitrate administration not only abrogated effects due to iron deprivation but also those due to the inflammatory cytokines, leading to complete rescue of erythroid differentiation. To address the underlying basis for erythroid cross-talk of iron and cytokine signaling, we screened pathways implicated in iron metabolism and inflammation. Two relevant pathways identified were Jun kinase (JNK) and calmodulin-associated kinase II (CAMKII), important in TNFα and IFNγ signaling, respectively. In particular, TNFα and iron deprivation synergized in the activation of JNK, and IFNγ and iron deprivation synergized in activating CAMKII. In both cases, isocitrate partially restored the activation to basal levels. As an important negative control, iron deprivation did not affect IFNγ activation of STAT1 phosphorylation, indicating that its effects were not due to upregulation of receptor expression or function. Altogether, these data suggest that among the various cytokines implicated in ACD, only IFNγ and TNFα synergize with iron deprivation in the inhibition of erythropoiesis. These actions occur through cross-talk between intracellular signaling pathways, specifically pathways involving aconitase and cytokine-activated kinases. The connection of aconitase/metabolism with inflammation is novel and has implications for clinical treatment of ACD, as well as for new understanding of erythroid and inflammatory signaling. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1574 Poster Board I-600 Introduction Acute myeloid leukemia (AML) is a heterogeneous disease which harbors various genetic alterations. Among theses genetic events, Mutations of FLT3, NPM1, MLL and other genes often predict prognosis, particularly in cases cytogenetic normal (CN-AML). Could these be criteria for risk stratification in Pediatric AML ? Patients and Methods 155 cases of de novo AML were diagnosed routinely according to morphology, immunology, cytogenetics, and molecular biology examination on bone marrow (BM) aspirates between Jan. 2002 and Dec. 2008. All patients received chemotherapy according to the AML-XH-99 protocol, which consist of Daunorubicin, Cytosine arabinoside, Etoposide, Homoharringtonine. For acute promyelocytic leukemia, all-trans retinoic acid and Arsenic trioxide were also included. Meanwhile, total RNA of leukemic cells form all diagnostic BM samples were extracted, and then reverse transcribed. MLL partial tandem duplication (MLL/PTD) fusion transcripts were screened by real-time quantitative polymerase chain reaction. FLT3 internal tandem duplication (FLT3/ITD), FLT3 tyrosine kinase domain mutation (FLT3/TKD) and NPM1 mutation were examined by High resolution melting analysis. Results Of the 155 children with de novo AML, 121(78.1%) had received chemotherapy for more than one week with data available for analysis. Among them, 55(45.5%) was cytogenetically normal (CN-AML). In this total cohort of patients 49(27.09%) had FLT3/ITD (32.70% in CN-AML), 14 (9.03%) had FLT3/TKD (7.30% in CN-AML), 62 (40%) had NPM1 mutation (49% in CN-AML), and additional 8 (5.16%) had MLL/PTD (5.50% in CN-AML). In this cohort of patients 98 (63.22%) had at least one mutation. The clinical outcomes were listed in table 1. Generally, patients with FLT3 mutation (ITD or TKD mutation) usually have worse results after chemotherapy, as reported previously by other researchers. Meanwhile, NPM1 mutations usually predict better prognosis in our cohort of AML patients. MLL/PTD always predicts the worst outcome in AML as other MLL rearrangements in leukemia. Among CN-AML patients, 5-year EFS and OS were similar to whole cohort of patients according to those mutations. Cox regression analysis in a univariate model revealed that the presence of FLT3/ITD and NPM1 was significant prognostic factor of EFS, (P

Description: Abstract 156 Platelet adhesion and aggregation at sites of vascular injury are key events required for haemostasis and thrombosis. It has been documented that von Willebrand factor (VWF) and fibrinogen (Fg) are required for platelet adhesion and aggregation. However, we previously showed that occlusive thrombi still form in mice deficient for both Fg and VWF (Fg/VWF−/−) via a β3 integrin-dependent pathway. Here, we have investigated novel, non-classical ligands of β3 integrin that may regulate platelet adhesion and aggregation. To identify potential ligand(s) of β3 integrin, latex beads were coated with purified human platelet β3 integrin and incubated with human plasma. Protein(s) specifically associated with β3 integrin were electrophoresed and apolipoprotein AIV (ApoA-IV) was identified by mass spectrometry. We found that ApoA-IV binds to the surface of stimulated platelets, but not to quiescent platelets or β3−/− platelets, and ApoA-IV/platelet association was blocked by the addition of a specific anti-β3 integrin monoclonal antibody. It appears that ApoA-IV binds to, but is not internalized by platelet β3 integrins. ApoA-IV-deficient (ApoA-IV−/−) mice exhibited enhanced platelet aggregation induced by ADP, Collagen, and TRAP in plasma (but not PIPES buffer) compared to wild type (WT) littermates. This enhancement was diminished when ApoA-IV−/− plasma was replaced by WT plasma, indicating that the reduction was due to plasma ApoA-IV and not an unrelated platelet effect. When platelets were incubated with FITC-Fg, ApoA-IV was able to reduce platelet/Fg association, indicating that ApoA-IV may act to displace pro-thrombotic β3 integrin ligand(s). In support of this, ApoA-IV reduced the number of adherent platelets on immobilized Fg in perfusion chamber assays and enhanced thrombus formation was observed when ApoA-IV−/− mouse blood was perfused over collagen. We found that addition of recombinant ApoA-IV inhibited platelet aggregation and thrombus formation in vitro, while the control apolipoprotein ApoA-I did not. Using intravital microscopy, we further demonstrated that early platelet deposition was increased, and the time for thrombus formation and vessel occlusion were shorter in ApoA-IV−/− mice, which can be corrected by recombinant ApoA-IV transfusion. Furthermore, recombinant ApoA-IV inhibited WT platelet aggregation, thrombus formation and enhanced thrombus dissolution both in vitro and in vivo. Our data demonstrate for the first time that ApoA-IV is a novel ligand of platelet β3 integrin that negatively regulates thrombosis. These new data are consistent with the reported association between ApoA-IV and reduced cardiovascular diseases, and establish the first link between ApoA-IV and thrombosis. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1575 Poster Board I-601 It has been proposed that if more than 2 mutations are required for leukemia to arise, then an elevation in the mutation rate would be necessary for these mutations to coincide in the same cell, based on the rarity of spontaneous mutations (Loeb 1991). Hypermutability could occur transiently, for example, due to mutagens, or permanently, due to inactivation of repair pathways. Genome-wide analysis has indicated that in acute lymphoblastic leukemia (ALL), more than 2 somatic mutations are often found (Mullighan et al 2007), suggesting that hypermutability is likely to occur at least in some cases. To investigate this hypothesis, we have used our method for the detection of rare cells that have acquired a spontaneous inactivating mutation in the PIG-A gene. Expanded populations of stem cells with PIG-A mutations in the setting of bone marrow failure are the hallmark of paroxysmal nocturnal hemoglobinuria (PNH), but under most other circumstances, PIG-A mutations are thought to be growth neutral in vitro and in vivo. PIG-A is on the X-chromosome, and therefore, a single inactivating mutation is sufficient to produce the PNH phenotype, characterized by a lack of glycosylphosphatidylinositol (GPI)-linked proteins and an inability to bind to the FLAER reagent. Furthermore, it is known from patients with PNH and from cell lines that a very broad spectrum of mutations can inactivate PIG-A. This combination of features makes PIG-A uniquely suited as a sentinel gene for spontaneous mutagenesis in humans, which is otherwise very difficult to quantitate. Here we have analyzed a panel of blast cells derived from ficolled marrow samples from patients with B-lineage ALL, obtained from the Children's Oncology Group ALL Cell Bank. To determine the frequency of spontaneous PIG-A mutations, samples were stained sequentially with an Alexa-488 conjugate of the FLAER reagent, a mixture of murine anti-CD55 and anti-CD59 antibodies, FITC-conjugated rabbit anti-mouse immunoglobulin secondary antibody, and PE-conjugated anti-CD45 antibody. The blast population was identified based on forward and side scatter, dim expression of CD45, and exclusion of propidium iodide. Control GPI (+) and GPI (-) cells from a patient with PNH served as a control. Among 19 cases of B lineage ALL studied, the frequency of blast cells with the PNH phenotype varied over a very wide range, and the distribution of mutant frequency values was seen to be bimodal. One group, representing about 40% of the cases of ALL, had a median mutant frequency of 6.8 × 10-6, which is very similar to the proportion of granulocytes that acquire the PNH phenotype due to spontaneous PIG-A mutations in normal individuals (Araten et al, 1999). These values are also close to the frequency of mutants reported for the HPRT, GPA, and XK genes in normal individuals. The other 60% of the samples had a median mutant frequency of 465 × 10-6, which is far above the range of this parameter in normal individuals. Among 6 established cell lines derived from T-ALL, the mutant frequency ranged from 4.1 ×10-6 to 265 × 10-6. Here as well, the distribution suggested that in this subtype of ALL, there may be two distinct phenotypes with respect to this parameter. Based on this data, we believe that an increase in inactivating mutations is not essential for the development of ALL, but that it is a common feature of this condition. We cannot rule out the possibility that samples with a low mutant frequency would demonstrate some form of genomic instability that would not be detected by our assay, such as an increase in gene duplications or translocations. Further investigations are needed to determine whether hypermutability as detected by this assay is associated with distinct cytogenetic abnormalities, clinical features at presentation, and outcome. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1547 Poster Board I-570 Introduction Levels of high-density lipoprotein cholesterol have been correlated with anti-inflammatory, anti-oxidative, anti-aggregation, anti-coagulant and pro-fibrinolytic activities. We hypothesized that lipoprotein cholesterol and triglycerides have important roles in sickle cell disease pathogenesis. Patients and Methods A prospective study of biochemical and hematological analyses of 152 steady-state children with sickle cell disease and 132 healthy subjects using immunochemistry, immunoassay and electronic cell counter respectively. Clinical data were collected from patient medical records. Data analyses were performed using Prism 5.01 (Graphpad Software, San Diego, CA), EPIinfo 6.04 (CDC, Atlanta, Georgia) and STATA SE 10 software (StataCorp, Texas, USA). Results There was a significant positive association of high-density lipoprotein cholesterol with hemoglobin (p

Description: Abstract 1554 Poster Board I-577 Introduction The International Prognostic Factor Project Score (IPS) is the most widely utilized risk stratification index for Hodgkin lymphoma (HL) (Hasenclever, N Engl J Med, 1998). Based on patients treated before 1992, it incorporates 7 adverse risk features (male gender, age ≥45 y, stage IV, hemoglobin

Description: Abstract 1553 Poster Board I-576 We evaluated survival for all patients diagnosed with Hodgkin lymphoma (HL) in Sweden 1973-2005 (n=6,136; 3,515 men and 2,621 women; median age 40 years). Patients were categorized into six age groups and four calendar periods (1973-1980, 1981-1988, 1989-1996, and 1997-2005). Relative survival ratios (RSRs) were computed as measures of patient survival. Cumulative relative survival improved over time in all age groups with the greatest improvement in patients 51-65 years. Also in patients 66-80 years, significant improvements were recorded in both five- and ten year RSRs. Importantly, a plateau in relative survival was observed after 5 years in patients below ≤ 35 years of age during the last calendar period suggesting a lack of long-term treatment-related mortality. The ten-year RSRs in this calendar period were 0.95, 0.95, 0.92, 0.80, and 0.51 for the age groups 0-18, 19-35, 36-50, 51-65, and 66-80, respectively. Thus, despite this progress, age at diagnosis remains an important predictor of outcome. We also found a significantly better survival for women when adjusted for age and calendar period. During the study period, refined treatment options for patients with limited and advanced-stage HL have contributed to an increasing cure rate. In addition, our findings support that long-term mortality of HL therapy has decreased. Elderly patients still do poorly and targeted treatment options associated with fewer side effects will advance the clinical HL field. Cumulative relative survival stratified by age at diagnosis and calendar period of diagnosis Cumulative relative survival stratified by age at diagnosis and calendar period of diagnosis Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1542 Poster Board I-565 Introduction Sickle cell disease (SCD) is characterized by increased oxidative stress playing an important role in the pathophysiology of hemolysis, vascular occlusion and organ damage in sickle cell patients. Sickle erythrocytes are both an important source and target of reactive oxygen species (ROS). Levels of both total and reduced form of glutathione (GSH), a major intracellular anti-oxidant, have been demonstrated to be decreased in sickle erythrocytes, despite the increased de novo synthesis of GSH in these cells. The mechanism leading to this depletion of intracellular glutathione in sickle erythrocytes is not known yet. After reaction with ROS, GSH is oxidized into its oxidized form (GSSG) and can be transported actively out of the erythrocyte. We questioned whether, during episodes of increased oxidative stress, GSSG efflux in sickle erythrocytes is higher than in normal erythrocytes. Materials and methods Erythrocytes of 10 homozygous sickle cell patients and 9 race-matched healthy controls were stimulated with 2,3-dimethoxy-l,4-naphthoquinone (DMNQ), which induces intracellular ROS generation, and hydrogen peroxide (H2O2) to stimulate GSH consumption. Intra- and extracellular levels of GSH and GSSG were measured at baseline and after 210 minutes of DMNQ and H2O2 stimulation. Results While both intra- and extracellular GSSG levels (μM) at baseline were comparable in sickle and control erythrocytes (14.5(11.5–22.7) vs. 14.3(11.6–16.3) and 0.05(0.00–0.19) vs. 0.07(0.00–0.20) respectively), GSSG levels were significantly higher in sickle erythrocytes after 210 minutes DMNQ stimulation (intracellular: 74.4(52.9–93.1) vs. 45.3(40.8–66.7),P=0.005; extracellular: 23.3(18.2–37.3) vs. 13.2(11.1–14.6),P=0.001) which suggests an increased generation of GSSG intracellularly and a resulting elevated efflux to the extracellular environment. These observations were confirmed with H2O2 stimulation of erythrocytes, showing that, while comparable at baseline, the GSSG levels were higher in sickle erythrocytes after 210 minutes stimulation (intracellular: 26.1(22.8–30.1) vs. 17.5(14.2–20.1),P=0.043; extracellular: 6.9(2.3–16.6) vs.1.2(0.6–1.6),P=0.008). In contrast to the control erythrocytes, where intracellular GSH levels remained unchanged, GSH levels decreased significantly in sickle erythrocytes during DMNQ stimulation, suggesting a limited anti-oxidative reserve capacity in SCD. Conclusion GSSG efflux in sickle erythrocytes is increased and results in net loss of intracellular glutathione, rendering sickle erythrocytes more susceptible to oxidative damage. The higher rate of GSH consumption during an episode of oxidative exacerbation in sickle erythrocytes suggests a reduced anti-oxidative reserve capacity in SCD. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1523 Poster Board I-546 Sickle cell anemia (SCA) is a genetic disorder characterized by recurring episodes of vaso-occlusive crisis (VOC) that can lead to hospitalization or sudden death. Hypoxia is an accepted trigger of sickling and degrees of nighttime hypoxia correlate with strokes and frequency of VOC. To better understand the mechanism of events leading to VOC, we simulated the occurrence of nocturnal hypoxia in SCA patients by administration of five breaths of 100% nitrogen. Tidal volume (Vt), arterial oxygen saturation, electrocardiogram (ECG), and microvascular perfusion (PU) by Laser-Doppler were continuously recorded. We had anticipated a drop in PU after each controlled episode of hypoxia. However, we observed multiple prominent drops in PU in SCA subjects (n=8) that were not as clearly evident in controls (CTL; n=9), and found no direct relationship between hypoxia and change in PU (p = NS). As deep breaths or sighs can trigger reflex peripheral vasoconstriction, we examined Vt respiratory tracings obtained simultaneously and observed that PU drops frequently followed sighs (see Figure) in SCA subjects, but rarely in CTL. A statistical algorithm was used to find all sighs and vasoconstrictive events (PU drops) during each 40-minute experimental session. PU drops were associated with sighs in 7 of 8 SCA patients and in 4 of 9 CTL subjects (P 〈 0.001, Poisson regression analysis). Five CTL and 1 SCA subjects had infrequent sighs and no association between sighs and PU drops. The likelihood ratio of sigh-associated PU drops was significantly higher in SCA than CTL subjects (median = 59.9 % vs. 〈 1 % for SCA vs. CTL, P = 0.008, rank-sum test) whereas the frequency of sighs was not significantly different between the two groups (median = 2.2 % vs. 1.3 % for SCA vs. CTL, P = 0.16, rank-sum test), indicating that SCA patients are much more likely to have sigh-associated peripheral vasoconstriction. Since the sigh-vasoconstrictor response is controlled by the autonomic nervous system (ANS), we measured heart rate variability (HRV) which is an accepted index of sympathetic/parasympathetic balance. These studies showed substantial reduction of parasympathetic modulation of HRV during hypoxia in SCA but not in CTL subjects (p 〈 0.01), indicating a marked abnormality of the ANS in SCA. In overview, the likelihood of coupling between spontaneous sighs and subsequent vasoconstrictive events (PU drops) is much higher in SCA patients than in CTL. Thus, we speculate that a drop in perfusion secondary to increased neural coupling between the lung and vasculature may be an initiating event in VOC. Hypoxia may secondarily promote VOC by altering ANS sensitivity and increasing the probability that a sigh will in turn lead to reflex peripheral vasoconstriction. In a background of HbS, transient decreases in perfusion may prolong red cell residence time in the microvasculature, leading to HbS polymerization, sickling and vascular occlusion. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1515 Poster Board I-538 BACKGROUND Acute chest syndrome (ACS) is a common cause of morbidity and mortality for individuals with sickle cell disease (SCD). The treatment of ACS is mainly supportive. Prior studies have shown that intravenous pulse-dose corticosteroids, such as dexamethasone, can decrease the duration and morbidity of ACS. However, such treatment may also precipitate ‘rebound‘ episodes of vaso-occlusive pain in some individuals that might negate the overall benefit of corticosteroids. Tapered corticosteroid therapy might decrease the rebound effect while maintaining therapeutic benefit. Therefore, we designed a prospective study to begin to test this hypothesis and to assess biomarkers that might clarify the therapeutic and toxic mechanisms of corticosteroids in SCD, predict outcome, and guide therapy. METHODS We conducted a multi-center, placebo-controlled pilot study to test the feasibility and safety of high-dose oral dexamethasone followed by a taper for ACS. Children and adults with SCD and ACS of any severity were randomized to dexamethasone (0.3 mg/kg q12h x 2, 0.3 mg/kg q24h x 2, 0.2 mg/kg q24h x 2, 0.1 mg/kg q24h x2, then stop) or placebo to start within 24 hours of diagnosis. All subjects received standard, protocol-directed supportive care for ACS. We defined the duration of ACS using a novel, objective tool that assessed rate and effort of breathing, oxygen saturation in room air, thoracic pain, and use of supplemental oxygen and ventilatory support. The primary outcomes were the duration of ACS and the duration of hospitalization for ACS. We also measured a panel of biomarkers before, during and after therapy. Inflammatory biomarkers included high sensitivity C-reactive protein and secretory phospholipase A2 (sPLA2). White cell and endothelial activation markers included sVCAM-1, sICAM-1, vWF:Ag and vWF:RCoF, sE-selectin, sP-selectin, sL-selectin, nitric oxide metabolites (NO), and whole blood tissue factor. We used generalized linear mixed models controlling for age to test for differences between treatment groups. RESULTS We enrolled 12 subjects (9 children, 3 adults; mean age 17.3 years, range 5 - 45) with homozygous sickle cell anemia at 4 centers and randomized 11 (1 drop-out) to either dexamethasone (N=5) or placebo (N=6). The objective ACS assessment tool was completed on all subjects without difficulty. In this pilot study, dexamethasone reduced the duration of hospitalization (41.5 vs 62.3 hrs; P=0.024), but not the duration of ACS (log of duration 2.4 vs 3.5 hrs; P=0.127), supplemental oxygen (17.5 vs 41.2 hrs; P=0.876), hypoxemia (13.8 vs 34.3 hrs; P=0.770), or total opioid usage in morphine equivalents (54.4 vs 68.8 mg; P=0.885). There were no statistically significant differences in adverse events between arms. However, 3 patients treated with dexamethasone had a painful event in the 2 weeks after hospital discharge (1 required re-hospitalization), compared to 1 patient in the placebo group (0 re-hospitalizations). No marked leukocytosis occurred in either treatment group, but the leukocyte count at 1-week follow-up had decreased less from baseline in the dexamethasone group compared to placebo (-17.7 vs -37.6%; P=0.028). The baseline sPLA2 concentration was 3 100 ng/mL in 4/5 and 4/6 patients in the dexamethasone and placebo arms. The white cell activation marker, sL-selectin was significantly decreased at 1-week follow-up in the dexamethasone group compared to placebo (573.8 ng/mL vs 742.8; change from baseline -121.2 vs 57.2; P

Description: Abstract 153 Outside-in signal transduction is one of several amplification pathways that platelets employ following their adhesion to components of the extracellular matrix or to multivalent ligands like fibrinogen and von Willebrand factor. Previous studies have shown that outside-in signal transduction initiated by the binding of fibrinogen to its receptor, the integrin αIIbβ3, results in the activation of β3-associated Src family kinases (J Cell Biol 157:265, 2002; J Cell Sci 121:1641, 2008) that phosphorylate key tyrosine residues within the cytoplasmic domain of the transmembrane immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor protein, Fcγ;RIIa (Blood 112:2780, 2008). “Activation” of FcγRIIa sets off a cascade of events that facilitate assembly and activation of other key signaling intermediates, including the tyrosine kinase Syk and phospholipase Cγ2. PLCγ2, through its lipase activity, generates lipid products that support a multitude of cellular activation responses, including cytoskeletal rearrangements leading to platelet shape change and spreading, secretion of platelet granules, and activation of additional cell surface integrins. Curiously, mice lack the gene encoding FcγRIIa, and murine platelets are notoriously poor at sending αIIbβ3-mediated outside-in signals into the cell. The purpose of the current investigation, therefore, was to determine whether enforced expression of FcγRIIa in mouse platelets might serve to amplify murine platelet activation downstream of ligand binding to αIIbβ3. We found that the spreading of platelets derived from FcγRIIa transgenic mice was more robust and extensive compared to that of their wild-type counterparts. ADP- and collagen related peptide-induced granule secretion, as measured by P-selectin exposure, was also enhanced, as was the degree of integrin of αIIbβ3 activation, as reported by the binding of the activation-dependent monoclonal antibody, JON/A. Taken together, these data provide further support for importance of FcγRIIa in transmitting αIIbβ3-mediated amplification signals into the cell, and help to explain the often-observed defect in αIIbβ3-mediated signal transduction in murine platelets. Disclosures: Newman: New York Blood Center: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Consultancy.

