ALBERT

Description: Cancer cells frequently overexpress tissue factor (TF) and become procoagulant. This conversion may be driven by genetic transformation, including through the expression of the oncogenic epidermal growth factor receptor (EGFR) and its mutant, EGFRvIII, present in glioblastoma multiforme (GBM). Here we show that the EGFRvIII-dependent GBM cell transformation is associated with the onset of the simultaneous overexpression of TF, protease-activated receptors 1 and 2 (PAR1 and PAR2), and ectopic synthesis of factor VII (FVII). Efficient generation of factor Xa by these cells still requires exogenous FVIIa. However, as a result of EGFRvIII-dependent transformation, GBM cells become hypersensitive to TF/PAR-mediated signaling and produce ample angiogenic factors (vascular endothelial growth factor and interleukin-8) on exposure to FVIIa and PAR1- or PAR2-activating peptides. Thus, oncogenes may cause complex changes in the ability of GBM cancer cells to interact with the coagulation system, thereby exacerbating its influence on angiogenesis and disease progression.

Description: Although much is known about extrinsic regulators of platelet function such as nitric oxide and prostaglandin I2 (PGI2), considerably less is known about intrinsic mechanisms that prevent overly robust platelet activation after vascular injury. Here we provide the first evidence that regulators of G-protein signaling (RGS) proteins serve this role in platelets, using mice with a G184S substitution in Gi2α that blocks RGS/Gi2 interactions to examine the consequences of lifting constraints on Gi2-dependent signaling without altering receptor:effector coupling. The results show that the Gi2α(G184S) allele enhances platelet aggregation in vitro and increases platelet accumulation after vascular injury when expressed either as a global knock-in or limited to hematopoietic cells. Biochemical studies show that these changes occur in concert with an attenuated rise in cyclic adenosine monophosphate levels in response to prostacyclin and a substantial increase in basal Akt activation. In contrast, basal cyclic adenosine monophosphate (cAMP) levels, agonist-stimulated increases in [Ca++]i, Rap1 activation, and α-granule secretion were unaffected. Collectively, these observations (1) demonstrate an active role for RGS proteins in regulating platelet responsiveness, (2) show that this occurs in a pathway-selective manner, and (3) suggest that RGS proteins help to prevent unwarranted platelet activation as well as limiting the magnitude of the normal hemostatic response.

Description: Constitutive expression of tissue factor (TF) by cancer cells triggers local activation of the coagulation cascade and promotes breast cancer progression through cell signaling involving protease activated receptor (PAR)2. In human breast cancer, TF and PAR2 are up-regulated and TF cytoplasmic domain phosphorylation is correlated with relapse. Here we show that cancer cell PAR2 signaling promotes angiogenesis independent of PAR2 phosphorylation at the recognized β-arrestin recruitment site. Similar to PAR2−/− mice, TF cytoplasmic domain–deleted (TFΔCT) mice have delayed spontaneous breast cancer development in the polyoma middle T model. Simultaneous deletion of PAR2 in TFΔCT mice did not further delay tumor appearance, consistent with overlapping roles of TF and PAR2 in promoting the angiogenic switch in early stages of breast cancer. In advanced carcinomas, tumor-associated macrophages were reduced in TFΔCT and TFΔCT/PAR2−/− mice, and increased tumor vessel diameters of TFΔCT mice were partially reversed by PAR2-deficiency, indicating that the TF cytoplasmic domain has additional roles that are interdependent with PAR2 signaling in regulating host angiogenic responses. These experiments demonstrate a crosstalk of tumor cell TF cytoplasmic domain and PAR2 signaling and provide a possible mechanism for the close correlation between TF phosphorylation and cancer recurrence of TF and PAR2-positive clinical breast cancer.

Description: Abstract 3783 Introduction: Dengue fever is the most prevalent mosquito-borne viral disease, and its clinical signs and symptoms can vary widely. Since hemorrhagic manifestations are common, hematologists are often included in the care of these patients, and face a difficult challenge trying to discriminate dengue fever from other more common viral infections. Laboratory tests used in this context are less than optimal. The expected findings of leukopenia and thrombocytopenia are absent in many cases, thus delaying the suspicion for dengue and the ordering of confirmatory serological tests. Leukocyte morphology changes in numerous medical conditions, including infections. The VCS technology available in the LH and DxH hematology analyzers is capable of automatically quantifying leukocyte morphologic characteristics: cell volume by voltage impedance (V); cytoplasmic and nuclear density by radio frequency conductivity (C); and cytoplasmic granularity and nuclear complexity by laser light scatter (S). The mean and standard deviation of each parameter is calculated. This data is collected for neutrophils, lymphocytes, monocytes and eosinophils separately. Previous studies have demonstrated the significant value of these VCS parameters in improving the laboratory diagnosis of malaria and septicemia, both in adults and neonates. The objective of this study was to compare the performance of the VCS parameters with that of traditional hematological tests used in the diagnosis of dengue fever. Methods: We included in this study 174 patients who were clinically suspected to have dengue fever, as indicated by the clinicians ordering serological testing. Of these, 92 patients were positive and 82 negative by serology. Using the Mann-Whitney U test, we compared the mean VCS values, as well as total leukocyte counts, leukocyte differential counts and platelet counts, between both groups. For those tests that showed a statistically significant difference between dengue positive and dengue negative patients, their diagnostic performance was compared by ROC curve. Finally, we separated patients with normal platelet and WBC counts, likely to be misdiagnosed based on these traditional laboratory tests, and evaluated the diagnostic performance of the VCS parameters in this subgroup. Results: Tests that showed a statistically significant difference (p

Description: MicroRNAs are small noncoding RNAs that regulate cellular development by interfering with mRNA stability and translation. We examined global microRNA expression during the differentiation of murine hematopoietic progenitors into megakaryocytes. Of 435 miRNAs analyzed, 13 were up-regulated and 81 were down-regulated. Many of these changes are consistent with miRNA profiling studies of human megakaryocytes and platelets, although new patterns also emerged. Among 7 conserved miRNAs that were up-regulated most strongly in murine megakaryocytes, 6 were also induced in the related erythroid lineage. MiR-146a was strongly up-regulated during mouse and human megakaryopoiesis but not erythropoiesis. However, overexpression of miR-146a in mouse bone marrow hematopoietic progenitor populations produced no detectable alterations in megakaryocyte development or platelet production in vivo or in colony assays. Our findings extend the repertoire of differentially regulated miRNAs during murine megakaryopoiesis and provide a useful new dataset for hematopoiesis research. In addition, we show that enforced hematopoietic expression of miR-146a has minimal effects on megakaryopoiesis. These results are compatible with prior studies indicating that miR-146a inhibits megakaryocyte production indirectly by suppressing inflammatory cytokine production from innate immune cells, but cast doubt on a different study, which suggests that this miRNA inhibits megakaryopoiesis cell-autonomously.