Description: Abstract 1520 Poster Board I-543 The adhesion of both red and white cells to the vessel walls of the microcirculation initiates vaso-occlusion in sickle cell disease (SCD). The chronic inflammatory nature of SCD leads to elevation of circulating cytokines in patients, which may contribute significantly to the activation of blood cells and their consequent adhesion. Nitric oxide (NO) has important inhibitory effects on cellular adhesive properties and drugs that enhance NO bioavailability or NO-cGMP-dependent signaling may hold potential for treatment of various aspects of SCD. This study aimed to observe the effect of cytokines, often found augmented in the plasma of SCD individuals, on the in vitro adhesive properties of neutrophils (neu) and red blood cells (RBC) from healthy control (CON) and steady-state SCD (SCD) individuals. Furthermore, the effects of BAY 73-6691, an inhibitor of the cGMP-hydrolyzing enzyme, phosphodiesterase 9A (PDE9A) and BAY 41-2271, a guanylate cylase activator, on non-stimulated and cytokine-stimulated cell adhesion were determined. Neus and RBCs were isolated from peripheral blood of CON and SCD individuals. Cell adhesion (5×106neu/ml in RPMI or 2×108RBC/ml in HBSS) to immobilized fibronectin (FN;20μg/ml) was assessed using static adhesion assays (30min, 37°C, 5%CO2) in the presence or absence of cytokines IL-8 (100-500ng/ml), TNF-alpha (0.1-1μg/ml) and GM-CSF (1-100ng/ml) and/or in the presence/absence of BAY 73-6691 (10-60μM), BAY 41-2271 (60-150nM) or DMSO vehicle (0.02%v/v). As previously demonstrated, SCDneu have a greater capacity to adhere to FN than CONneu (see Table). Stimulation of cells in vitro with all three cytokines significantly augmented both CONneu adhesion to FN and further increased SCDneu adhesion (Table). Co-incubation of both CONneu and SCDneu with BAY 73-6691, but not BAY 41-2271, significantly reduced their adhesions to FN (Table). Furthermore, BAY 73-6691, but essentially not BAY 41-2271, significantly inhibited CONneu and SCDneu adhesion stimulated by IL-8, TNF-alpha and GM-CSF (Table). DMSO vehicle had no significant effect upon neu adhesion (data not shown). As previously reported, SCD RBC have a greater capacity to adhere to FN, in vitro, compared to CON RBC (12.8±1.7% comp. 7.8±1.0%, n=7, p0.05) (1μg/ml TNF-alpha: CON RBC, 7.0±1.0%; SCD RBC, 11.4±1.9%, n=6, p〉0.05). Furthermore, BAY 73-6691 and BAY 41-2271 did not affect either basal CON RBC (data not shown) or SCD RBC adhesion (SCD RBC basal; 14.8±1.0%; 60μM BAY 73-6691, 16.4±1.3%; 150nM BAY 41-2271, 15.7±1.2%, n=6). Key SCD inflammatory mediators were found, at physiologically relevant concentrations, to augment the adhesive properties of neutrophils, but not RBC, from control and SCD individuals. Circulating inflammatory cytokines may play a role in the induction of leukocyte adhesive properties in SCD; in contrast factors other than inflammatory stimuli may be more important for induction of SCD RBC adhesion. Data suggest that elevation of intracellular cGMP may be an important approach for reducing SCD leukocyte, but not SCD RBC, adhesive properties, even in an inflammatory environment. PDE9A is highly expressed in hematopoietic cells and inhibition of this enzyme, with consequent augmentation of cGMP, may represent a tissue/cell-specific therapeutic drug target worthy of further in vitro and in vivo studies as a therapy for SCD. Table Effect of cytokine stimulation and cGMP-elevating agents upon adhesive properties of neutrophils from healthy control and steady-state SCD individuals % Neutrophil adhesion to Fibronectin (mean±SEM) Controls SCD Basal N=18 IL-8 (200ng/ml) N≥7 TNF-alpha (500ng/ml) N≥7 GM-CSF (100ng/ml) N≥7 Basal N=18 IL-8 (200ng/ml) N≥7 TNF-alpha (500ng/ml) N≥7 GM-CSF (100ng/ml) N≥7 Basal 16.1±1.5 26.6±2.8 27.4±5.5 21.4±1.6 21.4±1.9 32.5±3.5 29.2±3.0 25.2±2.2 * p

Description: Abstract 1508 Poster Board I-531 Developmental hematopoiesis satisfies the immediate needs of the embryo and provides the hematopoietic stem cells necessary for lifelong blood homeostasis. Recently, the mid-gestation mouse placenta was identified as an important definitive hematopoietic organ; data from us and others indicates that the human placenta functions analogously, harboring and potentially generating hematopoietic stem and progenitor cells. The function of the human placenta in embryonic hematopoiesis has not been addressed. We assessed the hematopoietic potential of placental tissues before the onset of feto-placental circulation and found robust de novo generation of clonogenic progenitors. Interestingly, pre-circulation progenitors were greatly enriched (69 ± 14% of total) in macrophage progenitors. Immunostaining demonstrated that these progenitors are generated in the chorionic plate. As development progresses, placental macrophages migrate to the villous stroma. Surprisingly, in the villi, placental macrophages associate closely with an unexpected population of extravascular, z-globin+ primitive erythroid cells, prompting the hypothesis that embryonic macrophages promote the maturation of primitive erythroblasts in the placenta. Concordantly, we found that human primitive erythroblasts enucleate, as has been recently demonstrated in mice. Interestingly, the first enucleated erythroid cells were found in the villous stroma; between 5-7 weeks developmental age, their relative frequency in the stroma was 2-4 fold higher than in lumens. We also observed free nuclei in the placental stroma; a similar population of ejected red cell nuclei, termed pyrenocytes, has recently been described in mouse embryos. Immunohistochemistry and FACS confirmed that these free nuclei in the placenta were pyrenocytes. Electron microscopy revealed placental macrophages containing ingested red cell nuclei. Together, this data suggests that placenta-derived macrophages promote the terminal maturation of primitive erythroblasts in the villous stroma, nominating the first trimester human placenta as a site of primitive hematopoiesis. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1511 Poster Board I-534 Background What determines the degree of hemolysis and of anemia in patients with hemoglobin SS is not fully known. The rate of hemolysis and severity of anemia are ameliorated by the presence of alpha thalassemia and by higher hemoglobin F percentage. Mild G6PD deficiency in the form of G6PD-202/-376 may be associated with episodic hemolysis in individuals of African descent, but past studies indicated little influence of G6PD-202/-376 on the degree of hemolysis and anemia in sickle cell disease patients (1,2). In this study we examined the roles of single and double α-globin deletions and G6PD-202/-376 on the degree of hemolysis and the hemoglobin concentration in hemoglobin SS patients. Methods Two hundred sixty two children and adolescents with hemoglobin SS were recruited at three tertiary medical centers and studied at steady state. Principal component analysis was used to develop a hemolytic component from concentrations of lactate dehydrogenase, aspartate aminotransferase and bilirubin. PCR was used to determine the presence of α-thalassemia and G6PD-202/-376. Multivariate models were employed to determine the independent effects of these genotypes on hemoglobin concentration and degree of hemolysis. Results Single a-globin deletion was associated with an estimated 0.4 g/dL increase in steady-state hemoglobin concentration and double α-globin gene deletion with a 0.8 g/dL increase (P = 0.005 for trend) due to, progressively lower degrees of hemolysis (P = 0.004). G6PD-202/-376 was associated with an estimated 0.7 g/dL decrease in the hemoglobin concentration (P = 0.003) (Figure 1a), but this observation could not be explained by increased hemolysis. Rather, the reticulocyte count was an estimated 22% lower with G6PD-202/-376 (P = 0.032) (Figure 1b). Discussion G6PD -202/-376 may be associated with lower hemoglobin concentration in sickle cell anemia and the mechanism is probably impaired erythropoiesis rather than hemolysis. A recent study (3) indicates that G6PD is needed for definitive erythropoiesis as well as for normal survival of red blood cells in the periphery. Our present findings raise the possibility that, in the setting of the markedly increased erythropoiesis of sickle cell anemia, G6PD-202/-376 may result in impairment in erythropoiesis that is discernible in the peripheral blood hemoglobin concentration. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1513 Poster Board I-536 Introduction Treatment of sickle cell anemia with hydroxyurea (HU) is associated with significant decreases in the frequency of painful crises, acute chest syndrome, morbidity, and mortality. Some patients, however, show no improvement even with prolonged HU therapy. Identifying treatment responders is important for predicting clinical improvements and for assessing the risk/benefit ratio of HU treatment for individual patients. The salutary effects of HU are thought to be the result of increasing the fetal hemoglobin (Hb F) level. NHLBI guidelines for sickle cell treatment define levels of 15%-20% Hb F as therapeutic endpoints. Research and reviews based on pediatric and adult patients have variously argued that levels from about 10% to 20% are beneficial. Patients and Methods Patients in this study were from the Multicenter Study of Hydroxyurea (MSH) in Sickle Cell Anemia, a randomized double-blind placebo controlled trial of HU. The N=299 adult patients were recruited from 21 sites across the U.S. and Canada, and were evenly distributed between males and females. Following randomization to placebo or HU, patients had biweekly follow-up visits until the trial was terminated early due to a significant reduction in painful crises (the primary study endpoint) in the HU arm. Levels of Hb F in MSH patients were assessed at baseline and again approximately 18-21 months after treatment began, with the level at each time being the average of two measurements. In the previously reported MSH study, patients were divided into quartiles of Hb F change as a measure of response to HU treatment. In this approach the bottom two quartiles showed either no or minimal positive change in Hb F levels, and fully overlapped with placebo group in the extent of change. We redefined HU patients as ‘responders’ or ‘nonresponders’ based on a 15% Hb F threshold; those with baseline HbF below 15% and follow-up above 15% were labeled ‘responders,’ while all others were labeled ‘nonresponders.’ The 15% level was chosen due to its frequent identification in previous publications as a level at which meaningful benefits could be expected. For both coding schemes, we compared the following outcomes between subgroups: rate of painful crises, proportion of days at home with pain and with opioid use, and average daily pain. Results Using the 15% rule, responders had significantly better outcomes than nonresponders on all outcome measures: rate of painful crises (p=.011), proportion of at-home days with pain (p=.025), proportion of days with analgesic use (p=.002), and average daily pain (p

Description: Abstract 1496 Poster Board I-519 Obesity and subsequent diabetes have emerged as major health problems in the U.S. While the consequences of elevated blood glucose levels on the cardiovascular system and other organs are well known, the direct effects on the hematopoietic system are more elusive. Similarly, the impact of gestational diabetes on embryonic hematopoiesis has not been examined in detail. The zebrafish has emerged as an important model system to study conserved regulators of organ development and homeostasis. In order to evaluate the role of elevated glucose levels on hematopoietic stem cell (HSC) production, zebrafish embryos were exposed to increasing doses of D-glucose from 5 somites to 36 hours post fertilization (hpf); HSC number, as indicated by in situ hybridization for the conserved markers runx1 and cmyb in the Aorta-Gonad-Mesonephros (AGM) region, was increased at 0.5, 1% and 2% glucose; results were confirmed by in analysis of CD41 expression. Quantification using FACS analysis of fluorescent HSC reporter embryos and qPCR revealed a 2-3-fold enhancement following 1% glucose treatment. Other mono, di-, and trisaccharide sugars had similar effects, causing increased numbers of HSCs, however, L-glucose had no impact. BrdU incorporation in the AGM region was elevated after 1% glucose treatment, while acridine orange staining revealed an inhibitory effect on apoptosis. To evaluate potential mediators of these glucose-responsive effects, embryos were injected with antisense morpholino oligonucleotides (MO) against both the insulin (insr), and insulin-like growth factor receptors (igfr); insr and igfr receptors can each bind insulin, released following elevations in blood sugar levels. MO knockdown of insra or igfrb, but not igfra, influenceded runx1+ HSCs substantially, indicating an important role of these endocrine regulatory signaling pathways in HSC formation. However, D-glucose completely reversed these effects, implying either functional redundancy, or a multi-step, multi-effector process of HSC regulation by endocrine factors. To further clarify when insr- and/or igfr-mediated activity was influencing HSC formation and to correlate that effect with elevated glucose exposure, embryos were treated for defined periods with either 1% glucose, insulin, or IGF; exposure from 10 somites to 24 hpf influences the formation and arterial/venous specification of dorsal aorta, the conserved site of initial definitive HSC production, while exposure from 24 to 36 hpf regulates HSC induction. IGF exerted a positive effect on HSCs only after the establishment of the hematopoietic niche (〉24hpf). Glucose treatment, however, positively influenced HSC formation at all time points examined, suggesting it works not only in the HSC niche to induce HSCs, but also prior to HSC formation. MO knockdown of the glucose transporter glut1 resulted in diminished HSC production, confirming a direct role of glucose in this process. To determine whether the effect of glucose elevation was mediated by changes in cellular energy production, embryos were exposed to chemical inhibitors of oxidative phosphorylation. Cyanide and oxaloacetate reversed the beneficial effects of D-glucose, indicating that energy production can modulate HSC formation. Investigation into the functional redundancy and cross-regulation of insulin and IGF on HSC self-renewal and the evolutionary conservation of the effects of energy metabolism on HSC production are ongoing; further studies will be needed to determine if glucose maintains an influential role on HSC homeostasis or bone marrow recovery following injury. These results could have an impact on methods for HSC modulation for therapeutic purposes, and may further unveil specific risks of obesity and diabetes for hematopoiesis and HSC homeostasis during gestation and in the adult. Disclosures: Goessling: Fate Therapeutics: Consultancy, Patents & Royalties. North: Fate Therapeutics: Consultancy, Patents & Royalties.

Description: Abstract 146 Antiphospholipid syndrome (APS) is characterized by thrombosis, recurrent fetal loss and the presence of the lupus anticoagulant, anticardiolipin antibodies or anti-beta-2 glycoprotein 1 antibodies. Sera from patients with APS contain polyclonal antibodies that bind to various plasma proteins including beta-2 glycoprotein 1. Although beta-2 glycoprotein 1 antibodies have been well-documented as a biomarker for the diagnosis of APS, their direct role in the pathogenesis of thrombosis is unknown. Here, we have demonstrated using intravital microscopy that purified anti-beta-2 glycoprotein 1 antibodies isolated from the serum of a patient with APS greatly amplify thrombus size following laser-induced vessel wall injury in live mice. A patient with systemic lupus and APS complicated by pulmonary embolism was studied. IgG was isolated from serum by affinity chromatography using a Protein A/G column. Anti-beta-2 glycoprotein 1 antibodies in the IgG fraction were affinity-purified using homogeneous beta-2 glycoprotein 1 covalently bound to CNBr-activated agarose beads. Purified anti-beta-2 glycoprotein 1 antibodies were eluted at low pH. Patient IgG depleted of anti-beta-2 glycoprotein 1 antibodies was obtained by repeated chromatography over the beta-2 glycoprotein 1 column. The effects of (a) purified anti-beta-2 glycoprotein 1 antibodies, (b) anti-beta-2 glycoprotein 1 antibody-depleted patient IgG, and (c) IgG from normal human sera on thrombus formation were studied quantitatively in the live mouse. Intravital microscopy was performed using the cremaster muscle as a vascular window, and thrombus formation was initiated by laser injury to the arteriolar wall. Five minutes prior to vessel wall injury, purified anti-beta-2 glycoprotein 1 antibodies, anti-beta-2 glycoprotein 1 antibody-depleted patient IgG, or normal human IgG were infused via a jugular catheter. Platelet thrombus size was determined by widefield microscopy and Alexa 647-conjugated Fab fragments of an anti-CD 41 monoclonal antibody. Up to 10 thrombi were generated per mouse, and the median integrated fluorescence for 25-30 thrombi determined. Infusion of anti-beta-2 glycoprotein 1 antibodies increased thrombus size in a dose-dependent manner. Infusion of purified anti-beta-2 glycoprotein 1 antibodies at 0.12 μg/g mouse and 0.40 μg/g mouse increased thrombus size by about 18-fold and 122-fold respectively over thrombi formed in untreated mice. However, anti-beta-2 glycoprotein 1 antibody-depleted patient IgG and normal human IgG did not affect platelet thrombus size. These results indicate that the anti-beta-2 glycoprotein 1 antibodies isolated from APS patient serum are responsible for markedly increased thrombus size in this thrombosis model. The target cellular antigen of the anti-beta-2 glycoprotein 1 antibodies and the mechanism of enhanced thrombus formation remain unknown. However, these results provide evidence that anti-beta-2 glycoprotein 1 antibodies are not only a marker but are directly involved in the pathogenesis of thrombosis. This in vivo animal model offers an approach to identifying inhibitors of anti-beta-2 glycoprotein 1-mediated thrombosis. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1455 Poster Board I-478 Sustained production of all mature blood cell types relies on the continuous proliferation and differentiation of a rare population of self-renewing, multipotent hematopoietic stem cells (HSCs). HSC maintenance and lineage differentiation are thought to be regulated by spatially confined niches, defined by cellular components, soluble regulators, and the extracellular matrix immediately surrounding stem cells. Identification of these microenvironments in which endogenous and transferred HSCs reside within the BM is a major challenge in stem cell biology with relevant clinical implications. Yet the extreme rarity of HSCs, their dynamic nature, and the lack of specific markers to identify them, have precluded an accurate definition of HSC niches to date. Quantitative imaging technologies such as Laser Scanning Cytometry (LSC) are designed for the automated analysis of large cell numbers at a single cell level with high resolution while preserving the morphological information lost in flow cytometry, therefore providing data of statistical significance even for rare cell populations such as HSCs. We have employed LSC to analyze the localization of both adoptively transferred and endogenous hematopoietic stem and progenitor cell (HSPC) populations inside whole longitudinal sections of murine femoral BM cavities. Our results indicate that, as previously suggested, purified HSPC (Lin−c-kit+Sca-1+) significantly accumulate in endosteal regions (ER) of BM cavities (within 100μm of inner bone surface) upon transplantation. Nevertheless, analysis of sufficient numbers of more differentiated cell subsets (Lin−c-kit+Sca-1− progenitors, pro B cells and mature B cells) indicated that these areas serve as homing sites for most hematopoietic cells, highlighting the limitations of any conclusions drawn on HSC niche identity from studies performed with transferred HSPC populations. Immunofluorescent staining of endogenous cell populations revealed a gradient in distribution of early hematopoietic progenitors (c-kit+), which accumulated in but were not restricted to ER regions. Of note, a vast majority (〉80%) of HSPC (Bmi-GFPhic-kit+, or Lin−c-kit+Sca-1+),were found inside ER, although not directly adjacent to endosteal surfaces. Our studies define endosteal areas as tissue regions where HSPC reside in close proximity, but not necessarily in direct contact with a dense vascular network, osteoblastic cells and other potential niche cell types and growth factors currently under investigation. Disclosures No relevant conflicts of interest to declare.

Description: A population-based cohort was used to determine the incidence and risk factors associated with development of venous thromboembolism (VTE) among Californians diagnosed with acute leukemia between 1993 to 1999. Principal outcomes were deep vein thrombosis in both the lower and upper extremities, pulmonary embolism, and mortality. Among 5394 cases with acute myelogenous leukemia (AML), the 2-year cumulative incidence of VTE was 281 (5.2%). Sixty-four percent of the VTE events occurred within 3 months of AML diagnosis. In AML patients, female sex, older age, number of chronic comorbidities, and presence of a catheter were significant predictors of development of VTE within 1 year. A diagnosis of VTE was not associated with reduced survival in AML patients. Among 2482 cases with acute lymphoblastic leukemia (ALL), the 2-year incidence of VTE in ALL was 4.5%. Risk factors for VTE were presence of a central venous catheter, older age, and number of chronic comorbidities. In the patients with ALL, development of VTE was associated with a 40% increase in the risk of dying within 1 year. The incidence of VTE in acute leukemia is appreciable, and is comparable with the incidence in many solid tumors.

Description: Currently, FLT3 tyrosine kinase inhibitors (TKIs) are emerging as the most promising drug therapy to overcome the dismal prognosis of acute myelogenous leukemia (AML) patients harboring internal tandem duplications (ITDs) of FLT3. However, up-front drug resistance occurs in approximately 30% of patients, and molecular mechanisms of resistance are poorly understood. Here, we have uncovered a novel mechanism of primary resistance to FLT3 TKIs in AML: an FLT3 receptor harboring a nonjuxtamembrane ITD atypically integrating into the β-2 sheet of the first kinase domain (FLT3_ITD627E) induces dramatic up-regulation of the anti-apoptotic myeloid cell leukemia 1 protein (MCL-1). Using RNA interference technology, deregulated MCL-1 protein expression was shown to play a major role in conferring the resistance phenotype of 32D_ITD627E cells. Enhanced and sustained binding of the adaptor protein GRB-2 to the FLT3_ITD627E receptor is involved in MCL-1 up-regulation and is independent from TKI (PKC412)–induced inhibition of the receptor kinase. Thus, we describe a new mechanism of primary resistance to TKIs, which operates by reprogramming local and distant signal transduction events of the FLT3 tyrosine kinase. The data presented suggest that particular ITDs of FLT3 may be associated with rewired signaling and differential responsiveness to TKIs.

Description: Abstract 3132 Poster Board III-69 Women with a past history of venous thrombosis are at higher risk for venous thromboembolism (VTE) during and after pregnancy. The highest risk period is the first four weeks post partum. For a woman whose previous event was secondary to a major transient risk factor the antepartum risk of recurrent VTE is low whereas for women whose previous event was idiopathic the antepartum risk is higher. In our institution, for women whose previous event was secondary to a major transient risk factor, standard treatment is to follow closely with no thromboprophylaxis antepartum and then receive either prophylactic low molecular weight heparin (LMWH) or warfarin for six weeks postpartum. For women whose previous event was idiopathic or who were on warfarin at the time of becoming pregnant they receive prophylactic LMWH antepartum with a scheduled delivery and are put back on warfarin if they have long term anticoagulation needs or LMWH for six weeks if they don't have longterm anticoagulation needs. We report the outcome for our patients from 1997 to 2008. All patients were followed for the duration of pregnancy and for 6 weeks postpartum for pregnancy outcome, recurrent VTE, and major bleeding in the thrombosis clinic of London Health Sciences Centre. There were a total of 90 women; 30 women (mean age 30.6 years) with a history of previous secondary thrombosis with 37 pregnancies and 60 women (mean age 29.5 years) with past idiopathic thrombosis with 99 pregnancies. For the secondary group there was 1 episode (2.70%; 95% CI 0.48-13.82) of antepartum recurrent VTE whereas for the idiopathic group there were 3 episodes (3.03%; 95% CI 1.04-8.53). There was no statistical difference between groups (p=1.0). There were no episodes of postpartum VTE recurrence or major hemorrhage for either group. For the secondary group there was 1 fetal loss at 23 weeks (2.7%; 95% CI 0.48-13.82) whereas for the idiopathic group there were 6 fetal losses at 8, 10, 10, 11, 22, 37 weeks gestation (6.06%; 95% CI 2.81-12.60). There was no statistically significant difference between groups (p=0.77). This retrospective review suggests that for pregnant women with a past history of VTE, a strategy of no antepartum prophylaxis for previous secondary thrombosis and antepartum prophylactic LMWH for previous idiopathic thrombosis as well as prophylactic LMWH or warfarin postpartum is efficacious and safe. Disclosures Off Label Use: low molecular weight heparin for prevention of VTE in pregnancy.

Description: Abstract 3003 Poster Board II-980 Calmodulin (CaM) is a calcium-sensing protein ubiquitously expressed in every eukaryotic cell type regulating biological processes such as cell proliferation, vesicular fusion, fertilization and apoptosis. CaM antagonists induce apoptosis in various tumor models and inhibit tumor cell invasion and metastasis, thus some of which have been extensively used as anti-cancer agents. Tamoxifen (TMX), a potent antagonist of CaM, has been in the center of management of hormone-sensitive breast cancer, and also represents the best example of chemo-prevention to reduce the incidence of invasive breast cancer. Furthermore, TMX is potentially useful in treatment of other kinds of cancer. However, TMX has some severe side effects, one of which is thrombocytopenia. Up to now, the pathogenesis of thrombocytopenia still remains unclear. In platelets, CaM has been found to bind directly to cytoplasmic domains of several platelet receptors. Incubation of platelets with CaM antagonists impairs the receptors-related platelet function. However, it is still unclear whether CaM antagonists, especially TMX, induce platelet apoptosis. Here, we show that CaM antagonists TMX and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7) dose-dependently induce apoptotic events in human platelets, including depolarization of mitochondrial inner transmembrane potential, caspase-3 activation, gelsolin cleavage and phosphatidylserine (PS) exposure. CaM antagonist did not incur platelet activation as detected by P-selectin surface expression and PAC-1 binding. However, ADP- and botrocetin-induced platelet aggregation and platelet adhesion and spreading on von Willebrand factor surface were significantly reduced in platelets pre-treated with CaM antagonist. Therefore, these findings indicate that CaM antagonists induce platelet apoptosis, which suggests a possible pathogenesis of thrombocytopenia in some patients treated with CaM antagonist drugs, and also may present as a novel mechanism for platelet clearance and dysfunction in vivo or in vitro. The elevation of the cytosolic Ca2+ level may involve in the regulation of CaM antagonist-induced platelet apoptosis. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3004 Poster Board II-981 Protein Kinase C-delta (PKCδ) is a novel PKC isoform that differentially regulates platelet dense granule secretion. PKCδ positively regulates Protease activated receptor (PAR)-mediated dense granule secretion, whereas it negatively regulates glycoproteinVI (GPVI)-mediated dense granule secretion in platelets. PKCδ, a serine/threonine kinase is phosphorylated on its tyrosine residues. There are nine potential tyrosine phosphorylation sites in the regulatory domain of PKCδ. Phosphorylation at different tyrosine residues regulates its substrate specificity. We have previously shown that the association of PKCδ with Lyn and SHIP-1 negatively regulates GPVI-mediated dense granule secretion. However, the event leading to the association between PKCδ and SHIP-1 is not known. We hypothesize that the differential tyrosine phosphorylation of PKCδ downstream of PARs or GPVI receptors result in the preferential association with SHIP-1. In the current study, we show that PKCδ is phosphorylated at tyrosine residues Y332, Y523, Y525 and Y565 upon PAR or GPVI stimulation. Y311 residue is predominantly phosphorylated upon stimulation of PARs, whereas Y155 residue is preferentially phosphorylated upon GPVI stimulation. PAR-mediated Y311 phosphorylation peaks at later timepoint, whereas GPVI-mediated Y155 phosphorylation peaks at an early timepoint. correlating with dense granule secretion. Furthermore, we show that agarose-conjugated Y155 phosphorylated PKCδ peptide associates with SHIP-1 upon GPVI stimulation, and not PARs. These data suggest that the phosphorylation of PKCδ at distinct tyrosine residues differentially regulate its association with SHIP-1. Therefore, we conclude that the GPVI-mediated phosphorylation of PKCδ at 155 is required for its association with SHIP-1. This study is supported by pre-doctoral fellowship to Ramya Chari from American Heart Association, Pennsylvania-Delaware affiliate. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2999 Poster Board II-968 The Gi-coupled ADP receptor, P2Y12, is the target of clopidogrel bisulfate (Plavix), currently the most successful anti-platelet strategy used in the clinic. In a recent study, we have shown that the Ca2+-sensing nucleotide exchange factor, CalDAG-GEFI, and P2Y12 represent the major signaling pathways leading to Rap1 and integrin activation in platelets (Cifuni et al., 2008, Blood). In the present study, we have further evaluated the importance of CalDAG-GEFI signaling and Rap1 activation for various aspects of platelet activation, and we have compared thrombus formation of CalDAG-GEFI−/− and WT/clopidogrel platelets under static and flow conditions in vitro. Our studies establish a revised model for platelet activation by collagen. In platelets activated with threshold concentrations of GPVI agonists, CalDAG-GEFI serves as a highly sensitive response element to Ca2+ that allows for the rapid activation of Rap1. CalDAG-GEFI-mediated Rap1 activation triggers a first wave of integrin activation and ERK (MAPK) signaling, followed by TxA2 release. TxA2 provides crucial feedback for the activation of PKC and granule/ADP release. ADP in turn triggers the second, P2Y12-dependent wave of Rap1-mediated signaling events, leading to the sustained activation of integrins and further release of TxA2. Higher concentrations of GPVI agonists lead to the concomitant activation of CalDAG-GEFI and PKC, facilitating platelet aggregation independent of feedback by endogenous TxA2. Under physiological flow conditions, CalDAG-GEFI-dependent platelet activation (clopidogrel-treated WT platelets) allowed for the formation of small but unstable thrombi, which rapidly disintegrated at high shear rates. In contrast, CalDAG-GEFI−/− platelets (P2Y12-dependent platelet activation) in anticoagulated blood firmly adhered to the thrombogenic surface but failed to form thrombi, even at high concentrations of collagen. Addition of exogenous TxA2 to anticoagulated CalDAG-GEFI−/− blood did not restore thrombus formation under flow. However, small thrombi were observed with non-anticoagulated CalDAG-GEFI−/− blood perfused at venous but not arterial shear rates, suggesting that a) locally generated thrombin facilitates the recruitment of free flowing CalDAG-GEFI−/− platelets to already adherent platelets, and b) the slow kinetics of P2Y12-dependent Rap1 activation only supports thrombin-induced platelet-platelet cohesion at low shear conditions. In conclusion, our studies demonstrate that CalDAG-GEFI/Rap1 signaling plays a critical role for the first wave of integrin activation and TxA2 generation important for platelet adhesion to a thrombogenic surface. Signaling by P2Y12/Rap1 is essential for sustained platelet activation/thrombus stabilization and partially compensates for CalDAG-GEFI/Rap1-mediated platelet adhesion under low flow conditions. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3000 Poster Board II-977 Human platelets synthesize and store functionally silent tissue factor (TF) which expresses procoagulant activity (PCA) after platelet activation. Fast activation of TF was elicited by VWF-Ristocetin (VWF-R) through GPIbαa activation and Src-Lyn transduction pathway (Blood, Nov 2008; 112:113). Given that GPVI, along with GPIb and TF have been found in “lipid rafts”, and the activated form of GPVI signals through Fyn, another member of the Src family, we tested if GPVI was involved in TF-initiated PCA. We also studied the time-course and pathway specificity of TF activation and the role of platelet FVII in PCA. Weak TF immunofluorescence and co-localization with GPIba were observed in non stimulated washed platelets. A mild increase of TF fluorescence was detected 2 min after TRAP activation, which augmented when the stimulus was VWF-R. Furthermore, striking enhancement of TF fluorescence occurred 2 min after depositing platelets over a VWF-coated surface, but not over fibrinogen or albumin. Platelets adherent to VWF matrix showed GPIb clustering and loss of co-localization with TF. Externalization of TF was confirmed by immunoprecipitation (Ip) of biotinylated membranes before and after platelet activation. Concomitantly, TF-dependent FXa generation increased 5-10-fold shortly after VWF stimulus. Washed platelets stimulated with VWF-R agglutinated normally when stirred in an aggregometer, and the fraction of platelets exposing anionic phospholipids (annexin V binding) was similar to parallel samples stimulated with TRAP. However, VWF-R induced null 14C-serotonin secretion and P-selectin exposure (flow cytometry) in washed platelets. In contrast, TRAP, collagen, ADP and convulxin induced full platelet aggregation, 14C-serotonin and P-selectin secretion at 2-5 min, but with no increase in FXa generation. Platelet PCA was inhibited by antibodies against TF, GPIba, FVIIa, as well as by SU6656 and PP2 (Src pathway inhibitors), but not by Gö6850 (a PKC inhibitor) or exogenous TFPI. p85, a subunit of PI-3K constitutively associated with GPIb complex, becomes strongly associated with TF after stimulation with VWF-R, though only weakly after TRAP activation, confirming the coordinate activation of GPIb and TF. FVII and FX were revealed in platelet membrane fractions by immunoblotting and both co-precipitate with TF in non-stimulated platelets. Two min after activation with VWF-R striking co-precipitations of TF with FVII and FX light chains were evidenced, denoting activation of platelet FVII and FX. When exogenous FX was added to the assay, the amount of FXa generated after 1 and 2 min stimulation was similar whether or not exogenous FVIIa was added. Platelets from four non-related patients with bleeding related to hereditary defect of GPVI had null aggregation and secretion with convulxin and collagen, less than 7% labeling of GPVI by flow cytometry and an immunoreactive membrane GPVI of Mr≈40kDa (native GPVI Mr=62kDa). All of them had normal agglutination with VWF-R and normal FXa generation. In summary, GPIb activation by VWF constitutes a unique and fast inducer of platelet TF-dependent PCA. This process requires anionic phospholipid exposure, but is independent of platelet GPIIb/IIIa and GPVI function. Platelet FVII can initiate FXa generation without need of plasma FVII. The associations of platelet FVII and FX with TF on membrane fractions, together with the large amount of FV in platelets, indicate that human platelets provide not just TF and a PCA phospholipid platform, but also all the components of the prothrombinase complex to trigger the clotting process. Taken together, our results underline the central role of platelets in the whole hemostatic process, unifying primary and secondary hemostasis and circumscribing thrombin generation and fibrin deposition where platelet plug is being formed. Platelet PCA should become a pharmacological target for preventing or managing bleeding and thrombotic disorders. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2988 Poster Board II-964 BACKGROUND: In pediatric venous thromboembolism (VTE), duration of hypercoagulability—which has implications for recurrence risk and duration of therapy–is not readily assessed despite a battery of laboratory assays for comprehensive thrombophilia testing. Global clotting assays offer the possibility to better understand duration of hypercoagulability on an individualized basis. OBJECTIVE: We sought to evaluate overall coagulability and fibrinolytic potential over time in children with acute VTE and to compare these findings with D-dimer and established thrombophilia traits. METHODS: The Clot Formation and Lysis (CloFAL) global assay was performed in platelet-poor plasma in 58 children enrolled in a single-institutional prospective inceptional cohort study of VTE at The Children's Hospital, Colorado, between March 2006 and June 2009. This spectrophotometric fibrin registration assay employing clotting activation with dilute tissue factor and phospholipid and fibrinolytic enhancement with tissue plasminogen activator has been previously shown analytically sensitive to physiologic and pathologic alterations in multiple components of the coagulation and fibrinolytic systems (Goldenberg et al., Thromb Res 2005). Hypercoagulability was defined by an area under the curve (AUC) of the CloFAL waveform that exceeded the upper limit of age-appropriate reference values established in healthy children (n=26) using the non-parametric method of Tukey, and hypofibrinolysis was similarly defined as a fibrinolytic index (FI, which relates to the slope of decline in absorbance over the period of 30 minutes following maximal amplitude of clot formation) that was below the lower limit of normal. Coagulation index (CI, measured as the AUC over the first 30 minutes of the assay, indexed to the pooled normal plasma standard) was also evaluated, and compared to established reference ranges. Analyses were grouped by period post-diagnosis, as follows: acute (0-1 month; n=10), subacute (1-3 months; n=12), early chronic (3-6 months; n=10), late chronic (≥1 year; n=32). Comprehensive testing for genetic and acquired thrombophilia states was performed in all subjects, along with serial assessment of D-dimer and automated euglobulin lysis time (ELT). RESULTS: Of the 58 children with VTE evaluated for global coagulative and fibrinolytic capacity, 25% underwent repeat testing in follow-up. A positive relationship with factor VIII activity (FVIII) was demonstrated for both CI and AUC (r=0.37, P=0.006 and r=0.52, P