Description: Therapeutic targeting of virus-encoded proteins using cellular immunotherapy has proved successful for Epstein-Barr virus (EBV)–associated posttransplant lymphoproliferative disease. However, the more limited repertoire and immunogenicity of EBV-encoded proteins in other malignancies such as Hodgkin lymphoma and extranodal natural killer (NK)/T lymphoma has been more challenging to target. The immunosubdominant latent membrane protein 2 (LMP2) is considered the optimal target in such Latency II tumors, although data relating to its expression in T/NK malignancies are limited. In addressing the validity of LMP2 as an immunotherapeutic target we found that LMP2-specific effector CD8+ T cells recognized and killed EBV-positive NK- and T-cell tumor lines, despite an apparent absence of LMP2A protein and barely detectable levels of LMP2 transcripts from the conventional LMP2A and LMP2B promoters. We resolved this paradox by identifying in these lines a novel LMP2 mRNA, initiated from within the EBV terminal repeats and containing downstream, epitope-encoding exons. This same mRNA was also highly expressed in primary (extra-nodal) NK/T lymphoma tissue, with virtually undetectable levels of conventional LMP2A/B transcripts. Expression of this novel transcript in T/NK-cell lymphoproliferative diseases validates LMP2 as an attractive target for cellular immunotherapy and implicates this truncated LMP2 protein in NK- and T-cell lymphomagenesis. This study is registered at clinicaltrials.gov as NCT00062868.

Description: There is increasing evidence that miRNA and transcription factors interact in an instructive fashion in normal and malignant hematopoiesis. We explored the impact of TEL-AML1 (ETV6-RUNX1), the most common fusion protein in childhood leukemia, on miRNA expression and the leukemic phenotype. Using RNA interference, miRNA expression arrays, and quantitative polymerase chain reaction, we identified miRNA-494 and miRNA-320a to be up-regulated upon TEL-AML1 silencing independently of TEL expression. Chromatin immunoprecipitation analysis identified miRNA-494 as a direct miRNA target of the fusion protein TEL-AML1. Using bioinformatic analysis as well as functional luciferase experiments, we demonstrate that survivin is a target of the 2 miRNAs. miRNA-494 and miRNA-320a were introduced to the cells by transfection and survivin expression determined by Western blot analysis. These miRNAs blocked survivin expression and resulted in apoptosis in a similar manner as TEL-AML1 silencing by itself; this silencing was also shown to be Dicer-dependent. miRNAs-494 and -320a are expressed at lower levels in TEL-AML1+ leukemias compared with immunophenotype-matched nonTEL-AML1 acute lymphoblastic leukemia subtypes, and within TEL-AML1+ leukemias their expression is correlated to survivin levels. In summary our data suggest that TEL-AML1 might exert its antiapoptotic action at least in part by suppressing miRNA-494 and miRNA-320a, lowering their expression causing enhanced survivin expression.

Description: Apc, a negative regulator of the canonical Wnt signaling pathway, is a bona-fide tumor suppressor whose loss of function results in intestinal polyposis. APC is located in a commonly deleted region on human chromosome 5q, associated with myelodysplastic syndrome (MDS), suggesting that haploinsufficiency of APC contributes to the MDS phenotype. Analysis of the hematopoietic system of mice with the Apcmin allele that results in a premature stop codon and loss of function showed no abnormality in steady state hematopoiesis. Bone marrow derived from Apcmin mice showed enhanced repopulation potential, indicating a cell intrinsic gain of function in the long-term hematopoietic stem cell (HSC) population. However, Apcmin bone marrow was unable to repopulate secondary recipients because of loss of the quiescent HSC population. Apcmin mice developed a MDS/myeloproliferative phenotype. Our data indicate that Wnt activation through haploinsufficiency of Apc causes insidious loss of HSC function that is only evident in serial transplantation strategies. These data provide a cautionary note for HSC-expansion strategies through Wnt pathway activation, provide evidence that cell extrinsic factors can contribute to the development of myeloid disease, and indicate that loss of function of APC may contribute to the phenotype observed in patients with MDS and del(5q).

Description: Abstract 2825 Mantle cell lymphoma (MCL) constitutes one of the lymphomas with poorest prognosis at relapse and there are no effective salvage. The activity shown by the combination Gemcitabine and Oxaliplatin in several types of limfoma, along with its “in vitro” synergistic effect, made this regimen an attractive regimen for salvaging patients with MCL. Against this background, we performed an off-label pilot study in order to assess efficacy and toxicity of the combination R-GemOx in relapsed or refractory patients with MCL. For this, 27 patients (70% male, median age 70 years) diagnosed with MCL between November 2004 and January 2010 were included in this study. Inclusion criteria were adequate performance status, confirmed diagnosis of MCL and relapse or refractoriness to the previous treatment. The regimen consisted of Rituximab 375 mg/m2 on day 1, Gemcitabine 1000 mg/m2 and Oxaliplatin 100 mg/m2 on day 2, every 14 days, up to 8 cycles. Dose and interval were adjusted according to hematological and extrahematological toxicities. Median number of previous regimens was 1 (range 1 to 3), being EPOCH-R (n=14), R-CHOP (n=6), and RFC (n=3) the most frequently used induction therapies. Twelve patients relapsed after prior CR, 10 progressed after achieving a PR, whereas 5 were refractory to therapy. At inclusion, 85% of patients were in advanced (III/IV) clinical stage, 40% had bone marrow infiltration, 28% gastrointestinal involvement, and 17% cavum infiltration. Median number of cycles administered was 8 (range, 3 to 8). Doses were reduced in 9 cycles and delayed in 15 cycles. Neutropenia grade 3–4 was observed in 9 cycles and thrombocytopenia grade 3–4 in 6. Hepatotoxicity grade 1–2 in 5 pts and grade 3 in 1, sensitive neurotoxicity grade 1–2 in 12 pts, and renal impairment grade 2 in 1 patient. After completion of the treatment, 21 pts (77%) were considered in CR/uCR, 1 (4%) achieved a PR, 1 a SD, whereas 4 PD. Six patients subsequently received a stem-cell transplantation (4 allogeneic, 2 autologous). Thirteen pts are still alive and out of progression. Ten pts have died (8 due to progression and 2 due to acute GVHD). With a median follow-up of 23 months (range: 3–57), PFS and OS at 2 years are 41% and 58%, respectively. The R-GemOx combination showed a significant activity in relapsed or refractory pts with MCL with a very acceptable toxicity profile. These results prompted us to conduct a multicenter phase II clinical trial that is now ongoing. Disclosures: López: Roche Farma: Research Funding, Travel Support. Bosch:Roche Farma: Research Funding.