Description: Abstract 3011 Poster Board II-987 Somatic mutation of the X- linked gene PIG-A results in partial or absolute deficiency of cell surface expression of glycosylphosphatidylinositol-anchored proteins (GPI-APs). In humans, this genetic event in a hematopoietic stem cell leads to the blood disease paroxysmal nocturnal hemoglobinuria (PNH). PNH has two major clinical presentations: predominantly hemolytic without overt marrow failure, referred to as classic PNH, and PNH clonal expansion in the setting of marrow failure, or the aplastic anemia/PNH syndrome (AA/PNH). The diagnosis of PNH is based on the presence of GPI-AP-deficient clones mainly within erythrocytes and granulocytes. Since PNH is a stem cell disorder, all hematopoietic lineages are involved and GPI-AP-deficient clones are found among platelets, monocytes and lymphocytes. Except for their different clinical presentations, classic PNH and AA-PNH have never been shown to have specific discriminating biological features. Indeed, we previously reported no difference in the expression profile of CD34 cells in hemolytic and aplastic forms of PNH (Chen G et al, Leukemia 2005; 19:862). As marrow failure in PNH, as in aplastic anemia, is immune-mediated, and T cells are affected by the PIG-A mutation, we hypothesized that the mutant subset of lymphocytes might differ in PNH. We compared by microarray the T cell compartment in both forms of the disease. We sorted peripheral blood GPI-AP-normal and -deficient pooled CD4 and CD8 T cells using CD59 and CD55 as GPI-AP markers, and subjected each subset to array hybridization and analysis. Samples were obtained from 1 patient with classic PNH, 3 patients with AA-PNH syndrome and 3 age-matched normal donors as controls. In preliminary control experiments, we determined using microarray of total RNA derived from healthy donors' T cells, with and without GPI-AP-specific antibodies, the absence of altered gene expression pattern or activation of specific genes due to selective antibody binding to normal cells. By principal component analysis (PCA), the phenotype of the PNH clone in classic PNH was divergent from the AA-PNH clone and from that of normal donors. By consistency test, only 15 probesets/genes resulted common in the comparison between GPI-AP-deficient and GPI-AP-normal cells between classic PNH and AA-PNH syndrome. Using Ingenuity Software, we found that the represented probesets/genes were involved in some major canonical pathways such as cell death, immune and lymphatic system development, immune response, and cell-to-cell signaling interaction. In order to investigate the different gene expression profile derived from specific subset of T cells, we sorted GPI-AP-deficient and GPI-AP-normal CD8 T cells using CD48 and CD52 as markers. When we compared GPI-AP-deficient and GPI-AP-normal cells between classic PNH and AA-PNH syndrome, we found that only 23 probesets/genes were up-regulated and 132 down-regulated confirming the divergent gene expression profile between the two diseases (Figure 1). We conclude that the restricted similarity in the gene expression profile in the GPI-AP-deficient T cells between classic PNH and AA-PNH syndrome leads to a specific “signature” of genes that might support the different clinical presentation and evolution of the two diseases.Figure 1.Divergent gene expression profile between GPI-AP-deficient and GPI-AP-normal cells in classic PNH and AA-PNH syndromeFigure 1. Divergent gene expression profile between GPI-AP-deficient and GPI-AP-normal cells in classic PNH and AA-PNH syndrome Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2995 Poster Board II-973 Introduction: The interaction of glycoprotein (GP) Ib-IX with subendothelial-bound von Willebrand factor (VWF) initiates circulated platelet transient adhesion on the injured vascular wall under flow conditions. VWF conformational changes in response to high shear stress are thought to be critical for initiating platelet adhesion, there is increasing evidences indicate that the interactions of intraplatelet proteins 14-3-3ζ and filamin A with the cytoplasmic domain of GPIbalpha also play key roles in the regulation of VWF binding function of GPIb-IX, whereas it is unclear whether their structural linkage has functional implication. This study was to explore the mechanism underlying the roles of 14-3-3ζ and filamin A in the regulation of the VWF binding function of GPIb-IX. Methods and Results: A truncation mutant of GPIbalpha (at residue 565) deleting the C-terminal 14-3-3ζ binding sites retains 14-3-3ζ binding function, in contrast, deletion of the C-terminal residues 551-610 of GPIbalpha totally abolished 14-3-3ζ binding, indicating that the residues 551-564 of GPIbalpha is important in the interaction between 14-3-3ζ and GPIb-IX. An antibody recognizing phosphorylated R557GpSLP561 sequence reacted with GPIbalpha suggesting phosphorylation of a population of GPIbalpha molecules at Ser559, and a membrane permeable phosphopeptide (MP-P), M-R557GpSLP561 corresponding to residues 557-561 of GPIbalpha eliminated the association of 14-3-3ζ with the truncation mutant of GPIbalpha . MP-P also promoted GPIb-IX association with the membrane skeleton, and inhibited ristocetin-induced platelet agglutination, VWF binding to platelets and platelet adhesion to immobilized VWF. Furthermore, a GPIb-IX mutant replacing Ser559 of GPIbalpha with alanine showed an enhanced association with the membrane skeleton, reduced ristocetin-induced VWF binding and diminished ability to mediate cell adhesion to VWF under flow conditions. Conclusions: These data suggest a phosphorylation-dependent binding of 14-3-3ζ to central filamin A binding site of GPIbalpha, and the dimeric 14-3-3ζ binding to both the C-terminal site and central RGpSLP site inhibits GPIb-IX association with the membrane skeleton and promotes the VWF binding function of GPIb-IX. Disclosures: No relevant conflicts of interest to declare.

Description: 2980 Poster Board II-954 Background. There are few studies that examine incidence trends of venous thromboembolism (VTE) among the elderly, and moreover data on changes in the prevalence over time of VTE risk factors such as hospitalization are limited. Objectives. Using the Province of Québec's administrative health claims (“RAMQ”) and hospitalization (“MED ECHO”) databases, we determined among individuals 65 years of age and older the trend in annual VTE incidence over a ten-year period, and examined the prevalence of hospital vs. community acquired VTE over time. Methods. Using RAMQ medical service and prescription claims data, we identified a cohort of elderly persons with incident VTE between 1983 and 1994. All individuals 65 years of age and older between January 1, 1994 and December 31, 2004 with at least one medical service (i.e. physician) claim linked to a VTE ICD-9 code in conjunction with a prescription claim for an anticoagulant in the subsequent 60 days, and who had no prior VTE-coded claim between 1983 and 1994 were included in the cohort. The first (incident) VTE-coded claim (index claim) during the period between January 1, 1994 and December 31, 2004 defined entry into the cohort. Using Québec population census data, we determined annual VTE incidence estimates, adjusting for the population's age and sex distribution as per 1999 census data. An index VTE event that occurred during a hospitalization and up to 90 days following hospital discharge was defined as hospital-acquired VTE, otherwise it was considered to be community-acquired. Results. A total of 27 758 persons were included in our cohort. The age and sex adjusted annual VTE incidence among individuals 65 years of age and older was 2.1, 2.7, and 2.8 per 1000 population in 1994, 1999 and 2004, respectively (p trend 〈 0.001 (across years)). The incidence rates increased with age and were slightly higher in men than women. Overall, 35% of VTE events were hospital-acquired (n=9598) and 65% occurred in the community (n=18160). There was a significant trend over time of an increase in the proportion of VTE associated with hospitalization with 32% of VTE being hospital acquired in 1994 and 39% in 2004. Conclusion. In a general population of individuals 65 years of age and older, VTE is a common problem, and its overall annual incidence has increased over time. Our results suggest that hospital admission and recent hospitalization are increasingly important risk factors for VTE occurrence in this population, and that efforts are needed to improve and optimize VTE preventative strategies in the hospitalized elderly. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3009 Poster Board II-985 Congenital dyserythropoietic anemia type II (CDA II) is an autosomal recessive disorder affecting the normal differentiation-proliferation pathway of the erythroid lineage. It comprises an anemia of variable severity, jaundice, and variable splenomegaly. Erythroid hyperplasia with binuclearity or multinuclearity involving late erythroblasts in the bone marrow (BM) is a key feature of the diagnosis (Iolascon, 2001). In addition, on electron microscopy, vesicles of endoplasmic reticulum (ER) appear to be running beneath the plasma membrane (Alloisio, 1996). The principal biochemical feature is the hypoglycosilation of several proteins, such as transferrin and band 3 (Anselstetter, 1977). Recently, we identified SEC23B as the CDA II causative gene (Schwarz, 2009). The SEC23B gene encodes the SEC23B component which is part of the cytoplasmic coat protein (COP)II complex. COPII coated vesicles bud from the endoplasmic reticulum to export newly synthesized proteins to the trans Golgi. In yeast, COPII coated vesicles form by the sequential binding of Sar1-GTP, the inner complex proteins Sec23-Sec24 and the outer complex components Sec13-Sec31 on the endoplasmic reticulum (ER) (Fromme, 2008). Our aim was to characterize the COPII complex in CD34+ progenitor cells during erythroid differentiation by gene expression analysis. Mononuclear cells from the peripheral blood of normal subjects were isolated on a Ficoll-Hypaque gradient and CD34+ progenitors were separated on immuno-affinity columns. For erythroid differentiation, CD34+ cells of three different pools were separately plated on plastic culture dishes in methylcellulose medium containing 3U/mL erythropoietin (EPO) (Pinho, 2008). The cells have been cultured for 7 and 14 days after EPO treatment. Quantitative real time (qRT)-PCR on CD34+ during erythroid differentiation was performed to assess the gene expression level. The relative gene expression was calculated by 2*(-ΔCt) method (Livak, 2001), using GAPDH gene as internal control (figure 1). Mammalian orthologues have been identified for each of the five proteins involved in COPII coat formation. In humans, two isoforms of Sec23, Sar1 and Sec31 and four mammalian isoforms of Sec24 have been reported (Kuge, 1994; Paccaud, 1996; Wendeler, 2007; Mancias, 2008; Shugrue, 1999; Tang, 2000; Stankewich, 2005). We already demonstrated that during normal erythropoiesis a SEC23A down regulation is associated to SEC23B upregulation. These data suggest that SEC23B mutants could disrupt the COPII complex in erythroid lineage, and consequently induce CDA II (Schwarz, 2009). On the contrary, the two isoforms of SAR1 showed the same trend during erythroid differentiation: however, SAR1A isoforms has an higher expression when compared to SAR1B isoform. Among the four SEC24 isoforms, SEC24A, B and C showed the same increased expression after EPO treatment; SEC24D, indeed, showed a decreasing trend during differentiation time. Only one form of mammalian SEC13 has been described (Shaywitz, 1995), and it showed an increased expression after EPO treatment. Between SEC31 mammalian isoforms, SEC31A showed overall an higher gene expression compared to SEC31B gene. The gene expression analysis of SEC12, the transmembrane guanine nucleotide exchange factor that catalyzes the COPII vesicle formation by GDP-GTP exchange on Sar1 (Sato, 2004), revealed an higher mRNA level at 14 days when compared to 7 days after EPO treatment. Here, we have identified the isoforms of COPII complex most expressed during erythroid differentiation. This study could allow us to clarify the role of each COPII gene in erythroid lineage, and to identify other genes potentially involved in CDA II pathogenesis.Figure 1.Gene expression profile during CD34+ erythroid differentiation. Data are presented as mean ± standard deviation. *p value 〈 0.05 calculated on day 0 by Student t test corrected by Bonferroni method.Figure 1. Gene expression profile during CD34+ erythroid differentiation. Data are presented as mean ± standard deviation. *p value 〈 0.05 calculated on day 0 by Student t test corrected by Bonferroni method. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2949 Poster Board II-925 Background: The efficacy of rituximab is dependent on a number of host immune interactions, including binding through excitatory (FcγRIIA, FcγRIIIA) as well as inhibitory (FcγRIIB) Fcγ receptors. In previous studies, we showed that polymorphisms in FcγRIIIA-158 predicted outcome to single agent rituximab therapy. Patients displaying L/H or L/R at FcγRIIIA-48 or at least one valine (V/V or V/F) at FcγRIIIA-158 demonstrated greater responses to rituximab versus those patients who expressed FcγRIIIA-48-L/L or FcγRIIIA-158 F/F, respectively (JCO 23:474). The predictive role of FcγRIIIA polymorphisms in patients receiving combination therapy with rituximab has not been addressed to date in WM. We therefore investigated the predictive role of FcγRIIIA-48, -158, as well as other important polymorphisms implicated in modulating IgG antibody binding and activation: FcγRIIA-27, -131, and FcγRIIB-187 in 65 patients with WM who received combination rituximab therapy. Patients and Methods: Sixty-four WM patients with a median age of 61, prior therapies of 0, IgM of 3,540 mg/dL, Hct of 32.3%, B2M of 2.7 g/L, who participated on a clinical study and whose outcomes have previously been reported were included in this analysis. Treatment included rituximab in combination with cyclophosphamide (n=43), thalidomide (n=14), or lenalidomide (n=7). Categorical responses for all patients were as follows: CR/VGPR 7 (11%); PR (n=30; 46.2%); MR (n=18; 27.7%); Non-Responders (n=9; 14.1%) for an overall response rate of 86%. Twenty seven patients have progressed with a median follow-up of 19.4 months. Polymorphic variants at FcγRIIA-27, -131, FcγRIIB-187, and FcγRIIIA-48, -158 were determined by Taq Man real time PCR analysis and sequencing, and impact on overall response, categorical response rates, and progression free survival determined. Results: The expression of H/H at FcγRIIA-131, or at least one valine (V/V or V/F) at FcγRIIIA-158 was associated with improved categorical response, particularly the attainment of CR/VGPR. For FcγRIIA-131, H/H was expressed in 2/9 (22.22%) WM patients who were non-responders; 13/38 (34.2%) patients attaining a major (≥ PR) response, and 4/7 (57.14%) patients who attained a CR/VGPR. For FcγRIIIA-158, the expression of at least one valine was observed in 3/9 (33.3%) WM patients who were non-responders; 20/38 (52.62%) patients attaining a major (≥ PR) response, and 5/7 (71.42%) patients who attained a CR/VGPR. Polymorphisms at FcγRIIA-27, and FcγRIIB-187 showed no association with response. The expression of L/H or L/R at FcγRIIIA-48 was observed in 3/7 (42.86%) patients with CR/VGPR, whereas 2/9 (22.22%) of patients who were non-responders expressed this polymorphism. Subset analysis showed that among patients who received cyclophosphamide based therapy, no differences in polymorphic variation for FcγRIIA-131, FcγRIIIA-48, and -158 were observed between non-responders, major responders and those achieving CR/VGPR. Conclusions: Taken together, the results of this study support a role for the use of FcγRIIA-131, FcγRIII-158, and possibly FcγRIIA-48 as determinants of better categorical responses in WM patients receiving combination therapy with an immunomodulatory agent. The combined use of cyclophosphamide with rituximab therapy appears to negate the inferior outcomes predicted by polymorphic variants in FcγRIIA-131, FcγRIIIA-48, and -158 which have previously been shown to be associated with lower response rates to single agent rituximab therapy. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 294 Endogenous serum erythropoietin (sEPO) less than 500UI/L and a transfusion requirement lower than 2 units per month are the best predictive factors for response to treatment by erythropoiesis-stimulating agents (ESA) in low/int-1 myelodysplastic syndromes (MDS). However, the highest response rate hardly reaches 60% suggesting that other factors may influence the response. To investigate the biological signature of response to ESA, we enrolled 100 low/int-1 MDS patients in a prospective study of erythropoiesis at diagnosis before they were treated with ESA. According to the IWG 2006 criteria, 43 patients were non-responders. These patients had significantly higher serum EPO level, higher number of transfusion per month, and lower number of bone marrow-deriving BFU-E and CFU-E than responders. Analysis of CD34+-deriving erythroid progenitors by in vitro liquid culture, demonstrated that all MDS patients (n=54) had an increased apoptosis and a delayed expression of erythroid marker, glycophorin A (GPA). A collapse of EPO-induced DNA synthesis was observed in non-responders, while EPO-dependent erythroid cell differentiation and survival to Fas-induced apoptosis was equivalent in the two groups. Thus, non-responders exhibited an early and isolated default in EPO-induced cell proliferation, suggesting a defect in EPO-R signaling. Immunofluorescence to p-ERK1/2 before and after EPO-R stimulation in immature erythroblasts was negative in 6/8 non-responders, and positive in all 11 responders. Immunohistochemistry to p-ERK1/2 on bone marrow biopsies in 5 non-responders was negative and positive in immature cells in 4 responders. By flow cytometry, p-ERK1/2 expression in the CD71+/GPA− bone marrow cell fraction corresponding to immature erythroblasts (n=30) was significantly lower in non-responders (n=16) than in responders (n=14; Wilcoxon-test: p

Description: Abstract 2991 Poster Board II-967 Background. The natural function of ACTH in blood is not well defined. However elevated plasma level was reported in patients with chronic inflammation, suggesting as one of inflammatory markers. Erythrocytes possess membrane-bound acetylcholinesterase (AChE) while plasma contains a less specific cholinesterase (ChE). We reported at a previous meeting [Blood 2008, 112(11) Abst #3849] that AChE activity of RBC-derived microparticles (RMP) is 6 times higher than those in PMP. In this study we measured plasma ACTH levels in several groups and encountered unexpectedly high plasma ChE activities in patients with TIA and other thrombosis. Methods. (i) Patient population. A series of n=108 consenting patients were recruited sequentially having various disorders For purpose of analysis, they were divided in 2 main groups: n=53 with thrombosis (TBS), F/M 27/26; and n=55 non-TBS, F/M 24/31. Thrombosis group consists of venous and arterial thrombosis including those with TIA. Non TBS group consists of anemias, thrombocytopenias including ITP, MDS. (ii) Assay was essentially by Ellman's method. In our system, milli-absorbance units/min (mA/min) x 0.065 = umols substrate cleaved/min. Values reported here are in unitsof mA/min per mL plasma. Normal controls (NC, n=14) had a cutoff of 3000 mA/min per mL plasma, = mean +2SD. (iii) Sample handling. Platelet-poor plasma (PPP) was prepared by centrifuging 10 min at 1800 xg, then frozen in aliquots. For assay, it was diluted 1:20 with saline, then 5 uL and 10 uL were used in 96-well microtiter plate. Results: (i) Significantly higher ChE activity was observed in thrombosis (TBS) patients compared to non-TBS. Mean value ±SD in TBS was 2928 ±773, and in Non-TBS was 1897 ±713 (units as above). This difference was significant, p3000) in 30% of patients, while the Non-TBS had elevated ChE in 7.8%. (iii) Further analysis among thrombosis group revealed that the subgroup with neurological TBS (TIA, minor strokes) had the most consistent elevations of ACTH; 10 of15 with TIA group (66%) had ChE activities 〉3000. Summary/ Discussion. Although ChE and AChE in blood has long been studied, to our knowledge, this is the first report to show a relation between plasma ChE and thrombosis. Its elevation is very pronounce in thrombosis of the CNS, manifesting as TIA. Further study is warranted to elucidate how ChE is related to AChE of the CNS and on red cells. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2924 Poster Board II-900 Introduction : Indoleamine 2,3-dioxygenase (IDO) is an enzyme that degrades the essential amino acid tryptophan along the kynurenine pathway. Pro-inflammatory cytokines, such as IFN-g, induce IDO during the inflammatory response in many human cell types. The induction of IDO is synergistic in the presence of TNF-a, IL-1 or IL-6, and might be mediated by a signaling pathway from NF-κB and/or MAPKs. Furthermore, some metabolites derived from tryptophan by IDO, such as L-kynurenine, block antigen-driven specific T-cell proliferation and induce T-cell death. Thus, IDO activity might play an important role in regulation of the immune response exerted by antigen presenting cells and also provide transformed cells with a potent tool to help escape from assault by the immune system. Indeed, we have previously reported that high serum L-kynurenine level is associated with poor prognosis of diffuse large B-cell lymphoma (DLBCL) (ASH 2008 abstract 2812). Here, we investigated the IDO expression of patients with DLBCL. Patients and methods : The study protocol comprised a prospective, consecutive entry design that was approved by our Institutional Review Board. We investigated 119 patients between December 2003 and June 2008 who were histologically diagnosed with DLBCL according to the WHO classification. We performed immunohistochemical (IHC) analysis for IDO expression by mouse anti-human IDO monoclonal antibody. Patients aged

Description: Abstract 299 Dysregulation of the cell cycle is a hallmark of cancer. However, targeting the cell cycle in cancer therapy has only been modestly successful since broad-spectrum cyclindependent kinase (CDK) inhibitors lack specificity and are highly toxic. The critical importance of controlling CDK4/CDK6 in cancer treatment is further exemplified by recent evidence of prominent CDK4/CDK6 dysregulation in human cancers, including breast cancer, metastatic lung adenocarcinoma, glioblastoma, mantle cell lymphoma and multiple myeloma (MM). To advance mechanism-based targeting of the cell cycle in cancer, we have developed a novel strategy that both inhibits cell cycle progression and enhances cytotoxic killing in tumor cells using PD 0332991(PD), the only known CDK4/CDK6-specific inhibitor that is also reversible, potent and orally bioavailable. We demonstrated by BrdU pulse-labeling that inhibition of CDK4/CDK6 with PD in primary bone marrow (BM) myeloma cells and human myeloma cell lines (HMCL) (IC50 60nM) leads to a complete early G1 arrest in the absence of apoptosis and upon release of the G1 block, synchronous cell cycle progression to S phase. Furthermore, prolonged early G1 arrest enhances cytotoxic killing of MM cells by diverse clinically relevant drugs at low dose, including bortezomib, carfilzomib (PR-171) and dexamethasone, and this is dramatically augmented during synchronous S phase entry. The enhancement of cytotoxic killing in either G1 arrest or synchronous S phase entry is sustained in the presence of BM stromal cells. This killing is caspase-dependent and triggered by the loss of mitochondrial outer membrane potential and activation of the intrinsic apoptosis pathway. Time course studies of cell cycle-specific gene expression by expression profiling, quantitative real time RT-PCR and immunoblotting further revealed that the expression of IRF-4, essential for normal plasma cell differentiation and myeloma cell survival, is strictly cell cycle-dependent: elevated in G1 and markedly declined in S phase. The IRF-4 protein is also markedly reduced (50%) by bortezomib treatment, resulting in a combined 5-fold reduction in S phase. This suggests that differential enhancement of cytotoxic killing in G1 arrest and S phase is mediated by cell cycle-dependent IRF-4 expression. Indeed, shRNA interference confirms that by antagonizing mitochondrial depolarization, IRF-4 is required to protect myeloma cells from cell cycle-dependent enhancement of bortezomib killing. By timely administration and discontinuation of PD treatment, we have further demonstrated in a human MM 1.S. xenograft myeloma model that it is feasible to induce sequential G1 arrest and synchronous S phase in vivo. This leads to synergistic tumor suppression through amplification of bortezomib killing of myeloma cells, but not normal BM cells. As PD is orally bio-available, specific and low in toxicity, our novel strategy has been implemented in the first phase I/II multi-center clinical trial targeting CDK4/CDK6 with PD in combination with bortezomib and dexamethasone in MM. Preliminary bone marrow immunohistochemistry demonstrates PD preferentially and completely inhibits CDK4/CDK6-specific phosphorylaton of Rb and DNA replication in tumor cells, but not other bone marrow cells in all patients. One patient achieved VGPR (12.5%) while 1 patient each achieved MR and SD respectively for an ORR 25% (Niesvizky et al, submitted). Collectively, our preclinical and clinical data indicate, for the fist time, that selective targeting of CDK4/CDK6 in combination therapy is a promising mechanism-based therapy for MM and potentially other cancers. Disclosures: Off Label Use: PD 0332991 is going to be used as a CDK4/6-specific inhibitor.. Chen:Pfizer, Inc.: Employment, Equity Ownership. Wilner:Pfizer, Inc.: Employment, Equity Ownership. Niesvizky:Millenium: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Seattle Genetics, Inc: Research Funding; Proteolix: Research Funding, data monitoring committee. Chen-Kiang:Pfizer Inc.: Research Funding.

Description: Abstract 2936 Poster Board II-912 Mantle cell lymphoma (MCL) pathogenesis is still partially unexplained. Although the overexpression of CyclinD1 dependent of t(11;14) is a distinctive molecular hallmark of this neoplasm, this event alone cannot account for the increased survival signaling that characterizes this lymphoma type. Here we investigate whether microRNA (miRNA) expression profile may help to explain the changes in the expression of gene pathways that are characteristic of MCL. Twenty-three frozen MCL samples, 11 frozen control tissues (7 lymph nodes and 4 tonsils), 8 MCL cell lines and 3 samples of CD19+IgD+CD27- cells obtained from tonsils, were studied for miRNAs and gene expression. MiRNA one color microarray data for 470 human miRNA were analyzed using SAM (Significance Analysis of Microarrays) algorithm. MiRNA targets were predicted by miRanda and TargetScan methods. Pathways identification and analysis was carried out by GSEA (Gene Set Enrichment Analysis) online resource. The analysis of 23 MCL cases compared to 11 control tissues showed a miRNA signature that included 117 miRNAs with FDR

Description: Abstract 2947 Poster Board II-923 Introduction: DLBCL occasionally presents in leukemic phase, and the prognostic significance of circulating lymphoma cells is unknown. We herein report characteristics and outcomes of newly diagnosed DLBCL presenting in leukemic phase at 2 Institutions. Methods: Flow cytometry database analysis and retrospective chart reviews were carried out with IRB-approval for cases accrued between 2001 and 2008. Leukemic phase DLBCL patients were matched on a 3:1 basis with control DLBCL with no circulating lymphoma cells based on IPI, year of diagnosis, and age ± 10 years. Results: 18 patients, median age 48 years (range 34-80), ECOG PS-1 (22%), 2(38%) and 3(40%), and IPI - 3(56%), 4(40%) and 5(4%) presented in leukemic phase. Extranodal sites included bone marrow (100%), spleen (83%), pleura (61%) and CSF (22%). 61% had B symptoms, and LDH was 6xULN (range, 1-56). WBC was 13,000/microL (range, 7,100-127,400), with 50% lymphoma cells (range, 2-92); these cells were immunophenotypically similar to those in the histologically confirmed DLBCL node, and co-expressed CD19, CD20, CD22, CD38, CD45, HLA-DR and FMC7 in 〉90% of cases, and kappa or lambda light chain restriction in 〉 50%. Karyotype was abnormal and complex in 61%. One patient expired before treatment began. Treatment consisted of R-CHOP (10), R-HCVAD (6), and single agent rituximab (1). 8 (44%) achieved CR (5 R-HCVAD and 3 R-CHOP), 5 (28%) PR, and 4 (22%) had resistant disease. 1 patient was autografted in CR1 and remains in remission. With a median follow-up of 32 months, 2 relapsed in leukemic phase, 1 of whom achieved CR2, but relapsed at the time of conditioning for a consolidative allograft. 10 (56%) patients died from progressive disease, 2 (11%) were lost to follow-up and 6 (33%) remain alive in remission. Overall (Panel A) and progression-free (Panel B) survival curves the 18 leukemic (solid line) and 54 non-leukemic phase (dashed line) DLBCL are depicted in the Figure. Conclusion: DLBCL presenting with circulating lymphoma cells is associated with chemo-resistance (44% CR) and poor outcomes with the exception of those who achieve complete remission. These patients are candidates for alternative therapies. Disclosures: Kaufman: Millenium: Consultancy; Genzyme: Consultancy; Celgene: Consultancy, Research Funding; Merck: Research Funding. Lonial:Millennium: Consultancy, Research Funding; Celgene: Consultancy; BMS: Consultancy; Novartis: Consultancy; Gloucester: Research Funding. Armitage:Eisa: Consultancy; Allo: Consultancy; Ziopharm: Consultancy.

Description: Abstract 134 The tumor microenvironment has been recognized as important in the pathobiology of follicular lymphoma (FL). Indeed, the role of extrafollicular lymphoma-associated macrophages (ef-LAMs) and FOXP3+ regulatory T-cells (Tregs) as predictors of overall survival in FL was previously reported by our group and others. We sought to further evaluate the role of specific T-cell subsets in our patient series. To this end we enumerated PD-1+ follicular helper T-cells (Tfhs), a potentially important regulator of immune response, in the tumor microenvironment in a series of 91 newly diagnosed FLs. Initial biopsies from 91 FL patients diagnosed between 9/1/85 and 12/13/02 were evaluated. FL grade 3B cases were excluded. Clinical data was obtained for gender, age, FLIPI risk group, presence of bulky disease, presence of B-symptoms and overall survival (OS). Immunohistochemical staining for PD-1 was performed on tissue microarray sections and the mean number of follicular PD-1+ cells per 9 high power fields (1000x, 3 follicles with 3 fields per follicle) was quantified. Recursive partitioning analysis was then performed to determine an optimal break-point for PD-1. BCL2 expression, ef-LAMs and FOXP3+ Tregs were evaluated as reported by us previously. The Cox proportional hazards model was used to determine significant univariate and multivariate prognostic risk factors, using P≤ 0.10 as the criterion for inclusion and retention in the multivariate analysis. The Kaplan-Meier method was used to summarize OS. 54% of patients were male with a median age at diagnosis of 58 years (range 24–89 years). The distribution of FLIPI scores was 35% low, 27% intermediate, and 37% high. The number of PD-1+ Tfhs was variable (median number of PD-1+ cells = 35.6, range 4.4 – 91.2). Number of PD-1+ cells correlated with number of Tregs (P=.01) and marginally with lack of BCL2 (P=.07) but not with other clinical or pathologic variables. On univariate analysis, age ≥ 55 (P=0.004), increasing FLIPI risk group (P=0.03), bulky disease (〉10 cm; P=0.05), lack of BCL2 (P=.01), high ef-LAMs (〉16.8/hpf, P=.003), and high PD-1+ cells (〉35.6/hpf, P=0.10) were all associated with decreased OS. On multivariate analysis, independent poor prognostic risk factors were: age ≥55 (hazard ratio[HR]=2.76, 95% confidence interval [CI] = 1.33–5.76, P=.007), high ef-LAMs (HR=2.13, 95%CI = 1.11–4.07, P=.02) bulky disease (HR=2.10, 95% CI 0.91–4.83, P=.08), and high PD-1+ cells (HR 2.04, 95% CI=1.11–3.75, P=.02). A prognostic model based on the number of risk factors present can be constructed with patients having 0, 1-2 or 3-4 risk factors defining 3 distinct prognostic groups (Figure 1). In summary, cellular constituents of the FL microenvironment play an important role in the biology of FL. A combination of standard clinical and biologic factors that include Tfhs and ef-LAMs appear to predict OS in FL. Given the putative role for Tfhs in down-regulating the immune response, these data support strategies aimed at modulating Tfh function in FL. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 292 Background: Allelic deficiency for the RPS14 gene impairs differentiation and survival of erythroid progenitors in del(5q) MDS (Nature 2008; 451:335). Nucleolar stress arising from disruption of ribosome assembly fosters MDM2 sequestration by free ribosome components resulting in p53 stabilization and erythroid hypoplasia (Nat Cell Biol 2009; 11:501). We recently reported that reduced gene dosage of the lenalidomide (LEN) inhibitable, haplodeficient phosphatases CDC25C and PP2Acα is a key determinant of drug sensitivity in del(5q) MDS (PNAS 2009; 106: 12974). We now show that shRNA suppression of these genes to levels commensurate with haplodeficiency reinforces p53 accumulation, and that treatment with LEN promotes MDM2-mediated p53 degradation to transition del(5q) clones to G2/M arrest. We hypothesized that emergence of resistance to LEN in del(5q) MDS arises from two possible mechanisms: (1) up-regulation of haplodeficient drug targets or compensatory isotypes, or (2) inactivating mutations of the TP53 or CDC25C genes. Methods: To investigate mechanisms of LEN resistance, we studied sequential bone marrow (BM) specimens obtained at baseline (BL), response to treatment (TR) and treatment failure (TF) from 12 LEN treated patients with Low/INT-1 risk, transfusion-dependent del(5q) MDS. Eleven patients achieved clonal suppression and transfusion independence; 7 patients developed clinical drug resistance with primary clonal recovery. Immunohistochemical (IHC) staining for cdc25-C, -A and -B; PP2A–Ca and p53 were performed using a biotin-streptavidin-horseradish peroxidase method and compared to 6 age-matched controls; intensity of cytoplasmic or nuclear staining in hematopoietic elements was recorded after blinded review. DNA and RNA were extracted from cryopreserved BM mononuclear cells (BM-MNC) or fixed paraffin blocks from BM clot and biopsy sections. Expression of CDC25C splice variants was assessed by RT-PCR and total gene expression by real time (QT)-PCR. Exonic DNA encoding the catalytic [exons 8–14] and nuclear export domains [exon 11] of CDC25C and the DNA-binding domain of TP53 [exons 4–9] was sequenced for gene mutation analysis. Differences in mean values were compared by paired t-test. Results: P53 immunostaining was significantly higher in del(5q) BL specimens compared to controls ( relative expression [RE] 9.6 vs. 0.25; P =0.007). An admixture of nuclear and cytoplasmic staining for p53 and each cdc25 isotype was observed at BL that was largely restricted to erythroid precursors, whereas at TR cdc25-C and -A expression was primarily cytoplasmic, consistent with drug-induced nuclear exclusion. At TR, RE of only cdc25C (BL, 75 vs. TR, 49; P=0.05) and PP2A (29.2 vs. 12.3; P=0.025) was significantly reduced; whereas at TF cdc25C (TR, 43 vs. TF, 166; P=0.003), cdc25A (42.4 vs. 150; P=0.006), PP2A (7.3 vs. 65.6; P=0.028) and p53 (0.92 vs. 25.4; P=0.024) RE significantly increased. Nuclear localization of cdc25C and p53 but not cdc25A predominated at TF, consistent with escape from cdc25C inhibition. QT-PCR confirmed transcriptional up-regulation of CDC25C at TF with a mean 8.8-fold increase in gene expression vs. BL. DNA sequencing revealed no acquisition of somatic mutations within the CDC25C and TP53 exons studied [n=5]. Conclusions: Secondary resistance to LEN in del(5q) MDS is associated with over-expression and activation of the haplodeficient drug-inhibitable phosphatases, cdc25C and PP2A, with consequent restoration of wt-p53 activation. Absence of gene mutations within the coding exons analyzed suggests that transcriptional compensation alone is responsible for drug resistance. Novel agents targeting transcriptional repression of CDC25C may restore LEN sensitivity and merit investigation in drug resistant del(5q) MDS. Disclosures: List: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Komrokji:Celgene: Research Funding, Speakers Bureau. Lancet:Celgene: Research Funding. Maciejewski:Esai: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Description: Abstract 2900 Poster Board II-876 Ph-negative myeloproliferative neoplasms: polycythemia vera (PV), essential thrombocythemia (ET) and primitive myelofibrosis (PMF) carry an acquired somatic mutation JAK2V617F in 95% (PV), and in 50 to 60% (ET or PMF) of the patients. Mutations of the TET2 gene have been observed with roughly similar frequencies in the three MPN, irrespective of the presence of JAK2V617F. Evolution to myelofibrosis or acute leukemia may occur with time in MPN patients. Although its molecular bases are poorly understood, the evolution is likely due to the acquisition of additional mutations. To investigate whether cytogenetic abnormalities are distributed differently according to type of transformation and to the JAK2 and TET2 statuses, the Groupe Francophone de Cytogénétique Hématologique has collected 82 patients with transformation of MPN. There were 66 (80%) acute myeloid leukemia or myelodysplastic syndromes (AML/MDS) and 16 (20%) myelofibroses (MF). Of note pipobroman (Pi) treatment seems to be associated with MF, and hydroxyuera (Hu) with AML/MDS evolution in our series. Statistical analyses of clinical, cytogenetic and molecular data are shown Table 1. On the cytogenetical point of view, several points are noteworthy. Some abnormalities were unevenly distributed: there were significantly more -7/del7q and -5/del5q in AML/MDS and tri1q and tri9 in MF. MF and PMF cytogenetic profile looked similar, suggesting a potential link between cytogenetic markers and the phenotype. Although the derivative chromosome der(1;7), observed in 9 patients, is responsible for a loss of 7q, it seemed different from patients with -7/del7q [excluding der(1;7)]. In the -7/del7q group, AML/MDS patients were more numerous than MF patients and the overall survival was shorter compared with the der(1;7) group (22/22 (100%) vs 6/9 (67%) AML/MDS, p=0.02; median: 4 vs 41 months, p=0.0007 respectively). Some specific associations could be observed, such as 17p deletions with 5q deletion (12/30, 40% vs 4/48, 8%, p=0.0007) and 20q deletion with der(1;7) (4/9 (44%) vs11/69 (16%), p=0.03). We detected 24/40 (60%) JAK2V617F and 8/25 (32%) TET2 mutations in transformed MPN, with all possible combinations between the wildtype and mutated forms of both genes. For one post-ET AML patient, JAK2V617F had been observed in a fraction of the granulocytes at the chronic phase. Analysis of blood cDNA obtained at chronic phase showed the same TET2 mutation as observed at acute phase. Because the blast cells were JAK2wt-TET2mut and carried a t(10;16)(q22;q23) affecting the CBFB gene, it is likely that the resulting non-MYH11 CBFB fusion gene transformed a JAK2wt-TET2 mutated progenitor that predominated in the chronic phase. In conclusion, no specific chromosomal abnormality was associated with TET2 or JAK2 mutations. Chromosomal abnormalities were associated with a type of transformation (AML/MDS or MF), suggesting a specific role in the process. In addition, association between some chromosomal abnormalities suggest a specific oncogenic cooperation.Table 1.n=82AML/MDS n=66 MF n=16 p univariate p multivariate Sex F39 (59%)5 (31%)nsnsPV/ET/PMF30/26/1013/3/0nsnsAge at diagnosis of MPN54 [20-82]55.5 [31-69]nsnsChronic Phase (duration, years)12 [2-34]14.5 [3-28]nsnsPrior treatments (n=73*)57*16..No treatment (n=6)60nsnsOne treatment (n=40)33 (58%)7 (44%)nsnsTreatments with Hu (n=57)48 (73%)9 (56%)0.03Treatments with Pi (n=41)26 (46%)15 (93%)0.00060.05Age at transformation66.5[37-92]68 [45-80]nsnsAbnormal karyotype62 (94%)16 (100%)nsnsComplex karyotype45 (68%)7 (44%)nsns-7/del7q28 (42%)3 (18%)0.07ns-7/del7q[without der(1;7)]22 (33%)00.0040.04-5/del5q28 (42%)2 (12%)0.03ns-13/del13q5 (8%)3 (19%)nsns-20/del20q11 (17%)4 (25%)nsns-17/del17p15 (23%)1(6%)nsns+1q14 (22%)9 (56%)0.01ns+95 (8%)4 (25%)0.04ns+811 (17%)3 (19%)nsnsdic17 (26%)3 (19%)nsnsder(1;7)6 (9%)3 (19%)nsnsAmplification MLL0 (0%)nsnsJAK2mut17/31 (55%)7/9 (78%)nsnsTET2mut6/19 (32%)2/6 (33%)nsnsMedian overall survival (months)448

Description: Abstract 2998 Poster Board II-976 Introduction: The platelet surface receptor glycoprotein (GP) IIbIIIa (integrin αaIIbβ3) mediates platelet aggregation and plays a key role in hemostasis and thrombosis. Numerous GPIIbIIIa antagonists have been designed and tested as inhibitors of platelet aggregation. Two of these antagonists, eptifibatide (Integrilin) and tirofiban (Aggrastat) have been approved by the U.S. Food and Drug Administration (FDA) and widely used for preventing and treating thrombotic complications in patients undergoing percutaneous coronary intervention and in patients with acute coronary syndromes. It has been reported, however, that some GPIIbIIIa antagonists, such as orbofiban and xemilofiban, promote apoptosis in cardiomyocytes by activation of the apoptosis executioner caspase-3, raising the possibility that platelets also may be susceptible to pro-apoptotic effects of eptifibatide and tirofiban. Over the past decade it has been well-documented that apoptosis occurs not only in nucleated cells but also in anucleated platelets stimulated with thrombin, calcium ionophores, very high shear stresses and platelet storage (Leytin et al, J Thromb Haemost 4: 2656, 2006; Mason et al, Cell 128: 1173, 2007). It has been further reported that platelet activation and apoptosis may be induced by different mechanisms and/or require different levels of triggering stumuli (Leytin et al, Br J Haematol 136: 762, 2007; Br J Haematol 142: 494, 2008). Recently, we have shown that injection of anti-GPIIb antibody induced caspase-3 activation in mouse platelets in vivo (Leytin et al, Br J Haematol 133: 78, 2006), suggesting that direct GPIIbIIIa-mediated pro-apoptotic signaling is able to trigger caspase-3 activation within platelets. Study Design and Methods: The current study aimed to examine, for the first time, the effect of eptifibatide and tirofiban on caspase-3 activation in human platelets. We studied the effects of eptifibatide and tirofiban on caspase-3 activation in resting platelets, which express GPIIbIIIa receptors in their non-active (“closed”) conformation, and in platelets stimulated with thrombin or calcium ionophore A23187, which induce transition of GPIIbIIIa receptors into active (“open”) conformation. Resting platelets were treated with control buffer, 0.48 μM eptifibatide or 0.48 μM tirofiban, and stimulated platelets were treated with 1 U/mL thrombin or 10 μM A23187, or preincubated with eptifibatide or tirofiban before treatment with thrombin or A23187. Caspase-3 activation was determined by flow cytometry using the cell-penetrating FAM-DEVD-FMK probe, which covalently binds to active caspase-3. Results and Discussion: We found that treatment of resting platelets with eptifibatide and tirofiban did not affect caspase-3 activation (P〉0.05, n=7). In contrast, a 2.3-2.7-fold increase of caspase-3 activation was observed in platelets after thrombin or A23187 stimulation (P