Description: Abstract 3498 Introduction: Oral mucositis is a complication of conditioning treatment that produces pain and morbidity in allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients. The rationale of this study was to evaluate the efficacy of a calcium phosphate mouth rinse (Caphosol) vs standard regimen in 40 adult patients undergoing allo-HSCT. Patients and Methods: 40 patients treated with allo-HSCT (31 from unrelated donors, 9 from siblings) performed in Hematology and BMT center in Katowice in 2009 were randomized and stratified by the conditioning regimen (busulfan-, treosulfan- or TBI- based), type of transplant (unrelated or related) and age into two equal groups. Treatment group received Caphosol washes 4 times daily from first day of conditioning until reaching ANC 0.2 G/l. Control group received standard topical mouth care with salvia, antibacterial and antifungal solutions. During the trial patients subjectively assessed level of pain in mouth and in pharynx using 0–10 scale and swallowing problems using 0–5 scale. Mucositis was judged by experienced physician. Nonparametric Mann-Whitney U-tests were used for statistical analysis. Results: Average oral toxicity in WHO scale in Caphosol vs control group was 0.9 vs 1.8 (p=0.02), duration of mucositis was 3.2 vs 7.1 days (p=0.02). Total parenteral nutrition (TPN) due to mucosits was required in Caphosol vs control group in 0 vs 6 pts, average duration of TPN was 0 vs 1.9 days (p=0.009). Analgetics were required, respectively, in 3 vs 9 pts and analgesy lasted for 1.1 vs 3.4 days (p=0.047). Average subjective peak pain in mouth was 0.85 vs 1.75 (p=0.005) and in pharynx 1.95 vs 2.2 (NS) in Caphosol vs control group, average pain intensity was lower in Caphosol group throughout the whole period of mucositis. Intensity of swallowing problems tended to be lower in Caphosol group (NS). Acute GVHD was observed in 7 vs 9 pts in Caphosol vs control group and its average degree was 0.5 vs 0.9 (NS). Conclusions: Caphosol mouth rinse in the allo-HSCT recipients is associated with decrease of oral toxicity, lower peak pain due to mucositis and its shorter duration. In consequence, comfort of life is improved and the incidence of acute GVHD is reduced, as well as the requirement of TPN and analgetics. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4517 Oral mucositis is a common toxicity associated with many cancer therapies and has been correlated with risk for infection, mortality, and extended hospital stay. Prophylactic use of a supersaturated calcium phosphate mouth rinse (SSCPR, Caphosol..) was found in a phase III study to reduce the frequency, intensity, and duration of oral mucositis in patients (pts) undergoing allogeneic or autologous hematopoietic stem cell transplantation (Papas, et al. Bone Marrow Transpl 2003;31:705). That study also found a faster time to recovery of an absolute neutrophil count (ANC) ≥0.2×109/l, but not for other engraftment endpoints, possibly a result of the mixed pt population studied. We performed a single-center retrospective review of a uniform population of pts

Description: To define a role for hematopoietic stem cell transplantation (HSCT) in infants with acute lymphoblastic leukemia and rearrangements of the mixed-lineage-leukemia gene (MLL+), we compared the outcome of MLL+ patients from trial Interfant-99 who either received chemotherapy only or HSCT. Of 376 patients with a known MLL status in the trial, 297 (79%) were MLL+. Among the 277 of 297 MLL+ patients (93%) in first remission (CR), there appeared to be a significant difference in disease-free survival (adjusted by waiting time to HSCT) between the 37 (13%) who received HSCT and the 240 (87%) who received chemotherapy only (P = .03). However, the advantage was restricted to a subgroup with 2 additional unfavorable prognostic features: age less than 6 months and either poor response to steroids at day 8 or leukocytes more than or equal to 300 g/L. Ninety-seven of 297 MLL+ patients (33%) had such high-risk criteria, with 87 achieving CR. In this group, HSCT was associated with a 64% reduction in the risk of failure resulting from relapse or death in CR (hazard ratio = 0.36, 95% confidence interval, 0.15-0.86). In the remaining patients, there was no advantage for HSCT over chemotherapy only. In summary, HSCT seems to be a valuable option for a subgroup of infant MLL+ acute lymphoblastic leukemia carrying further poor prognostic factors. The trial was registered at www.clinicaltrials.gov as #NCT00015873 and at www.controlled-trials.com as #ISRCTN24251487.

Description: Mantle cell lymphoma (MCL) is a mostly incurable malignancy arising from naive B cells (NBCs) in the mantle zone of lymph nodes. We analyzed genomewide methylation in MCL patients with the HELP (HpaII tiny fragment Enrichment by Ligation–mediated PCR) assay and found significant aberrancy in promoter methylation patterns compared with normal NBCs. Using biologic and statistical criteria, we further identified 4 hypermethylated genes CDKN2B, MLF-1, PCDH8, and HOXD8 and 4 hypomethylated genes CD37, HDAC1, NOTCH1, and CDK5 when aberrant methylation was associated with inverse changes in mRNA levels. Immunohistochemical analysis of an independent cohort of MCL patient samples confirmed CD37 surface expression in 93% of patients, validating its selection as a target for MCL therapy. Treatment of MCL cell lines with a small modular immunopharmaceutical (CD37-SMIP) resulted in significant loss of viability in cell lines with intense surface CD37 expression. Treatment of MCL cell lines with the DNA methyltransferase inhibitor decitabine resulted in reversal of aberrant hypermethylation and synergized with the histone deacetylase inhibitor suberoylanilide hydroxamic acid in induction of the hypermethylated genes and anti-MCL cytotoxicity. Our data show prominent and aberrant promoter methylation in MCL and suggest that differentially methylated genes can be targeted for therapeutic benefit in MCL.

Description: The neutrophil nicotinamide adenine dinucleotide phosphate-oxidase is a multisubunit enzyme (comprising gp91phox, p22phox, p67phox, p40phox, p47phox, and Rac) that plays a vital role in microbial killing. The recent discovery of a chronic granulomatous disease patient who expresses a mutant p40phox subunit, together with the development of mouse models of p40phox function, indicate phosphatidylinositol 3-phosphate binding to the PX domain of p40phox is an important signal for oxidase activation. However, the presence of other conserved residues and domains in p40phox suggest further regulatory roles for this protein. To test this, we introduced wild-type and mutated versions of p40phox into fully differentiated mouse neutrophils by retroviral transduction of p40phox−/− bone marrow progenitors and repopulation of the bone marrow compartment in radiation chimaeras. Phosphorylation of p40phox on threonine 154, but not serine 315, was required for full oxidase activation in response to formylated bacterial peptide fMLP, serum-opsonized S aureus, and immunoglobulin-opsonized sheep red blood cells. A functional SH3 domain was not required for oxidase activation, and deletion of the entire domain resulted in enhanced oxidase responses. Phosphorylation of threonine 154 in response to S aureus was mediated by protein kinase Cδ and was required for full translocation of p47phox to phagosomes. These results define an important new element in the physiological activation of the oxidase.

Description: Our previous work has shown that axon guidance gene family Nogo-B and its receptor (NgBR) are essential for chemotaxis and morphogenesis of endothelial cells in vitro. To investigate NogoB-NgBR function in vivo, we cloned the zebrafish ortholog of both genes and studied loss of function in vivo using morpholino antisense technology. Zebrafish ortholog of Nogo-B is expressed in somite while expression of zebrafish NgBR is localized in intersomitic vessel (ISV) and axial dorsal aorta during embryonic development. NgBR or Nogo-B knockdown embryos show defects in ISV sprouting in the zebrafish trunk. Mechanistically, we found that NgBR knockdown not only abolished its ligand Nogo-B–stimulated endothelial cell migration but also reduced the vascular endothelial growth factor (VEGF)–stimulated phosphorylation of Akt and vascular endothelial growth factor–induced chemotaxis and morphogenesis of human umbilical vein endothelial cells. Further, constitutively activated Akt (myristoylated [myr]Akt) or human NgBR can rescue the NgBR knockdown umbilical vein endothelial cell migration defects in vitro or NgBR morpholino-caused ISV defects in vivo. These data place Akt at the downstream of NgBR in both Nogo-B– and VEGF-coordinated sprouting of ISVs. In summary, this study identifies the in vivo functional role for Nogo-B and its receptor (NgBR) in angiogenesis in zebrafish.

Description: Telomerase reverse transcriptase (TERT) is a good candidate for cancer immunotherapy because it is overexpressed in 85% of all human tumors and implicated in maintenance of the transformed phenotype. TERT-based cancer vaccines have been shown to be safe, not inducing any immune-related pathology, but their impact on tumor progression is modest. Here we show that adoptive cell therapy with the use of high-avidity T lymphocytes reactive against telomerase can control the growth of different established tumors. Moreover, in transgenic adenocarcinoma mouse prostate mice, which develop prostate cancer, TERT-based adoptive cell therapy halted the progression to more aggressive and poorly differentiated tumors, significantly prolonging mouse survival. We also demonstrated that human tumors, including Burkitt lymphoma, and human cancer stem cells, are targeted in vivo by TERT-specific cytotoxic T lymphocytes. Effective therapy with T cells against telomerase, different from active vaccination, however, led to autoimmunity marked by a consistent, although transient, B-cell depletion in primary and secondary lymphoid organs, associated with alteration of the spleen cytoarchitecture. These results indicate B cells as an in vivo target of TERT-specific cytotoxic T lymphocytes during successful immunotherapy.

Description: Adaptive immune responses are characterized by substantial restructuring of secondary lymphoid organs. The molecular and cellular factors responsible for virus-induced lymphoid remodeling are not well known to date. Here we applied optical projection tomography, a mesoscopic imaging technique, for a global analysis of the entire 3-dimensional structure of mouse peripheral lymph nodes (PLNs), focusing on B-cell areas and high endothelial venule (HEV) networks. Structural homeostasis of PLNs was characterized by a strict correlation between total PLN volume, B-cell volume, B-cell follicle number, and HEV length. After infection with lymphocytic choriomeningitis virus, we observed a substantial, lymphotoxin (LT) β-receptor–dependent reorganization of the PLN microarchitecture, in which an initial B-cell influx was followed by 3-fold increases in PLN volume and HEV network length on day 8 after infection. Adoptive transfer experiments revealed that virus-induced PLN and HEV network remodeling required LTα1β2-expressing B cells, whereas the inhibition of vascular endothelial growth factor-A signaling pathways had no significant effect on PLN expansion. In summary, lymphocytic choriomeningitis virus-induced PLN growth depends on a vascular endothelial growth factor-A–independent, LT- and B cell–dependent morphogenic pathway, as revealed by an in-depth mesoscopic analysis of the global PLN structure.

Description: Abstract 3562 Aim of the present study was to evaluate the clinical outcome of a large series of younger patients with symptomatic multiple myeloma (MM) who were enrolled in two subsequent clinical trials of thalidomide-dexamethasone (thal-dex) incorporated into double autologous stem-cell transplantation (ASCT) to support high-dose melphalan (200 mg/m2). In both studies, thal (100 mg/day for the first 14 days and then 200 mg/day) and pulsed dex (between 480 and 160 mg per cycle), were administered from the onset until the second ASCT. The analysis was performed on an intention-to-treat basis on a total of 593 patients who were followed for a median of 36 months. The best VGPR and CR rates were 69% and 35%, respectively. The median duration of CR was 66 months. Median TTP and PFS were 53 and 44 months, respectively. The 5-year projected rates of TTP and PFS were 46% and 38%, respectively, while the corresponding value for OS was 67%. More than 80% of the patients were screened at diagnosis for the presence of cytogenetic abnormalities by FISH analysis. Forty-five percent of patients had del(13q), while t(4;14) and del(17p) were found in 16 % and 7 % of patients, respectively. The presence of del(17p) and/or t(4;14) was associated with a significantly shorter 5-year projected TTP, PFS and OS in comparison with the absence of these abnormalities, indifferently from the presence or absence of del(13q) (TTP: 30% vs 53%, respectively P=0.0000; PFS: 28% vs 45%, respectively, P=0.0000; OS: 53% vs 69%, respectively, P=0.0000). OS and PFS curves of patients carrying del(13q) alone were almost superimposable to those of patients without cytogenetic abnormalities, while TTP was significantly shorter for patients with del(13q) alone (5-year projected rates: 40% vs 53%, respectively, P=0.04). Patients carrying del(17p) in the absence of t(4;14) had similar 5-year projected TTP and PFS as compared with t(4;14) positive but del(17p) negative patients. However, OS was significantly shorter for the subgroup with del(17p) and absence of t(4;14) in comparison with that of patients carrying t(4;14) without del(17p) (5 year projected rates: 18% vs 70%, respectively, P=0.03). In a multivariate analysis, presence of del(17p) and high beta2-m at baseline were the most important variables adversely influencing TTP (HR: 2.3, P=0.001 and HR: 1.8, P=0.002, respectively), PFS (HR: 2.0, P=0.001 and HR: 1.9, P=0.001, respectively), and OS (HR: 3.9, P=0.000 and HR: 2.0, P=0.005, respectively). Additional variables predicting for shorter TTP and PFS were the presence of t(4;14) (HR: 1.8, P=0.004) and of del(13q) (HR: 1.6, P= 0.009). Also the quality of best response to the overall treatment program influenced clinical outcomes. In particular, patients achieving CR had a significantly longer PFS and OS than those achieving a VGPR (PFS: median 68 vs 40 months, respectively, P=0.007; 5-year projected OS rates: 84% vs 70%, respectively, P=0.01). In conclusion, incorporation of thal-dex into double autotransplantation failed to overcome the poor prognosis conferred by del(13 q), t(4;14) and del(17p). In a multivariate Cox regression analysis, del(17p) and high levels of serum beta2-m at diagnosis were the strongest variables adversely influencing PFS and OS. In comparison with the presence of t(4;14) but absence of del(17p), patients carrying del(17p) without t(4;14) had a significantly shorter OS, possibly due to their worst outcome after relapse. Presence of del(13q) alone conferred a significantly shorter TTP, but did not have an adverse impact on OS due to the favorable role of effective salvage therapies incorporating either bortezomib or lenalidomide. Disclosures: Off Label Use: use of first line thalidomide in preparation for ASCT. Cavo:Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, no; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, no; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, no.

Description: Determining how normal and leukemic stem cells behave in vivo, in a dynamic and noninvasive way, remains a major challenge. Most optical tracking technologies rely on the use of fluorescent or bioluminescent reporter genes, which need to be stably expressed in the cells of interest. Because gene transfer in primary leukemia samples represents a major risk to impair their capability to engraft in a xenogenic context, we evaluated the possibility to use gene transfer–free labeling technologies. The lipophilic dye 3,3,3′,3′ tetramethylindotricarbocyanine iodide (DiR) was selected among 4 near-infrared (NIR) staining technologies. Unfortunately we report here a massive transfer of the dye occurring toward the neighbor cells both in vivo and in vitro. We further demonstrate that all lipophilic dyes tested in this study (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine perchlorate [DiI], DiD, DiR, and PKH26) can give rise to microenvironmental contamination, including when used in suboptimal concentration, after extensive washing procedures and in the absence of phagocytosis or marked cell death. This was observed from all cell types tested. Eventually, we show that this microenvironmental contamination is mediated by both direct cell-cell contacts and diffusible microparticles. We conclude that tracking of labeled cells using non–genetically encoded markers should always be accompanied by drastic cross validation using multimodality approaches.