Description: Abstract 1291 Poster Board I-313 Introduction In approximately 25% of hemophilia A patients, inhibitory anti-FVIII antibodies develop following treatment with FVIII protein infusion therapy. This is currently the most serious treatment-related complication in this population. While the presence of these antibodies has been evaluated by a functional clotting assay for the past 30 years (the Bethesda Assay), there is a growing appreciation that the anti-FVIII immune response may also include the appearance of non-neutralizing antibodies in some patients. The prevalence, natural history and clinical relevance of these antibodies are the subject of this study. Methods Plasma samples from 602 hemophilia A patients formed the study material for this project. These samples had been stored at –80C since the conversion from plasma-derived to recombinant FVIII (rFVIII) concentrates in Canada. The results of prior Bethesda assay testing were available for 392 of these samples. The ELISA-based test for non-neutralizing anti-FVIII antibodies utilized three different rFVIII products as antigens: two full-length rFVIII products and one B domain-deleted rFVIII. Each microtiter plate run included six negative and one positive control sample. All samples were tested in duplicate and samples with positive results (absorbance 〉mean + 3SD) were subjected to serial dilution testing. 93 of the samples were also tested by the same assay in a second laboratory. Results Anti-FVIII antibodies were documented using this ELISA-based assay in 11.5% of this population. 11.5% and 8.8% of the patients demonstrated non-neutralizing antibodies to the two full-length rFVIII concentrates Advate® and Kogenate®, respectively. In contrast, antibodies to the B domain-deleted concentrate, Xyntha®, were only found in 4.6% of patients. 2.8% of patients had antibodies to all three concentrates while 3.1% of patients had antibodies to only Advate (2.8%) or Kogenate (0.3%). Twenty four of the 392 patients (6.1%) tested with the Bethesda assay had inhibitory anti-FVIII antibodies (range 0.6 – 267 BUs). A third of these Bethesda +ve cases demonstrated positivity in this assay to all three rFVIII products, while another 30% had antibodies against one or two of the rFVIII concentrates. 48 of the 368 cases that tested negative in the Bethesda assay were positive for non-neutralizing anti-FVIII antibodies (13%). To date, 20 of the samples testing positive for non-neutralizing antibodies have been studied by serial dilution analysis and 9 of these samples were only positive at the initial dilution of 1:50. Seven cases had positive dilution titers of 1:200 (3 with inhibitory antibodies ranging from 0.6 to 98 BUs) and two samples were positive at a dilution of 1:400, both of which had positive Bethesda results of 30 and 115 BUs, respectively. To assess the inter-laboratory consistency of this testing protocol, 93 selected samples were assayed with the identical methodology in a second laboratory. These studies showed positive results for the presence of antibodies to the two full-length rFVIIIs in 32-45% of cases. In contrast, the range of positive results for the two laboratories with the B domain-deleted rFVIII was between 19 and 37%. Conclusions This study of a large Canadian hemophilia A population has demonstrated the presence of non-neutralizing anti-FVIII antibodies in 11% of the population. Antibodies to the B domain-deleted rFVIII were significantly less frequent. When evaluated, inhibitory antibodies were present in 6% of this population and in the Bethesda negative cases, non-neutralizing antibodies were found in 13% of patients. Finally, many of these non-neutralizing antibodies appear to be of low titer, and their clinical significance must await further study with clotting factor recovery and half-life analysis. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1268 Poster Board I-290 Purpose There are reports of an increased risk of skin cancers in patients with B-Chronic Lymphocytic Leukemia (CLL). These skin cancers include basal cell and squamous cell carcinoma. This analysis was performed to more completely define the prevalence of skin cancers in patients in the Mayo Clinic Rochester CLL database and to look for contributing factors. Methods The Mayo Clinic Rochester CLL Database includes all patients with a diagnosis of CLL since January 1995 seen in the Division of Hematology and who have signed institutional review board approved consents for research. For this study, 2240 patients were analyzed to compare differences in characteristics between CLL patients with and without skin cancer. Chi-square statistics were used to compare qualitative variables (age categories, gender, referral status, ALC categories, CD38, ZAP-70, IgVH gene mutation status, FISH categories, Rai stage), and t-tests were used for quantitative variables (age at diagnosis, ALC values). Overall survival (OS) and time to first treatment (TFT) analyses were performed with results being displayed using Kaplan-Meier curves and p-values calculated using a log-rank test. Prevalence of melanoma among CLL patients was compared to the age-adjusted prevalence of melanoma in individuals in the Iowa SEER registry. Results Median follow-up for the 2240 patients diagnosed between 1/1/1995 and 8/11/2009 was 4.6 years. In aggregate, 293 (13.1%) patients were found to have non-melanoma skin cancer (squamous cell carcinoma or basal cell carcinoma) cancer. The diagnosis of non-melanoma skin cancer occurred before the CLL diagnosis in 39% and at or after the diagnosis of CLL in 61%. There were 57 (2.5%) cases of melanoma in association with CLL. The diagnosis of melanoma occurred before the CLL diagnosis in 38% and at or after the diagnosis of CLL in 62%.The prevalence of non-melanoma skin cancer and melanoma skin cancer were both higher in non-referred (geographically regional) CLL patients than referred CLL patients (16.6% vs. 11.4%, p

Description: Abstract 1261 Poster Board I-283 Ki-67 is a nuclear antigen that is expressed in all stages of the cell cycle except G0 and is widely used as a marker of cellular proliferation in human tumors. We recently demonstrated that levels of plasma circulating Ki-67 (cKi-67) are significantly higher in patients with newly diagnosed acute lymphoblastic leukemia (ALL) than in healthy control subjects, and that elevated levels of cKi-67 are associated with a shorter survival in ALL patients. Here we examined the associations of cKi-67 levels with laboratory and clinical variables in patients with chronic lymphocytic leukemia (CLL). The study included 194 patients with CLL and 96 healthy control subjects. The cKi-67 levels in plasma were determined using electro-chemiluminescence-based immunoassay using the Mesoscale Discovery platform. Since usually Ki-67 is used as an index of tumor cell proliferation, we took into account the lymphocyte count of the CLL patients in peripheral blood and normalized the levels of the cKi67 to the absolute number of lymphocytes in the peripheral blood establishing plasma cKI-67 index (cKi-67 level ng/1000 circulating lymphocytes/μL plasma). Median (range) levels of absolute cKi-67 were significantly higher in patients with CLL than in control subjects (914.65 [102.0-4975.12] ng/mL vs 353 [35.76-2830.65] ng/mL; P

Description: Abstract 1321 Poster Board I-343 Wiskott-Aldrich syndrome is characterized by immune deficiency and thrombocytopenia. There is also a qualitative platelet defect in Wiskott-Aldrich syndrome, typically presenting on aggregometry as a failure of the second wave of aggregation. The pathophysiology of this defect has not been studied in detail. To investigate this phenomenon, we enumerated dense bodies and measured serotonin content in the platelets of 7 patients with Wiskott-Aldrich syndrome. Patients ranged in age from 14 to 45 years old. Five of 7 patients had had splenectomy. In these patients, the platelet count ranged from 120,000/mcL to 521,000/mcL. Platelet counts were 20,000/mcL and 46,000/mcL in the 2 patients who had not had splenectomy. The latter 2 patients had occasional petechiae, as did 2 of the splenectomized patients. Three patients had no clinical evidence of platelet dysfunction. One of these 3 had tolerated abdominal surgery without untoward bleeding. Blood specimens were taken in the course of routine follow-up, when patients were otherwise well. Platelets were prepaed for electron microscopy by the whole mount technique. For serotonin content, platelets were counted and lysed in distilled water. Serotonin in the lysate was chemically acetylated and then measured by ELISA. Serotonin levels were normalized to ng of serotonin per 10 9 platelets. The average ± standard deviation dense body count in our patient group was 1.1 ± 0.4 dense bodies per platelet (normal count 6-8 dense bodies/platelet). Serotonin content was measured in three patients, who showed 273 ± 103 ng serotonin per 10 9 platelets (normal value 621-1064 ng/10 9 platelets). Our results are the first quantitative analysis of platelet dense body counts and serotonin level in patients with Wiskott-Aldrich syndrome. In addition to further elucidating the pathophysiology of this disease, these data have implications for understanding the role of the Wiskott-Aldrich syndrome protein in the genesis of dense bodies. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1245 Poster Board I-267 Introduction Chronic lymphocytic leukemia (CLL) patients with del(11q) by fluorescence in situ hybridization (FISH) have a poor prognosis. These patients often have younger age of onset, bulky lymphadenopathy, and short clinical response to treatment with purine analogues. Deletion of 11q in CLL is thought to involve loss-of-function of ataxia-telangiectasia mutated (ATM), a protein kinase central to DNA damage responses. As part of a larger study of early-intermediate stage, high risk CLL patients, we applied whole genome copy number variation (CNV) analysis to characterize the genomic alterations in CLL patients with and without del(11q) by FISH. Patients and Methods We studied 17 patients aged 29-67 with early-intermediate stage, untreated CLL who had high risk for disease progression based on evaluation of molecular and immunophenotypic markers. Of these patients, six had del(11q) by FISH analysis. No patients in this study had del(11q) as a sole FISH abnormality. CLL cells and normal cells were separated by magnetic bead selection from patient peripheral blood samples with absolute lymphocyte counts that ranged from 7.4 to 106 × 109/L. CNV analysis was performed on purified genomic DNA from the CLL cells and from normal cells for each patient in order to distinguish acquired CNVs in malignant cells from polymorphic CNVs in the human genome. We used the Illumina human660w-quad beadchip, a single nucleotide polymorphism (SNP)-based microarray for whole-genome genotyping and CNV analysis that contains more than 550,000 tag SNPs and approximately 100,000 additional markers that target regions of common CNV. CNV data was analyzed using CNV partition (Illumina Genome Studio software) and PennCNV. Results CNV analysis reveals hemizygous deletions of 11q in all 6 patients positive for del(11q) by FISH. The size of the deletion varies from ∼980 Kb to ∼44 Mb. All deletions include the region of the ATM gene at 11q22.3. No large homozygous deletions of 11q were detected. The three largest interstitial deletions (∼39 Mb, ∼41 Mb, ∼44 Mb) span 11q14.1-q23.3. A slightly smaller deletion of ∼33 Mb spans 11q14.2-q23.3. One deletion of ∼8.6 Mb covers 11q22.1-q22.3. The smallest deletion of ∼980 Kb is found within 11q22.3, centered on the ATM gene. Three out of 6 patients show complex interstitial deletions of 11q that consist of two or more discontinuous segments of loss of heterozygosity (LOH) separated by short undeleted regions. Five out of 6 patients with 11q deletions also show 13q hemizygous deletion by CNV analysis and by FISH. In 4 of these patients, the 11q and 13q interstitial deletions are the only acquired CNV events detected in the CLL genome. In one patient with 11q and 13q interstitial deletions, CNV analysis also shows chromosome 12 copy number (CN)=3 (also detected by FISH), CN=3 at 3q24-telomere (tel), and LOH events consistent with hemizygous deletion at 11p13 and 7p15.2-tel. Most noteworthy are 2 patients negative for any FISH abnormality who show copy-neutral LOH of most of 11q. The region of copy-neutral LOH includes 11q12-tel in one patient and 11q13.1-tel in the other patient. The copy-neutral LOH event involves close to 100% of the CLL cells in one patient, but only about 50% of the CLL cells in the other patient. Conclusions Whole genome CNV analysis by SNP-based microarrays greatly expands our ability to detect acquired genomic events in CLL cells and provides a powerful tool for CLL clinical research. Applying this new genomic technology to CLL patients with 11q deletions reveals marked complexity in the size and structure of hemizygous deletion events, which include the region of the ATM gene at 11q22.3 and can be discontinuous, indicative of complex genomic events. We identified copy-neutral LOH of 11q in 2 patients, a finding not previously described in CLL cells. Copy-neutral LOH of 11q is a genetic mechanism by which biallelic mutations of ATM, in cooperation with the loss of other tumor suppressor genes on 11q, may contribute to poor outcomes in some CLL patients without detectable FISH abnormalities. Additional studies, including sequencing of the ATM gene in 11q copy-neutral LOH patients, are required to confirm this hypothesis. Disclosures Shanafelt: Genentech, Hospira, Polyphenon E International, Celgene, Cephalon, Bayer Health Care Pharmaceuticals: Research Funding. Kay:Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding; Biogenc-Idec, Celgene, Genetech, Genmab: Membership on an entity's Board of Directors or advisory committees. Zent:Genentech: Research Funding; Bayer: Research Funding; Genzyme: Research Funding; Novartis: Research Funding.

Description: Abstract 1239 Poster Board I-261 Background Purine analogues, in particular fludarabine, are considered the gold standard of treatment of CLL. However, fludarabine therapy is sometimes complicated by autoimmune haemolytic anemia (AHA). The mechanism of this side effect is not clear but it is conceivable that a fludarabine-induced suppression of some regulatory systems, including T-reg, is responsible for this phenomenon. In addition, we have observed that patients affected by autoimmune diseases such as AHA or PTI have a reduced number of T lymphocytes bearing the CD200 antigen that is considered a tolerogenic molecule. In this perspective, we evaluated the variation of T-reg and CD200+ T lymphocytes induced by incubating in vitro peripheral blood mononuclear cells (PBMC) of CLL patients and normal subjects with purine analogues and other drugs active against CLL. Method PBMC obtained from patients with chronic lymphocytic leukaemia (CLL) (n=9) and from normal adult (n=6) were isolated by density gradient and cultured in RPMI supplemented with 10% FBS and 1% of penicillin streptomicyn. Cells were then incubated for 24 hours with drugs at two concentrations: bendamustine (1 and 50 μg/ml) campath (1 and 5 μg/ml) prednisone ( 1 and 10 nM), fludarabine (0,25 and 10 μg/ml) pentostatin (3 and 60 μg/ml). The cytotoxicity was evalutated after 24 hours by trypan Blue and flow cytometry. T-reg cells were identified as CD4+/CD25+/FoxP3+ T cells and expressed as a percentage of the CD4+T-cell population. Results Although all of these drugs induced lymphocytes cytotoxicity, fludarabine, prednisone, and campath reduced also the percentage of T-reg and CD200+ T –lymphocytes, while bendamustin and especially pentostatin induced the same cytotoxicity but spared T-reg populations and CD200+ T-lymphocytes. Table I indicates results obtained with the highest concentration of drugs. In conclusion, pentostatin and bendamustine seems to be active drugs against CLL and their usage shouldn't be complicated by autoimmune phenomena. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1275 Poster Board I-297 Core binding factor (CBF) leukemias, characterized by translocations t(8;21) or inv(16)/t(16;16) targeting the core binding factor, constitute acute myeloid leukemia (AML) subgroups with favorable prognosis. However, 40-50% of patients relapse, and the current classification system does not fully reflect the heterogeneity existing within the cytogenetic subgroups. Therefore, illuminating the biological mechanisms underlying these differences is important for an optimization of therapy. Previously, gene expression profiling (GEP) revealed two distinct CBF leukemia subgroups displaying significant outcome differences (Bullinger et al., Blood 2007). In order to further characterize these GEP defined CBF subgroups, we again used gene expression profiles to identify cell line models similar to the respective CBF cohorts. Treatment of these cell lines with cytarabine (araC) revealed a differential response to the drug as expected based on the expression patterns reflecting the CBF subgroups. In accordance, the cell lines resembling the inferior outcome CBF cohort (ME-1, MONO-MAC-1, OCI-AML2) were less sensitive to araC than those modeling the good prognostic subgroup (Kasumi-1, HEL, MV4-11). A previous gene set enrichment analysis had identified the pathways Caspase cascade in apoptosis and Role of mitochondria in apoptotic signaling among the most significant differentially regulated BioCarta pathways distinguishing the two CBF leukemia subgroups. Thus, we concluded that those pathways might be interesting targets for specific intervention, as deregulated apoptosis underlying the distinct subgroups should also result in a subgroup specific sensitivity to apoptotic stimuli. Therefore, we treated our model cell lines with the Smac mimetic BV6, which antagonizes inhibitor of apoptosis (IAP) proteins that are differentially expressed among our CBF cohorts. In general, sensitivity to BV6 treatment was higher in the cell lines corresponding to the subgroup with good outcome. Time-course experiments with the CBF leukemia cell line Kasumi-1 suggested a role for caspases in this response. Interestingly, combination treatment of araC and BV6 in Kasumi-1 showed a synergistic effect of these drugs, with the underlying mechanisms being currently further investigated. Based on the promising sensitivity to BV6 treatment in some cell lines, we next treated mononuclear cells (mostly leukemic blasts) derived from newly diagnosed AML patients with BV6 in vitro to evaluate BV6 potency in primary leukemia samples. Interestingly, in vitro BV6 treatment also discriminated AML cases into two distinct populations. Most patient samples were sensitive to BV6 monotherapy, but about one-third of cases were resistant even at higher BV6 dosage. GEP of BV6 sensitive patients (at 24h following either BV6 or DMSO treatment) provided insights into BV6-induced pathway alterations in the primary AML patient samples, which included apoptosis-related pathways. In contrast to the BV6 sensitive patients, GEP analyses of BV6 resistant cases revealed no differential regulation of apoptosis-related pathways in this cohort. These results provide evidence that targeting deregulated apoptosis pathways by Smac mimetics might represent a promising new therapeutic approach in AML and that GEP might be used to predict response to therapy, thereby enabling novel individual risk-adapted therapeutic approaches. Disclosures Vucic: Genentech, Inc.: Employment. Deshayes:Genentech, Inc.: Employment.