Description: Abstract 4236 Anemia is one of the most common blood disorders in several diseases including cancer, heart failure, chronic kidney disease (CKD) and Myeloid Dysplastic Syndromes (MDS) associated with a negative outcome. Administration of recombinant Erythropoietin (EPO) represents the most common treatment for anemia. However, a significant number of people remain hypo or non-responsive to EPO treatment, and in some cases its use has been linked to tumor growth, cardiovascular disease and poorer survival. The members of TGFβ super family of ligands (Activins, GDFs and BMPs) and receptors (Type I and II) regulate more than 500 target genes transcriptionally by Smad phosphorylation and are involved in many cellular functions including cell growth, adhesion, migration, differentiation and apoptosis in a concentration and context dependent manner. Members of the TGFβ family have also demonstrated a role in erythropoiesis. ACE-536, a non-ESA agent is a soluble human Fc fusion chimera of a modified Activin Type IIb receptor with a mutation in its extracellular domain. Surface Plasmon Analysis (Biacore) analysis and cell based reporter assays revealed that this mutation disrupted its binding to Activin A but not to GDF11 or GDF8. ACE-536 acts as a decoy receptor for TGFβ signaling and demonstrated potent increase in red blood cells in all the tested animals (mice, rats and monkeys). Subcutaneous administration of ACE-536 (10mg/kg) to C57BL/6 mice resulted in a significant increase in hematocrit, hemoglobin and red blood cells (RBC) over the TBS treated vehicle group after 4 days. These observations were seen even in the presence of an EPO neutralizing antibody; suggesting that EPO is not directing the initial RBC response to ACE-536 treatment. There were no increase in BFU-E or CFU-E colony formation from bone marrow and spleen after 48hrs treatment with ACE-536 over TBS treated group demonstrating that it does not have effect on erythroid progenitor population. Differentiation profiling of bone marrow and splenic erythroblasts by flow cytometric analysis revealed that ACE-536 promotes maturation of developing erythroblasts. ACE-536 treatment for 72 hours resulted in a decrease in basophilic erythroblasts and an increase in late stage poly, ortho chromatophilic erythroblasts in bone marrow and spleen compared to the TBS treated mice. Treatment of Sprague-Dawley rats with a murine analogue of ACE-536 (RAP-536; 10mg/kg) increased the reticulocyte formation in peripheral blood over vehicle treated group. ACE-536 (10mg/kg) treatment combined with recombinant human EPO (1800 units/kg) for 72 hours increased RBC, hematocrit and hemoglobin by approximately 23% over TBS treated vehicle group and 12% over EPO treatment alone. Consistent with its role in proliferation, EPO increased splenic basophilic erythroblast formation. However, ACE-536 treatment combined with EPO significantly promoted maturation of late stage erythroblasts; demonstrating a novel mechanism during erythroid differentiation. To gain further insights into its mechanism of action, C57BL/6 mice were administered with or without RAP-536 (10mg/ml twice a week) pre treated for a week with neutralizing anti-Activin A (10mg/kg) or ActRIIa (10mg/ml) or ActRIIb (10mg/ml) (does not bind ACE-536) antibodies. Anti-ActRIIa but not anti-Activin A or anti- ActRIIb antibody pre-treatment inhibited the RBC increase by RAP-536 suggesting that ActRIIa or its ligands are necessary for transducing the signal. To summarize, ACE-536 treatment results in a rapid increase in red blood cells by a novel mechanism promoting maturation of late stage erythroblasts. The efficacy of ACE-536 molecule was tested in several acute and chronic anemia animal models including blood loss anemia, chemotherapy induced anemia, chronic kidney disease (5/6 Nephrectomy) and Myeloid Dysplastic Syndrome (MDS) and found that ACE-536 treatment prevents or decreases anemia in all these models. Furthermore, unlike EPO, ACE-536 did not promote tumor progression (in Lewis Lung Carcinoma model) thus offering strong promise as alternate treatments for anemia. Disclosures: Suragani: Acceleron Pharma: Employment. Cadena:Acceleron Pharma: Employment. Mitchell:Acceleron Pharma: Employment. Sako:Acceleron Pharma: Employment. Davies:Acceleron Pharma: Employment. Tomkinson:Acceleron Pharma: Employment. Devine:Acceleron Pharma: Employment. Ucran:Acceleron Pharma: Employment. Grinberg:Acceleron Pharma: Employment. Underwood:Acceleron Pharma: Employment. Pearsall:Acceleron Pharma: Employment. Seehra:Acceleron Pharma: Employment. Kumar:Acceleron Pharma: Employment.

Description: The cellular and molecular mechanisms orchestrating the complex process by which bone marrow megakaryocytes form and release platelets remain poorly understood. Mature megakaryocytes generate long cytoplasmic extensions, proplatelets, which have the capacity to generate platelets. Although microtubules are the main structural component of proplatelets and microtubule sliding is known to drive proplatelet elongation, the role of actin dynamics in the process of platelet formation has remained elusive. Here, we tailored a mouse model lacking all ADF/n-cofilin–mediated actin dynamics in megakaryocytes to specifically elucidate the role of actin filament turnover in platelet formation. We demonstrate, for the first time, that in vivo actin filament turnover plays a critical role in the late stages of platelet formation from megakaryocytes and the proper sizing of platelets in the periphery. Our results provide the genetic proof that platelet production from megakaryocytes strictly requires dynamic changes in the actin cytoskeleton.

Description: In neutrophils, the phosphoinositide 3-kinase/Akt signaling cascade is involved in migration, degranulation, and O2− production. However, it is unclear whether the Akt kinase isoforms have distinct functions in neutrophil activation. Here we report functional differences between the 2 major Akt isoforms in neutrophil activation on the basis of studies in which we used individual Akt1 and Akt2 knockout mice. Akt2−/− neutrophils exhibited decreased cell migration, granule enzyme release, and O2− production compared with wild-type and Akt1−/− neutrophils. Surprisingly, Akt2 deficiency and pharmacologic inhibition of Akt also abrogated phorbol ester-induced O2− production, which was unaffected by treatment with the phosphoinositide 3-kinase inhibitor LY294002. The decreased O2− production in Akt2−/− neutrophils was accompanied by reduced p47phox phosphorylation and its membrane translocation, suggesting that Akt2 is important for the assembly of phagocyte nicotinamide adenine dinucleotide phosphate oxidase. In wild-type neutrophils, Akt2 but not Akt1 translocated to plasma membrane upon chemoattractant stimulation and to the leading edge in polarized neutrophils. In the absence of Akt2, chemoattractant-induced Akt protein phosphorylation was significantly reduced. These results demonstrate a predominant role of Akt2 in regulating neutrophil functions and provide evidence for differential activation of the 2 Akt isoforms in neutrophils.

Description: In Burkitt lymphoma/leukemia (BL), achievement of complete remission with first-line chemotherapy remains a challenging issue, as most patients who respond remain disease-free, whereas those refractory have few options of being rescued with salvage therapies. The mechanisms underlying BL chemoresistance and how it can be circumvented remain undetermined. We previously reported the frequent inactivation of the proapoptotic BIM gene in B-cell lymphomas. Here we show that BIM epigenetic silencing by concurrent promoter hypermethylation and deacetylation occurs frequently in primary BL samples and BL-derived cell lines. Remarkably, patients with BL with hypermethylated BIM presented lower complete remission rate (24% vs 79%; P = .002) and shorter overall survival (P = .007) than those with BIM-expressing lymphomas, indicating that BIM transcriptional repression may mediate tumor chemoresistance. Accordingly, by combining in vitro and in vivo studies of human BL-xenografts grown in immunodeficient RAG2−/−γc−/− mice and of murine B220+IgM+ B-cell lymphomas generated in Eμ-MYC and Eμ-MYC-BIM+/− transgenes, we demonstrate that lymphoma chemoresistance is dictated by BIM gene dosage and is reversible on BIM reactivation by genetic manipulation or after treatment with histone-deacetylase inhibitors. We suggest that the combination of histone-deacetylase inhibitors and high-dose chemotherapy may overcome chemoresistance, achieve durable remission, and improve survival of patients with BL.

Description: Abstract 750 The non-malignant immune infiltrate comprises the bulk of pathologic tissue in classical Hodgkin lymphoma (CHL). This microenvironment has the potential to induce both malignant cell suppression and support. Increased macrophage infiltration assessed by CD68 expression has been shown to confer adverse prognostic significance (Steidl et al. N Engl J Med, 2010; 362:875-85) while increased FOXP3 expressing T cells are beneficial in this disease (Tzankov et al. Haematologica, 2008; 93: 193–200). However no histological score routinely leads to modification of treatment. Assessing outcomes by the parameters of overall survival (OS), disease specific survival (DSS) and freedom from first line treatment failure (FFTF) at 5 years in a cohort of patients treated at St Bartholomew's Hospital (Barts), London we developed a prognostic score based upon expression of both FOXP3 and CD68 in the CHL microenvironment which defined poor and good risk groups in both early (Stages I and IIA) and advanced stage disease, including a 'poor risk' early stage group with 25% FFTF and a ‘good risk' advanced stage group with 90% FFTF. Immunohistochemical analysis was performed on tissue microarrays (TMAs) from previously untreated patients' diagnostic lymph node biopsies in whom clinical outcome was available. From all 1056 adult patients with HL diagnosed at Barts between 1972 and 2005, high quality formalin-fixed paraffin-embedded tissue was available for 122 (12%), with characteristics representative of the whole group. Median age of the 122 patients was 30 (range 18–80) years, 35% female, 71% advanced stage with median follow up 16.5 (range 2–40) years. Triplicate cores were made from areas of high cellularity, containing malignant cells and avoiding fibrotic, acellular portions, arrayed onto glass slides and stained immunohistochemically for FOXP3 or CD68. Absolute and proportional numbers of FOXP3+ nuclei and CD68+ cell bodies were counted across all intact cores using an automated image analysis system (Ariol), confirmed by expert histopathologists, and means calculated and corrected to a single high powered field (hpf). Recursive partitioning was used to generate optimal cutoff values discriminating prognostic groups and comparisons performed by the chi-square test. Using cutoffs of 15% to define low, intermediate and high CD68 density, 3 prognostic groups were defined, the favourable group having the lowest CD68+ density. OS for low, intermediate and high groups were 89%, 80% and 65% respectively (p=0.02), with FFTF 82%, 64% and 29% (p=0.001). Prognostic significance was maintained in subgroups presenting with advanced stage (FFTF 73%, 63% and 33%, p=0.03), as well as early stage disease (FFTF 92%, 70% and 20%, p=0.01). Using cutoffs of 50 nuclei/hpf to define low, intermediate and high FOXP3+ cell density, 3 prognostic groups were defined, the favourable group having the highest FOXP3+ density. OS for low, intermediate and high groups were 68%, 80% and 94% respectively (p=0.006), with FFTF 50%, 62%, and 84% (p=0.002). Prognostic significance was maintained for both advanced (FFTF: 48%, 60% and 72%, p=0.04) and early stage disease (FFTF: 57%, 67% and 100%, p=0.04). A combined ‘FOXP3/CD68 score' derived from the patient's prognostic group for both markers, for which suitable cores were available on 98 patients, further improved the predictive value of each individually (See Figure). In this model, favourable, intermediate and unfavourable groups had 5 year FFTF of 93%, 62% and 47% (p=0.0002), DSS 93%, 82% and 63% (p=0.03) and OS 93%, 82% and 59% (p=0.002). The score retained prognostic significance for 5 year FFTF and OS in the subgroup of patients presenting with advanced (FFTF: 90%, 59% and 46%, p=0.008; OS: 90%, 80% and 54%, p=0.004) as well as early stage disease (FFTF: 100%, 71% and 25%, p=0.005; OS: 100%, 82% and 75%, trend only, p=ns). We conclude that FOXP3 and CD68 are important independent factors, which in combination have considerable predictive power and will now be validated prospectively. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 313 Background: While advanced PV and ET patients at high thrombotic risk are managed primarily with HU, patients who are intolerant or refractory to HU have limited therapeutic options. Identification of a dominant gain-of-function mutation in the JAK2 kinase, V617F, in myeloproliferative neoplasms (MPNs), including PV and ET, provided a key rationale for the development of a molecularly targeted therapy for these diseases. Long term follow-up data from an ongoing trial of INCB018424, a selective JAK 1/ JAK 2 inhibitor, in PV and ET patients refractory or intolerant to HU are presented. Methods: Study 18424-256 is an uncontrolled open-label Phase 2 study being conducted at 6 sites in the United States and Italy. An initial 8-week run-in evaluation established 10-mg and 25-mg twice daily as starting doses for expansion cohorts in PV and ET, respectively; dose adjustments for safety and efficacy are allowed so that each subject is titrated to their most appropriate dose. For PV, response is defined based on Hct control in the absence of phlebotomy, improvement or elimination of palpable splenomegaly when present, and normalization of leukocytosis and thrombocytosis. For ET, response is defined based on improvement or normalization of WBC and platelet counts and, when present, elimination of palpable splenomegaly. PV results (n=34; median 108 months from diagnosis): After a median follow-up of 15 months (range 8–21), 97% of enrolled subjects achieved Hct control to 15×109/L was present in 47% of subjects and improved (≤ 15×109/L) or normalized (≤ upper limit of normal) in 88% and 63%, respectively. Thrombocytosis 〉 600×109/L was present in 38% of subjects and improved (≤ 600×109/L) or normalized (≤ upper limit of normal) in 92% and 69%, respectively. 59% of subjects achieved phlebotomy independence, resolution of splenomegaly and normalization of leukocytosis and thrombocytosis. 6 patients discontinued therapy (3 due to AEs, 2 withdrew consent, 1 for no response). Grade 3 AEs potentially related to study medication included thrombocytopenia (2 patients), neutropenia (1), renal tumor (1), asthenia (1), viral infection (1), and atrial flutter (1). No Grade 4 drug-related AEs have occurred. ET results (n=39; median 84 months from diagnosis): After a median follow-up of 15 months (range 4–21), 49% of enrolled subjects normalized platelet counts to ≤ upper limit of normal after a median of 0.5 months and for a median duration of 3.5 months. 82% maintained platelet counts 〈 600×109/L, for a median duration of 9.8 months. Of 14 patients with baseline platelet counts 〉 1000×109/L, 13 have experienced 〉 50% reduction. 88% maintained normal WBC (median duration 14.5 months). Palpable spleens resolved in 3 of 4 subjects; 1 reduced 〉50% from baseline. 49% of subjects achieved normalization of WBC and platelet counts in the presence of non-palpable splenomegaly. 9 patients discontinued therapy (4 due to AEs, 2 withdrew consent, 3 for no response). Grade 3 AEs potentially related to study medication included leukopenia (2 patients), GI disorder (1), and peripheral neuropathy (1). No Grade 4 drug-related AEs have occurred. Both patient groups demonstrated reductions in patient-reported symptom scores for pruritus, night sweats, and bone pain. Of 26 PV patients reporting pruritus at baseline (median score of 6 on a 10-point scale), 24 reported scores of 0 after a median duration of 1 month and for a median duration of 7 months. 42% of PV and 56% of ET patients had at least a 20% decrease in JAK2V617F allele burden; 6% of PV and 12% of ET had 〉50% decrease. Clinical responses were unrelated to the presence/absence of JAK2V617F mutation at entry or to the allele burden changes following treatment. Conclusions: Rapid and durable clinical benefits (normalization of hematological parameters, resolution of splenomegaly and alleviation of symptoms) have been demonstrated in advanced PV and ET patients with 〉1 year of follow-up. In this study, INCB018424 continues to be a well tolerated, effective therapy in patients with PV and ET refractory or intolerant to hydroxyurea. Disclosures: Verstovsek: Incyte Corporation: Research Funding. Levy:Incyte Corporation: Employment, Equity Ownership. Bradley:Incyte Corporation: Employment. Garrett:Incyte Corporation: Employment. Vaddi:Incyte corporation: Employment. Huber:Incyte Corporation: Employment, Equity Ownership. Schacter:Pfizer Corporation: Employment. Vannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees.