Description: Abstract 1270 Poster Board I-292 3-Indole-acetic acid (IAA) is a growth hormone found in plants that may be oxidized by horseradish peroxidase (HRP) generating cytotoxic molecules capable of inducing injury in mammal cells, including a variety of human tumors. The aim of this study was to establish an assay based on targeting HRP to hematopoietic tumor cells using antibodies and induce apoptosis by incubation with IAA. To set up the best experimental conditions, we used two human lineages of hematologic tumors: NB4, derived from acute promyelocytic leukemia (APL) and Granta 519, from Mantle Cell Lymphoma (MCL). The targeting of HRP was performed with anti-CD33 or anti-CD19 antibodies (depending on the origin of the cell), followed by incubation with goat anti-mouse antibody conjugated with the enzyme. Eight groups of cells were incubated for 2, 8, 18, 24 and 48 hours: control, HRP targeted, HRP targeted with IAA at 1, 5 and 10 mM, cells not targeted with IAA at 1, 5 and 10 mM. Apoptosis was analyzed by flow cytometry, with anexin V-FITC and propidium iodide. The best experimental conditions for both NB4 and Granta 519 cells were achieved with incubation for 24 h with 10 mM IAA. For NB4, the group targeted with HRP and treated with 10 mM IAA for 24 hours presented 44.1% of apoptosis, whereas the control groups achieved 18.2 to 24.6% (P

Description: Abstract 1216 Poster Board I-238 Background: PD-1 (Program Death-1), an immune inhibitory receptor and its ligands PD-L1 and PD-L2, participate in peripheral tolerance and play a key role in immune suppression and evasion mechanisms in cancer and chronic infectious diseases. PD-1 inhibits activation signals and functions as a pro-apoptotic receptor in effector lymphocytes, and consequently regulates the extent and duration of specific adaptive and innate immune responses. CT-011, a humanized antibody, blocks the function of PD-1, resulting in increased activities of T and NK cells in vitro and in enhanced tumor immunity in experimental tumor models. At the molecular level, the antibody enhances PI3K-mediated survival and trafficking signals, attenuates cell death in effector/memory (CD4+CD45RO+) cells, and enhances trafficking in response to Stromal Cell-derived Factor-1 (SDF-1). We hypothesized that CT-011 would enhance effector/memory cells in patients with DLBCL after AuSCT and delay recurrence. Methods: We treated 41 patients (pts) with DLBCL from 30-90 days after AuSCT with CT-011 and now report data on effector/memory and memory lymphocytes in the first 30 pts. CT-011 was given at a dose of 1.5mg/kg for 3 doses, 6 weeks apart. The primary endpoint was to determine the proportion of patients who have not relapsed or died within 18 months following autologous PBSCT, and it is too early for that analysis. Our secondary endpoint was to measure the number of effector /memory and memory lymphocytes before and after treatment, and those data are the subject of this report. Results: Flow cytometry analyses (Table) on pre (baseline: BL) and post-treatment blood samples from the first 30 pts enrolled show elevated levels of specific effector/memory and memory CD4+ T lymphocytes following treatment with CT-011; the median absolute number (ABS) of effector/memory CD4+CD45RO+CD62L-CCR7- cells was increased by +49% from BL (p

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 2887 Poster Board II-863 Between November 2002 and April 2006, 72 patients with Waldenstrom's Macroglobulinemia (WM) were enrolled into this multicenter trial of primary treatment with DRC which consisted of dexamethasone 20 mg IV followed by rituximab 375mg/m2 IV on day 1 and oral cyclophosphamide 100 mg/m2 bid on days 1 to 5 (total dose 1000 mg/m2). DRC courses were repeated every 21 days for six courses and then patients without progressive disease were observed without treatment. Patient characteristics, toxicity and response data have been reported previously (Dimopoulos et al, J Clin Oncol 2007; 25:3344): 83% of patients achieved a response including 7% complete, 67% partial and 9% minor responses. In June 2009 we updated this study (minimum follow-up 〉3 years) in order to assess time to progression, time to next treatment, type of second-line treatment and response to this, overall survival (OS) and cause-specific survival (CSS) in which deaths unrelated to WM or complications of treatment were censored. Second line treatment was administered to patients who experienced progressive disease and also met criteria for treatment requirement based on consensus recommendations (Kyle et al, Sem Oncol 2003; 30:116). As of June 2009, 42 patients fulfilled the criteria for progressive disease (Kimby et al, Clin Lymphoma Myeloma 2006; 6:380) but 14 patients have not yet required second line treatment. The median time to progression was 35 months (95% Confidence Interval: 22-48 months) and the median time to next treatment requirement was 51 months. Among several factors who were analyzed for their possible correlation with shorter time to progression, only lymphadenopathy was significant (p

Description: Abstract 2877 Poster Board II-853 Background and Objective: In newly diagnosed multiple myeloma (MM) patients, treatment with lenalidomide plus high-dose dexamethasone (RD) was superior to high-dose dexamethasone in terms of both response rates and 1-year progression-free survival (PFS) (Zonder JA et al, Blood 2007;110:77). Preliminary results suggest that the combination lenalidomide plus low-dose dexamethasone (Rd) compared to the RD regimen yields significantly better 2-year overall survival (OS) (Rajkumar SV et al, J Clin Oncol 2008;26:8504). The combination of melphalan, prednisone, and lenalidomide (MPR) has been investigated in a phase I/II study showing promising results (Palumbo A et al, J Clin Oncol 2007; 25:4459-4465). The goal of this case –control study was to compare the efficacy and the toxicity of the lenalidomide/dexamethasone (len/dex) combination vs MPR as primary therapy for newly diagnosed elderly MM patients, to determine the additive value of melphalan compared to a regimen of lenalidomide plus corticosteroid. Patients and methods: Data from 51 newly diagnosed MM patients enrolled in Italy in a phase I/II dose-escalating trial, from January to October 2005, with MPR, were analyzed. For comparison of their outcome, 37 patients were identified among newly diagnosed patients seen at the Mayo Clinic from March 2005 to December 2008 who received len/dex as primary therapy and were enrolled in phase II or III trials. Patients treated with MPR received 9 monthly cycles of oral melphalan (doses ranging from 0.18 to 0.25 mg/kg on days 1-4), prednisone (2 mg/kg on days 1-4) and lenalidomide (doses ranging from 5 to 10 mg/day on days 1-21). After 9 cycles, patients started maintenance with lenalidomide alone (10 mg, days 1-21) until relapse or progression. Patients treated with len/dex received oral lenalidomide (25 mg/day, days 1-21) plus dexamethasone, either at low-dose (n=17) (40 mg orally days 1, 8, 15, 22) or at high-dose (n=21) (40 mg orally on days 1-4, 9-12, and 17-20). Treatment was continued until progression, relapse or unacceptable toxicity, or could be stopped at the physician's discretion. Patients (n=13) were allowed to receive transplant if they wished and were deemed eligible. Outcome was analyzed on an intention-to-treat basis. The Chi-square or the rank sum tests were used to compare variables. Time-to-event analysis was performed using the Kaplan-Meier method and comparisons were determined by the log-rank test and the Cox proportional hazards model. Results: On intention-to-treat analysis, 15.7% versus 23.7% patients, respectively in the MPR and in the len/dex group, (p=0.342) achieved a complete response, and 43.2% vs 47.4%, (p=0.691) achieved at least a very good partial response. Time-to-progression (TTP) (median: 24.7 vs 27.5 in MPR and len/dex groups, respectively; HR 1.04; 95% CI 0.55-1.98; p=0.903), PFS (median: 24.7 vs 27.5 in MPR and len/dex groups, respectively; HR 1.03; 95% CI 0.55-1.92; p=0.926) and OS (2-year OS: 86.2% in MPR group vs 89.1% in len/dex, HR 0.86; 95% CI 0.38-1.98; p=0.730) were not significantly different between the 2 groups. No significant differences in TTP, PFS and OS were reported when MPR patients were compared with the subgroup of patients treated with low-dose dexamethasone plus lenalidomide. Similar results were found when the analysis was restricted to MPR patients and len/dex pair mates receiving lenalidomide plus low/dose dexamethasone, matched according to age and sex, and who did not received transplant. The toxicity profile was different in the two groups. Hematologic grade 3-4 toxicities were more common with MPR compared with len/dex, in particular neutropenia (66.7% vs 21.1%, p 〈 0.001) and thrombocytopenia (31.4% vs 2.6%, p 〈 0.001), respectively. Grade 3-4 gastrointestinal events (13.2% vs 2.0%, p= 0.080), thrombotic events (13.2 vs 5.9, p= 0.279) and fatigue (10.5% vs 3.9%, p= 0.395) were more common with len/dex compared with MPR. Conclusion: Results of this case-control study show that both MPR and Rd are efficacious regimens for elderly MM patients. Data need however to be carefully evaluated and randomized control trials are needed to confirm these results. Disclosures: Off Label Use: research drug in combination to standard of care. Kumar:celgene: Research Funding; millenium: Research Funding; bayer: Research Funding; novartis: Research Funding; genzyme: Research Funding. Dispenzieri:celgene: Research Funding. Gertz:celgene: Honoraria; genzyme: Honoraria; millenium: Honoraria; amgen: Honoraria. Lacy:celgene: Research Funding. Musto:celgene: Honoraria. Fonseca:medtronic: Consultancy; genzyme: Consultancy; celgene: Consultancy; amgen: Consultancy; BMS: Consultancy; otsuka: Consultancy. Petrucci:celgene: Honoraria; Janssen Cilag: Honoraria. Greipp:celgene: Research Funding. Boccadoro:jansen Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; pharmion: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Palumbo:Janssen-Cilag: Honoraria; Celgene: Honoraria.

Description: Abstract 288 Interaction between the members of the BH3 domain family of proteins plays an important role in the development, progression, and prognosis of various subtypes of B-cell lymphoma. Therapies that selectively induce a pro-apoptotic environment are an attractive strategy to overcome chemotherapy-resistance in B-cell lymphoma. The proteasome is an important regulator of various members of Bcl-2 family proteins. We previously demonstrated that obatoclax, a novel BH3 mimetic, was able to enhance the anti-tumor activity of rituximab and chemotherapy agents and induced both apoptosis and autophagy in B-cell lymphoma. In an attempt to increase the therapeutic options for B-cell lymphoma patients we studied the biological effects of obatoclax in combination with bortezomib in a panel of rituximab-[chemotherapy]-sensitive (RSCL) and rituximab-[chemotherapy]-resistant cell lines (RRCL), as well as primary tumor cells isolated from 45 NHL patient biopsy samples with various subtypes of B-cell lymphoma: (ie including, DLBCL, follicular lymphoma (FL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), and Hodgkin lymphoma (HL)]. Patient-derived primary tumor cells were isolated from fresh biopsies by negative selection using magnetic beads. Transient knock-down of p53 and Noxa were performed to determine the role of p53 in the anti-tumor activity of bortezomib or obatoclax, respectively. NHL cells were exposed in vitro to escalating doses of obataclox (0, 2, 5, 10 and 20mM) and/or bortezomib (0, 2, 10 and 20nM) for 24 and 48 hrs. Cell death was determined by the cell glow luminescent assay and DNA synthesis was evaluated by standard [3H]-Thymidine incorporation assays at 24 and 48 hrs. Changes in mitochondrial potential and cell proliferation were determined by alamar blue reduction using a kinetic assay measuring activity at 4 hr intervals for 24 and 48 hrs. In vitro exposure of RRCL, RSCL, and primary tumor cells to the combination of obatoclax plus bortezomib demonstrated significant synergistic activity compared to controls. Patients with DLBCL (n=15) and FL (N=12) demonstrated significant sensitivity to this combination. Of note, activity was observed in patients with either de novo or relapsed/refractory germinal B-cell (GBC) or activated B-cell (ABC) DLBCL (as characterized by the Han's criteria). Additionally, cell death induced by obatoclax or bortezomib could be inhibited by transient knock-down of p53 or Noxa, respectively. In summary, deregulation of apoptosis by BH3 inhibition with obatoclax and bortezomib results in cell death and antiproliferation not only in RSCL and RRCL, but also in primary tumor cells derived from “treatment-naïve or refractory” DLBCL and FL patients. Data strongly suggests that both p53 and Noxa have pivotal roles in response to obatoclax and bortezomib. The combination of obatoclax plus bortezomib has the potential of becoming a novel and potent therapeutic strategy in the treatment of B-cell lymphoma in the future. Research, in part, supported as part of a subproject on NIH PO1 grant CA103985-1 awarded to the Garden State Cancer Center, Belleville, NJ and NHI R-01 grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2868 Poster Board II-844 Background and Objective: In newly diagnosed multiple myeloma (MM), the combination of lenalidomide plus high-dose dexamethasone (RD) is superior to high-dose dexamethasone (Zonder JA et al, Blood 2007;110:77). Preliminary results show that lenalidomide plus low-dose dexamethasone (Rd) has better 2-year overall survival (OS) compared with RD (Rajkumar SV et al, J Clin Oncol 2008;26:8504). The addition of clarithromycin (Biaxin) to lenalidomide and low-dose dexamethasone (BiRd) has been investigated in a phase II study, demonstrating a high response rate and 2-year OS (Niesvizky R at al, Blood 2008;111:1101-1109). However, the additive value of clarithromycin is not known. No randomized trials have compared Rd versus BiRd, none are planned. The objective of this case–matched study was to compare the efficacy and the toxicity of BiRd vs Rd as initial therapy for newly diagnosed MM. Patients and methods: Data from 72 newly diagnosed MM patients treated at the New York Presbyterian Hospital–Cornell Medical Center, from December 2004 to December 2006, with BiRD, were analyzed. For comparison, an equal number of pair mates were selected among newly diagnosed patients seen at the Mayo Clinic who received Rd, from March 2005 to December 2008. Case matching was performed with respect to age, gender, and transplant status. Patients treated with BiRd received oral lenalidomide (25 mg/day, days 3-21 of cycle 1; days 1-21 of subsequent cycles); dexamethasone (40 mg days 1, 2, 3, 8, 15, 22 of cycle 1; days 1, 8, 15, and 22 of each subsequent cycle); clarithromycin (500 mg twice daily, from day 2 of cycle 1). Patients treated with Rd received oral lenalidomide (25 mg/day, days 1-21) plus low-dose dexamethasone (40 mg days 1, 8, 15, 22). In both groups patients were allowed to discontinue treatment to pursue transplant, but treatment until progression, relapse or unacceptable toxicity was permitted at the physician's discretion. Outcome was analyzed on an intention-to-treat basis. The Chi-square or the rank sum tests were used to compare variables. Time-to-event analysis was performed using the Kaplan-Meier method; comparisons were determined by the log-rank test and the Cox proportional hazards model. Results: Median duration of treatment was 11.8 months in the BiRd group vs 6 months in the Rd group. On intention-to-treat analysis, complete response was significantly higher with BiRd compared to Rd (45.8% vs 13.9%, respectively, p