Description: Cyclic adenosine monophosphate (cAMP)-dependent signaling modulates platelet function at sites of vascular injury. Here we show that thrombospondin-1 (TSP-1) prevents cAMP/protein kinase A (PKA) signaling through a CD36-dependent mechanism. Prostaglandin E1 (PGE1) induced a robust inhibition of both platelet aggregation and platelet arrest under physiologic conditions of flow. Exogenous TSP-1 reduced significantly PGE1-mediated inhibition of both platelet aggregation and platelet arrest. TSP-1 prevented PGE1-stimulated cAMP accrual and phosphorylation of PKA substrates, through a mechanism requiring phosphodiesterase3A. TSP-1 also inhibited VASP phosphorylation stimulated by the nonhydrolyzable cAMP analog, 8-bromo-cAMP, indicating that it may regulate cAMP-mediated activation of PKA. The inhibitory effect of TSP-1 on cAMP signaling could be reproduced with a peptide possessing a CD36 binding sequence of TSP-1, while the effects of TSP-1 were prevented by a CD36 blocking antibody. TSP-1 and the CD36 binding peptide induced phosphorylation of Src kinases, p38 and JNK. Moreover, inhibition of Src kinases blocked TSP-1–mediated regulation of cAMP concentrations and the phosphorylation of VASP, indicating that TSP-1 modulated the cAMP/PKA signaling events through a tyrosine kinase-dependent pathway downstream of CD36. These data reveal a new role for TSP-1 in promoting platelet aggregation through modulation of the cAMP-PKA signaling pathway.

Description: Axl is an oncogenic receptor tyrosine kinase that plays multiple roles in tumorigenesis and metastasis of many cancers. This study is the first to demonstrate that Axl is induced in Kaposi sarcoma and Kaposi sarcoma herpesvirus (KSHV) transformed endothelial cells. Conditionally, expression of one KSHV latency protein vFLIP induces Axl expression in endothelial cells. This induction can be blocked by nuclear factor-κB inhibitor, consistent with the known vFLIP mechanism of action. KS cell lines lacking KSHV also have elevated Axl expression, which probably resulted from hypomethylation of AXL promoter. Axl activation activates downstream phosphoinositol-3 kinase signaling, and Axl knockdown by siRNA impairs phosphoinositol-3 kinase signaling. Furthermore, Axl knockdown inhibits KS cell growth and invasion. To explore the potential for translation of these findings, we generated monoclonal antibodies to block the biologic functions of Axl. MAb173, which induces receptor degradation, showed activity in vitro to inhibit KS cell invasion. Moreover, in vivo xenograft studies with KS cells with or without KSHV infection showed that MAb173 reduced tumor growth, increased tumor cell apoptosis, and markedly decreased Axl protein level in tumors. Axl thus has a potential role in KS pathogenesis and is a candidate for prognostic and therapeutic investigations.

Description: Abstract 1071 Background: Treatment of children with acute myeloid leukemia (AML) is associated with considerable toxicity. Children's Oncology Group (COG) AAML0531 trial adopted a modified AML Medical Research Council backbone and reviewed adverse event reports in real time to maximize accurate toxicity data. As of March 31, 2010, AAML0531 had randomized 968 patients with de novo AML to gemtuzumab ozogamicin (GMTZ) versus no GMTZ. Accurate description of toxicities with the backbone regimen as well as increment toxicities of new agents is important to optimize supportive care. Objectives were to describe hepatic, cardiac and infectious toxicities and duration of neutropenia in all patients, and to compare toxicities between treatment arms. Methods: AAML0531 included those ≥ 1 month to ≤ 30 years with de novo AML and used a 5 cycle chemotherapy regimen: Induction (Ind) I and II: cytarabine, daunorubicin, and etoposide (ADE 10+3+5 and 8+3+5); Intensification (Int) I: cytarabine and etoposide; Int II: mitoxantrone and high-dose cytarabine; and Int III: high-dose cytarabine and L'asparaginase. Subjects were randomized to receive or not receive GMTZ 3 mg/m2 during Ind I and Int II. Common Terminology Criteria for Adverse Events v3.0 toxicities were collected prospectively. Hepatic (ALT, bilirubin and veno-occlusive disease (VOD)), cardiac (left ventricular systolic dysfunction (LVSD)) and infection (sterile site bacterial and fungal) toxicities were targeted. All grades of cardiac and VOD toxicities were captured; all other severe (≥ grade 3) toxicities were recorded. Cumulative incidence (CI) of toxicities was calculated only during protocol chemotherapy. Duration of neutropenia was reported from beginning of cycle to absolute neutrophil recovery ≥ 500/uL for patients alive during the cycle. Results: Table describes the prevalence of severe (≥ grade 3) toxicities and illustrates that severe high ALT occurred in 3–10%, high bilirubin was less common and VOD was reported in ≤ 1% of cycles. Severe LVSD also was rare although compliance with suggested cardiac monitoring ranged from 50–80% per course of treatment. In terms of grade 1 or 2 LSVD there were no differences in any course except for Int II where grade 1 or 2 LVSD was higher with GMTZ (8.0% versus 2.4%; P

Description: Abstract 899 Introduction: The classic definition of acute (aGVHD) and chronic graft-versus-host disease (cGVHD) was based on a cut-off day 100 after transplantation, but this did not reflect that aGVHD can occur later and that symptoms of aGVHD and cGVHD can occur simultaneously. In 2005 a NIH consensus classification was proposed which included 1) classic aGVHD, occurring before day 100, 2) persistent, recurrent or late aGVHD occurring thereafter, 3) classic cGVHD and 4) an overlap syndrome with simultaneous features of aGVHD and cGVHD. Only few studies have evaluated this classification and no studies have determined the differential impact of reduced intensity (RIC) and myeloablative conditioning (MAC). Method: We retrospectively analyzed 202 AML patients who were transplanted between 1999 and 2008. 102 patients received RIC (generally 6×30 mg/m2 FLU, 4×4 mg/kg BU, 4×10 mg/kg ATG) and immunosuppression with CSA/MMF and 100 patients received MAC (generally 6×2 Gy TBI and 2×60 mg/kg CY) and CSA/MTX. Donors were HLA-matched related (n=82), -matched unrelated (n=88) or -mismatched (n=32). Result: Leukocyte recovery was faster after RIC than after MAC (14 vs. 19 days, P