Description: Abstract 2861 Poster Board II-837 Bcl-2 protein family has the unique capability to balance between the cell survival and death by regulating the expression of its individual members. AT-101 is a BH3 mimetic and a potent inducer of noxa and puma, the natural ligands of BH3 family proteins. It is known to bind and inhibit the anti-apoptotic functions of Bcl-2 family members Bcl-2 and Bcl-XL and Mcl-1. In vitro it has been shown to induce apoptosis in several tumor models systems including multiple myeloma. In this report we investigated the effect of AT-101 on a model cell line, BCWM.1 (a known WM cell line, BCWM.1/WT), representing Waldenström Macroglobulinemia. This disease is characterized by the presence of lymphoplasmacytic cells in the bone marrow and the secretion of IgM monoclonal protein into the serum. Several conventional therapies are available but the disease remains incurable. Therefore there remains a need to develop new therapies for this orphan disease. Recently, bortezomib (a proteasomal inhibitor) has shown promising anti-WM activity with enhanced responses when combined with traditional therapies. But continued treatment with bortezomib result in the development of resistance in the clinic. We developed an in vitro model of bortezomib resistance from BCWM.1 (hereafter referred as BCWM.1/BR). These cells also developed cross resistance to conventional therapies used for WM such as fludarabine and doxorubicin. Biological characterization of this cell line demonstrated Bcl-2 as a potentially important therapeutic target. We therefore assessed the effect of AT-101 to identify preclinically if this could be a potential clinical strategy in future. AT-101 induced a dose and time dependent inhibition in the viability of both BCWM.1/WT as well as BCWM.1/BR cells. Cell death was observed at as low as 1uM concentration of AT-101 and at 10uM a maximum of 50-70% death was observed by 24h. While BCWM.1/WT cells showed a significant death at 12h, treatment with AT-101 induced cell death in BCWM.1/BR cells as early as 6h. These results indicate that AT-101 induced a potent and quick inhibition in viability in BCWM.1/BR cells as compared to their parental wild type cells. Investigation into the mechanism of cell death showed that AT-101 induced apoptosis in a mitochondrial dependent pathway in these cells. A comparative analysis of the signal transduction pathways operated in BCWM.1/WT and BCWM.1/BR cells showed that many of the cellular activation and survival pathways such as AKT, ERK1/2 that are present in BCWM.1 cells are inhibited in the resistant cells. Interestingly, BCWM.1/BR cells expressed a fivefold increase in the Bcl-2 protein as compared to BCWM.1/WT cells suggesting a Bcl-2 dependent survival of these cells in the absence of other cellular activation and survival signals. Increased susceptibility of BCWM.1/BR cells to AT-101 thus can be understood to be a direct consequence of an increased expression of Bcl-2 and a dependence of the resistant cells on Bcl-2 family of anti-apoptotic proteins for their survival. Results presented in this report suggest that AT-101 has a unique therapeutic potential against Waldenström Macroglobulinemia that is independent of resistance to bortezomib. These observations highlight bcl-2 as a potential target, and AT-101 as possible therapeutic avenue for WM patients. Disclosures: Chanan-Khan: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Immunogen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Description: Abstract 2829 Poster Board II-805 Introduction: We could recently show that the heat shock proteins (HSP) HSP90 and HSP70 are frequently overexpressed in multiple myeloma (MM), stabilize as molecular chaperones various oncogenic proteins and contribute to survival of MM cells. Currently, several clinical Phase I/II studies are under way to evaluate the concept of pharmacological HSP90 blockade in human cancer. Under cellular stress conditions the heat shock transcription factor 1 (HSF1) has a key regulatory role for the up-regulation of HSP. Importantly, it has been observed that treatment with the proteasome inhibitor bortezomib, a clinically effective anti-MM agent, induces up-regulation of HSP90, HSP70 and HSP27. Furthermore, it has recently been demonstrated that HSF1 can protect cells from oncogene-driven malignant transformation. We therefore analyzed the role of HSF1 for the malignant growth of MM cells. Methods: Western analyses were performed to determine HSF1 expression and regulation in different human MM cell lines. To examine the expression of HSF1 and different HSP like HSP90, HSP70 and HSP27 in situ, samples from 60 bone marrow biopsies obtained from MM patients were immunohistochemically stained. To analyze the role of HSF1 for the survival of MM cells, HSF1 was either selectively depleted by siRNA-mediated knockdown using a pSUPER-based siRNA expression vector or targeted by treatment with a novel pharmacological HSF1 inhibitor triptolide. In addition, pharmacological inhibition of HSF1 was combined with concomitant pharmacological inhibition of either HSP90 (with the novel inhibitor NVP-AUY922) or bortezomib. Furthermore, gene expression analyses with an Affymetrix GeneChip were performed to identify HSF1 target genes in MM cells. Results: Here, we show that HSF1 is frequently overexpressed in MM cell lines in vitro and in the majority of the analyzed MM biopsies in situ, but not in MGUS or in normal plasma cells. Blockade of HSF1 either by siRNA-mediated knockdown or treatment with the novel pharamacological HSF1 inhibitor triptolide led to a strong induction of apoptosis in cells of the MM cell lines INA-6 and MM.1s. Importantly, also primary MM cells showed apoptosis induction after triptolide treatment. HSF1 inhibition led to downregulation of HSP70, HSP27 and HSP90. Gene expression analysis revealed a number of additional molecular targets of HSF1 involved in apoptosis regulation. Furthermore, initial experiments indicated that the apoptotic effect of pharmacological HSF1 inhibition is enhanced by the concomitant pharmacological inhibition of either HSP90 or the proteasome. Conclusion: We demonstrate that HSF1 is overexpressed in MM, contributes to the survival of MM cells and controls the activity of oncogenic HSP like HSP90, HSP70 and HSP27. Targeting HSF1 may therefore represent an attractive potential therapeutic strategy in MM, in particular in combination with HSP90 or proteasome inhibitors. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2843 Poster Board II-819 Multiple myeloma (MM) is an incurable B-cell malignancy characterized by accumulation of malignant plasma cells in bone marrow (BM) and recurrent or persistent infections. Toll like receptors (TLRs) are essential in the host defense against infections. In MM, recurrent infections could promote tumor growth and favor escape from standard therapies. TLRs initiated responses in B cells include proliferation, antiapoptosis and immune escape. In this study, we first screened four MM cell lines, MM1-r, MM1-s, RPMI8226, and U266, for the expression of major TLRs (including TLR 1–2,4-7and 9) by RT-PCR. Surprisingly, all the MM cell lines expressed multiple TLRs. We focused on TLR4, which had the most strongly expressed mRNA. Consistent with the RT-PCR result, fluorescence-activated cell sorting (FACS) analysis revealed that TLR4 protein was also present on the surface of MM cell lines. By FACS analysis, we found that MM1-s and MM1-r cells had increased TLR4 co-receptor CD14 expression when induced by lipopolysaccharide (LPS). We next asked if TLR4 was functional in MM cells. To activate TLR4 in MM cells (MM1-r, MM1-s, RPMI8226, and U266), we incubated the cells with LPS, the natural ligand for TLR4 and measured cell proliferation by [3H]thymidine incorporation. Proliferation of MM1-s and MM1-r cells increased significantly, but that of U266 and RPMI8226 cells did not. As mechanisms involved in the resistance to apoptosis play a major role in MM escape to therapies, we sought to determine the capacity of TLR4 ligand to promote the survival of MM cells. We pretreated MM cells with lipopolysaccharide followed by induction by adriamycin. Our results showed that LPS could save MM1-s cells from adriamycin induced apoptosis. In addition to proliferation and apoptosis, we would like to learn whether TLR4 ligand could change cell cycle, We found that MM1-s and MM1-r MM cells showed decreased number in G0/G1 phase but increased number in S phase when induced by LPS. To explore whether MAPK pathways were implicated in MM1-s and MM1-r cells to LPS stimulation, we investigated the role of ERK1/2, p38, and JNK MAPKs in LPS-stimulated cells by using specific inhibitors of MAPK pathways, Firstly, time-dependent MAPKs phosphorylation was measured to assess the activation of these kinases upon treatment with LPS. ERK1/2, p38, and JNK phosphorylation and NF-κB were significantly up-regulated following LPS treatment, with the maximum phosphorylation occurring at 20min after stimulation. It is interesting to note that JNK inhibitors inhibited phosphorylation of JNK and inhibited phosphorylation of p-38 simultaneously. Moreover, inhibitors of ERK1/2 inhibited phosphorylation of ERK1/2, increased phosphorylation of p-38 simultaneously, suggesting that ERK, JNK and p38 pathways were associated. Our findings demonstrated that LPS-induced cell proliferation dependent on JNK, ERK and p38 signaling, suggesting that ERK and p38 pathways were involved in MM1-s and MM1-r cells proliferation. Since TLRs activate innate and adaptive immune responses, we further study immunoregulatory factor IL-6, IL-12 and IL-18 secretion in the culture supernatants induced by LPS. LPS increased the production of IL-6 in RPMI8226 and U266 cells, and increased the production of IL-18 in MM1-r and MM1-s cells. To further examine the effect of LPS on immune escape, MM1-s cells were co-cultured with T cells from donors and 3H incorporation was examined for T cells proliferation. Our results showed that T cells proliferation was decreased when the cells were treated by LPS, suggesting that TLR4 ligand LPS facilitates MM1-s cells' evasion of immune surveillance. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2828 Poster Board II-804 Studies including thalidomide showed a rate of severe infection that can be life-threatening complication or compromise compliance to therapy ranging from 6% to 22%. Therefore, antibacterial prophylaxis has become a routine clinical practice despite its role in the new-drugs era has to be defined. We performed a post-hoc analysis of patients treated with thalidomide based combinations within controlled trials in order to assess time, type and outcome of infections. We analysed the main demographic and disease related variables to search for factors affecting onset of infections during induction and build a risk model in order to perform targeted prophylaxis. Two hundred and twenty four patients were eligible for this study. Median age was 70 years (range 31-90 years) and 141 patients (63%) had more than 65 years. Fifty three percent of patients had de novo MM whereas the remaining had received thalidomide as second or subsequent lines of therapy. ISS stage 2-3 and renal impairment were present in 156 (69%) and 38 (17%) of patients, respectively. Induction therapy consisted in the following protocols: ThaDD (160 patients: 71.5%), ThaDD-V (42 patients: 19%), VMPT (9 patients: 4%), TD (8 patients: 3.5%) and VTD (5 patients: 2%). Prophylaxis for infections was administered to 168 patients (75%) and consisted of quinolones (72%) or thrimethoprim-sulphamethoxazole (28%). Eighty six patients (38.5%) developed an infection resulting of grade 3-4 in 39 of them (17.5%) (12% grade 3, 5.5% grade 4). Probability of infection at six months was 39% although that of severe infection was 20% (18% at 4 months and just 2% from 4 to 6 months). Among the 39 patients with severe infection, 23 (59%) developed pneumonia, 9 FUO (23%), 6 bacteremia (1 septic shock) and 1 an orbital abscess. Aetiology of severe infection was recognized in 7 patients (4 Gram-negative bacteria, 1 Gram-positive bacteria, 1 CMV and 1 probable fungal infection). Eighty percent of severe infections occurred during the first 3 courses of induction therapy and only 12% during neutropenia. Fifteen percent of patients undergoing antibiotic prophylaxis developed infection vs 25% of patients who did not (p= 0.084). There were no difference between quinolones and thrimethoprim-sulphamethoxazole prophylaxis regarding incidence of infections. The majority of infections were empirically treated and cured with wide spectrum antibiotic therapy except when a specific aetiology was recognized. Only one patient died because of septic shock during neutropenia and 2 patients withdrawn from protocol because of infection. In univariate analysis monoclonal component 〉 2 g (p=0.021), platelets 〈 130.000/ml (p= 0.005), newly diagnosed MM (p=0.083) and antibiotic prophylaxis (p=0.061) were factors predicting severe infection development whereas age, sex, ECOG performance status, MM type, D-S stage, plasmacell infiltration in bone marrow, haemoglobin concentration, serum b2-microglobulin, serum albumin, ISS, serum C-Reactive Protein, serum creatinine, previous stem cell transplantation were not. Cox regression analysis selected monoclonal component 〉 2 g (p=0.015 HR= 1.8) and platelets 〈 130.000/ml (p=0.003 HR= 2.3) as covariates associated to severe infection. The 25 patients without adverse factors, the 125 with 1 and the 74 with 2 adverse factors had a probability of severe infection equal to 4%, 17% and 32 % (p= 0.023), respectively. This model remains useful apart from prophylaxis since the probability of severe infection in patients with at least 1 risk factors receiving prophylaxis is 17% vs 4% in patients without risk factors. Of note, patients developing severe infection had a significantly higher incidence of deep venous thrombosis (DVT) compared with patients who did not (20.5% vs 9%: p= 0.041). DVT occurred after a median time of 0.9 months (range 0.1-5 months; 75% within 2 months) from infection onset. In conclusion, despite antibiotic prophylaxis, patients receiving thalidomide combination therapy can develop severe infections particularly pneumonia. Wide spectrum antibiotic therapy is effective in the majority of cases since viral or fungal infections are very rare. Patients with large size of disease, represented by high MC and low platelets count, are at higher risk of severe infection that in turn significantly increase the risk of DVT. Therefore, these patients at high-risk should receive more suitable antimicrobial prophylaxis. Disclosures: Off Label Use: Thalidomide, Bortezomib and Doxil.

Description: Abstract 2854 Poster Board II-830 Targeting the cell cycle in combination with cytotoxic killing is a rational approach to cancer therapy. Progression in multiple myeloma (MM) stems from both loss of apoptotic control in the bone marrow (BM) microenvironment and dysregulation of the cyclindependent kinases (CDK)4 and CDK6, which precedes uncontrolled proliferation of myeloma cells in vivo in particular during relapse. This reinforces the critical importance of targeting CDK4/CDK6 in MM. Through selective and reversible inhibition of CDK4/CDK6 with PD 0332991, the only known CDK4/6-specific inhibitor, we have recently developed a novel strategy to sensitize primary myeloma cells for cytotoxic killing by diverse cytotoxic drugs. These include carfilzomib (PR-171), an irreversible selective inhibitor of the chymotrypsin-like activity of the proteasome, and PR-047, an orally bioavailable analog of carfilzomib. We showed that induction of prolonged early G1 arrest following inhibition of CDK4/CDK6 markedly enhances cytotoxic killing of primary BM myeloma cells by either carfilzomib or PR-047 despite protection by BM stromal cells. The enhancement of cytotoxic killing is further augmented during synchronous S phase entry upon removal of PD 0332991 subsequent to induction of prolonged G1 arrest in myeloma cell lines. In both cases, the enhancement in carfilzomib (or PR-047) mediated killing is not associated with cell cycle regulation of the proteasome activity. It is caspase-dependent, requiring only a brief (one hour) exposure to the proteasome inhibitor at concentrations as low as 30 nM. This killing is mediated by synergistic and rapid induction of mitochondrial membrane depolarization and activation of downstream caspase-9. Further, it is apparently initiated by cell cycle-dependent expression of the pro-apoptotic BH3-only proteins, which neutralize the anti-apoptotic Bcl-2 family proteins upstream of mitochondrial depolarization. Bim is upregulated during early G1 arrest to neutralize the anti-apoptotic MCL-1 and Bcl-2. By contrast, Noxa is silenced in G1 but dramatically upregulated in S phase, in particular when combined with carfilzomib. Importantly, targeting CDK4/CDK6 with PD 0332991 in combination with either carfilzomib or PR-047 leads to complete eradication of myeloma cells ex vivo, in contrast to the combination of PD 0332991 with other proteasome inhibitors. Selective inhibition of CDK4/CDK6 in combination with carfilzomib (or PR-047), therefore, not only halts tumor cell proliferation but also potently induces synergistic killing that is likely to profoundly inhibit cell cycle reentry and self-renewal in MM. PD 0332991 is a small molecule with bio-availability and proven tumor suppressing activity in both human myeloma xenograft and immunocompetent mouse myeloma models. It is well tolerated in humans as indicated by the ongoing Phase I/II clinical trials in myeloma and previous phase I trials in mantle cell lymphoma and solid tumors. Evidence from Phase 2 trials of carfilzomib indicates that it is also well tolerated, in fact, the peripheral neuropathy that is commonly observed with the proteasome inhibitor bortezomib appears to be less severe and possibly less frequent. Mechanism-based targeting of CDK4/6 in combination with selective proteasome inhibitors, like carfilzomib and PR-047, thus represents a new and promising therapeutic strategy for multiple myeloma and potentially other hematopoietic malignancies. Disclosures: Off Label Use: PD 0332991 is going to be used as a CDK4/6-specific inhibitor. Parlati:Proteolix, Inc.: Employment, Equity Ownership. Aujay:Proteolix, Inc.: Employment, Equity Ownership. Niesvizky:Millenium: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Seattle Genetics, Inc: Research Funding; Proteolix: Research Funding, data monitoring committee.

Description: Abstract 2842 Poster Board II-818 In addition to full length (FL) transcripts, clinically significant HAS 1 splice variants (Va, Vb and Vc) have been previously identified in multiple myeloma (MM) and Waldenstrom's macroglobulinemia. Increased HAS1Vb expression correlates with poor survival in a cohort of MM patients. Here, we show that directed mutation of HAS1 intron 3 alters HAS1 splicing and generates a pattern of HAS1 variant expression that mimics patterns detected in MM patients. This suggests that hypermutation of HAS1 and consequent expression of HAS1 splice variants may contribute to oncogenesis in MM. HAS1FL comprises of 5 exons (2089 bp); Va skips exon 4 (133 bp); Vb skips exon 4 and partially retains 59 bp of intron upstream of exon 5 (+59); Vc has all 5 exons and partially retains 26 bp of intron downstream of exon 4. In MM, frequent intronic mutations have been observed in introns 3 and 4, suggesting possible contributions to HAS1 alternative splicing. We have utilized a mammalian expression system to analyse HAS1 splicing by fusing a minigene extending from exon 3 to exon 5 (g345) with the upstream cDNA sequences. HAS1 expression is determined by transfection and RT-PCR using appropriated primer sets. This study focuses on identification of intronic mutations that may affect HAS1 splicing. We target mutations on (A/U)GGG motif because of its high abundance in HAS1 intron 3. The (A/U)GGG repeat was also shown to enhance the splicing of alternative intron in chicken β-tropomyosin (Sirand-Pugnet, P, et al, NAR, 1995, 23, 3501) and intronic G runs could work in a combinatorial way to control the selection of the proper 3' splice site in human thrombopoietin (Marcucci, R, et al, NAR, 2006, 35, 132). A 580 bp long human HAS1 intron 3 is GC-rich and comprises of 28 (A/U)GGG motifs (sequentially identified as G1, G2.., G28). HeLa cells transfected with an unmutated intron 3 construct mainly produce FL with a small amount of HAS1Va, a profile that is similar to CD40L/IL-4 activated normal B cells. Site directed mutagenesis of all 28 (A/U)GGG motifs (G1-28) abolished FL expression, but not HAS1Va, suggesting that these sequence alterations are highly unfavorable for constitutive splicing. It may be due to the loss of essential cis-acting element(s) and/or undesirable conformational changes that prevent spliceosome formation. Mutagenesis of G1-18 is shown to eliminate constitutive expression by increasing the usage of multiple alternative donor sites. Mutagenesis of G19-28 produces more HAS1Va than FL, presumably due to increased exon 4 skipping events. An increased Va/FL ratio could also be achieved by mutagenesis of G25-28 or G27-28, suggesting that this subregion is important for pathway selection. Mutagenesis was also studied in del1 construct, a unique derivative of HAS1 minigene that partially deletes intron 4. Similar to g345, del1 produces FL and HAS1Va as well as promotes expression of novel HAS1Vd, an isoform that includes +59 bp (like Vb) and exon 4. Alteration of HAS1 splicing profile caused by mutagenesis shown in g345 series is also observed in del1 series. Additionally, there is a shift from Vd to Vb expression in all constructs analysed (del1/G1-28, del1/G1-18, del1/G19-28, del1/G25-28 and del1/G27-28), a pattern of aberrant splicing that found in MM patients. Thus, in del1, increased exon 4 skipping events promote both Va and Vb expression. Sequencing of HAS1 intron 3 in a cohort of 50 MM patients indicates that recurrent mutations are found in the G repeat regions and that new repeats are generated by recurrent MM-specific HAS1 mutations. This suggests that mutation of the HAS1 construct mimics HAS1 mutation events that occur in MM patients themselves, and contributes to the clinically significant aberrant HAS1 splicing we have reported in MM (Adamia et al. Blood, 2008, 112, 5111; Blood, 2005, 105, 4836). Overall, critical mutations that could alter HAS1 expression and the ratio of HAS1 variants to FL were identified in intron 3. In intron 4, critical mutations that increase the usage of alternative splice site (+59) remain to be studied. We speculate that cumulative mutations within these two intronic sequences could bring the two events together to promote HAS1Vb splicing. While trans-acting elements are likely to regulate RNA splicing and its pathway, our studies clearly suggest that intronic mutations play an important role in the aberrant splicing of human HAS1, with probable contributions to disease progression in MM. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2839 Poster Board II-815 Multiple myeloma (MM) remains largely incurable. The existence of the putative myeloma stem cell population may account for drug-resistance and relapse. Previous investigation showed that the CD138-negative (CD138-) MM cell fraction contains the putative MM stem cells. However, little is known about the molecular characteristics of MM stem cells, which makes it difficult to specifically target such cells. In this study, by comparing gene expression profiles (GEP) of CD138+ and CD138- cells isolated from 10 MM cell lines, we discovered that RARα is the top one over-expressed gene in CD138- MM stem cells. RARα has two major isoforms, RARα1 and RARalpha2. Real-time PCR detected significantly higher expression of RARalpha2 but not of RARalpha1 in CD138- MM cells compared with CD138+ cells. We recently reported that increased RARalpha2 expression at diagnosis resulted in a significantly shorter overall-survival (P = 0.003); importantly, ATRA selectively killed RARalpha2-positive myeloma cells, but not RARalpha2-negative cells. These results suggested that ATRA could be used to eradicate specifically RARalpha2-over-expressing MM stem cells. Indeed, we found that ATRA selectively killed CD138- MM stem cells (P 〈 0.01) from ARP-1 and OCI-MY5 human myeloma cell lines and 5T33 murine myeloma cells but spared the CD138+ tumor cells from these cell lines. WNT and Hedgehog (HH) signaling pathways were activated in CD138- stem cells. To our surprise, ATRA treatment (10-6M) further increased WNT and HH signaling pathways in CD138- myeloma cells, based on increased protein levels of β-catenin and cleaved Shh/Ihh in ATRA-treated cells. Stimulation of these signaling pathways by LiCl (5 mM) and/or SHH(20 μg/mL) partially abrogated ATRA-induced cytotoxities on CD138- MM cells, demonstrating that stimulation of WNT and HH signaling induced partial ATRA-resistance in CD138- cells. A combinational treatment of ATRA (1 μM), a COX-2 inhibitor celecoxib (shown to inhibit WNT signaling, 50 μM) and a HH signaling inhibitor cyclopamine (10 μM) induced synergistic effects on cell death and growth inhibition of CD138- and RARalpha2-over-expressing MM cells in-vitro. In the 5T33 murine myeloma model, the combination of ATRA (20mg/Kg), celecoxib (10mg/Kg) and cyclopamine (20mg/Kg) induced synergistic inhibition of tumor growth in-vivo. Thus, our study provides novel approaches to specifically target MM stem cells. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2880 Poster Board II-856 Background: MM is increasing in incidence and remains incurable. .NK cells have modest killing activity against MM tumor cells in part because of inhibitory KIR receptors which recognize HLA class 1 antigens on MM tumor cell targets. However, experimental and clinical data in the allogeneic transplant setting suggest that NK cell stimulation by a mismatch between donor KIR and patient KIR ligand may improve outcomes for MM after a reduction of tumor burden by previously administered treatments. To mimic this effect with a pharmaceutical agent, 1-7F9/IPH2101, a fully human IgG4 anti-KIR mAb specific for KIR2DL1/2/3 (HLA-C specific KIRs) was generated (Romagne et al., Blood June, 2009). 1-7F9/IPH 2101 enhances patient NK cell cytotoxicity against autologous MM tumor cells in vitro. We present the interim results of the human phase I trial of this agent in patients with relapsed/refractory MM. Methods: An open-label, single-agent, dose-escalation, multiple dose safety and tolerability study of IPH2101 is being conducted in heavily pre-treated patients with relapsed/refractory MM. Dose escalation with IPH2101 (0.0003, 0.003, 0.015, 0.075, 0.3, 1, 3 mg/kg as IV infusion) is being studied using a 3+3 scheme. Re-dosing criteria (1/month x 4 months) are based on safety data from previous dosing. KIR occupancy, pharmacokinetics (PK), pharmacodynamics, effects on NK cell maturation, and biological effects of IPH 2101 are being monitored in all patients. Results: Currently, dose escalation is entering the final (3 mg/kg) cohort. Data from the first 22 treated patients are available. No Dose Limiting Toxicity (DLT) has been observed. 1 pt (at DL1) has been replaced and 3 additional pts have been enrolled (at DL4) due to an SAE an acute renal failure possibly related to drug. Related Adverse Events were seen in 4/22 patients (18%). 12/22 pts received at least 2 doses (6pts had 2, 1 pt had 3 and 5 pts had 4 cycles-median 2). KIR full occupancy (〉 90%) for at least 3 weeks is reached at 1mg /kg. In accordance with the pre-clinical PK/PD model there is a clear relationship between exposure (Cmax) and KIR occupancy. No deleterious effect on NK cell maturation has been seen. IPH 2101 has been well tolerated to date. In the cohorts accrued to date, two heavily pre-treated patients, both with high-risk cytogenetics, showed evidence to suggest disease stabilization while receiving IPH-2101. Conclusions: IPH 2101 improves autologous NK cell killing of MM tumor cells by blocking inhibitory KIR. In the on-going clinical trial, the antibody appears safe and well tolerated at the doses tested. Updated study results will be presented at the time of the meeting This immunotherapeutic approach may hold promise as treatment for MM and further study is warranted. Disclosures: Squiban: Innate pharma: Employment. Marzetto:Innate Pharma: Employment. Andre:Innate Pharma: Employment. Tollier:Innate Pharma: Employment.