Description: Abstract 3045 Background: Lenalidomide is an oral IMiD® immunomodulatory compound with a dual mechanism of action, namely tumoricidal and immunomodulatory activity, and established clinical efficacy and safety in patients with multiple myeloma (MM). Lenalidomide plus dexamethasone (Len + Dex) was well tolerated and demonstrated significant improvements in response and favorable overall survival (OS) compared with Placebo + Dex in 2 pivotal phase 3 registration trials in patients with relapsed/refractory MM (RRMM; Weber et al, NEJM 2007; Dimopoulos et al, NEJM 2007). Previously, in a phase 3, multicenter, single-arm, open-label, expanded-access study (MM-018), Len + Dex demonstrated a predictable safety profile that can preserve patient quality of life (QoL) (Yong et al Haematologica 2010 [abstract #0944]). Here we report efficacy, safety, and QoL data for patients enrolled in the Spanish cohort of MM-018. Methods: Patients with progressive disease after 〉 2 cycles of antimyeloma treatment, or after relapse from treatment, with ECOG performance status ≤ 2 received 28-day cycles of Len (25 mg/day, D 1–21) plus Dex (40 mg/day, D 1–4, 9–12, and 17–20 for cycles 1–4; D 1–4 in subsequent cycles). Endpoints included overall response (≥ partial response [PR] by European Group for Blood and Marrow Transplantation criteria) and QoL assessments measured by EORTC QLQ C-30 and EORTC QLQ MY-20 questionnaires at baseline and week 24. All prophylaxis was administered at the investigator's discretion. Results: Sixty-three patients receiving ≥ 1 dose of Len + Dex were evaluated for efficacy, safety, and QoL. Median age was 62 years (21 [33.3%] were 〉 65 years). Prior therapies included thalidomide (n = 15, 24%) and bortezomib (n = 37, 59%). Additionally, 5 (8%) patients had a history of deep vein thrombosis (DVT), and 23 (37%) had a history of peripheral neuropathy. A PR or better was observed in 49 (78%) patients, including complete response (CR) in 13 (21%), very good partial response (VGPR) in 13 (21%), and PR in 23 (37%) patients. Median time to first response and best response was 2.7 and 4.5 months, respectively. Median duration of response was 18.4 months. Response depth improved after long-term treatment with Len + Dex, and 32/63 (51%) patients received 〉12 cycles of therapy. Beyond 12 cycles of therapy, 8 patients achieved VGPR and 12 patients achieved CR; compared with 5 patients and 1 patient, respectively, prior to 12 cycles. Median time to progression and progression-free survival were both 13.3 months; median OS has not yet been reached. Forty-two (67%) patients remained on study at 6 months. Compliance to QoL assessment questionnaires was ≥ 80%. Patient-reported improvements in QoL and disease symptoms measured by both questionnaires were observed in nearly all scales. EORTC QLQ C-30 scores revealed clinically meaningful improvement (〉 5 points) for global QoL (n = 15, 40%), fatigue (n = 16, 42%), emotional function (n = 15, 40%), physical function (n = 12, 32%), role function (n = 11, 29%), social function (n = 11, 29%), cognitive function (n = 10, 26%), and pain (n = 9, 24%) at 6 cycles compared with baseline. Preservation of QoL in role function, emotional function, social function, and pain scores was observed at 6 cycles when compared with baseline in responders (≥ PR). EORTC QLQ MY-20 results revealed no relevant median change (〉 5 points) from baseline in all scales for all patients completing questionnaires at baseline and 6 cycles, except for a meaningful improvement in future perspective scores (median 11.1-point change). Adverse events observed in this study were consistent with those previously reported with Len + Dex. Grade 3/4 hematologic events were experienced by 40 (64%) patients, and included neutropenia (n = 32, 51%), thrombocytopenia (n = 11, 17%), anemia (n = 10, 18%), and febrile neutropenia (n = 4, 6%). DVT (all grades) was experienced by 5 (8%) patients, and only one grade 3/4 new-onset peripheral neuropathy was observed after 6 cycles of treatment. Conclusions: Len + Dex treatment in this expanded-access study demonstrated efficacy and predictable safety, consistent with that of previously published trials for patients with RRMM. More patients achieved VGPR and CR after long-term therapy compared with those receiving 〈 12 cycles of therapy. Furthermore, QoL assessments at baseline and 6 months revealed that patients treated with Len + Dex showed meaningful improvements in certain QoL and symptom scores. Disclosures: Oriol-Rocafiguera: Celgene: Consultancy; Janssen-Cilag: Consultancy; Novartis: Consultancy. García-Laraña:Celgene: Consultancy; Janssen-Cilag: Consultancy. Mateos:Celgene: Honoraria. Cibeira:Celgene: Honoraria for Lectures; Janssen-Cilag: Honoraria for Lectures; Pharmion: Honoraria for Lectures. Knight:Celgene: Employment. Rosettani:Celgene Corporation: Employment.

Description: Iron-refractory, iron-deficiency anemia (IRIDA) is a familial disorder characterized by iron deficiency anemia unresponsive to oral iron treatment but partially responsive to intravenous iron therapy. Previously, we showed that IRIDA patients harbor loss-of-function mutations in TMPRSS6, a type II transmembrane serine protease primarily expressed by the liver. Both humans and mice with TMPRSS6 mutations show inappropriately elevated levels of the iron-regulatory hormone hepcidin, suggesting that TMPRSS6 acts to negatively regulate hepcidin expression. Here we investigate the relationship between Tmprss6 and the bone morphogenetic protein (BMP)–Smad signaling pathway, a key pathway promoting hepcidin transcription in hepatocytes. We show that livers from mice deficient for Tmprss6 have decreased iron stores and decreased Bmp6 mRNA, but markedly increased mRNA for Id1, a target gene of Bmp6 signaling. In contrast, mice deficient for both Tmprss6 and hemojuvelin (Hjv), a BMP coreceptor that augments hepcidin expression in hepatocytes, showed markedly decreased hepatic levels of hepcidin and Id1 mRNA, markedly increased hepatic Bmp6 mRNA levels, and systemic iron overload similar to mice deficient for Hjv alone. These findings suggest that down-regulation of Bmp/Smad signaling by Tmprss6 is required for regulation of hepcidin expression and maintenance of systemic iron homeostasis.

Description: Protein vaccines for T-cell immunity are not being prioritized because of poor immunogenicity. To overcome this hurdle, proteins are being targeted to maturing dendritic cells (DCs) within monoclonal antibodies (mAbs) to DC receptors. To extend the concept to humans, we immunized human immunoglobulin-expressing mice with human DEC205 (hDEC205) extracellular domain. 3D6 and 3G9 mAbs were selected for high-affinity binding to hDEC205. In addition, CD11c promoter hDEC205 transgenic mice were generated, and 3G9 was selectively targeted to DCs in these animals. When mAb heavy chain was engineered to express HIV Gag p24, the fusion mAb induced interferon-γ– and interleukin-2–producing CD4+ T cells in hDEC205 transgenic mice, if polynocinic polycytidylic acid was coadministered as an adjuvant. The T-cell response was broad, recognizing at least 3 Gag peptides, and high titers of anti-human immunoglobulin G antibody were made. Anti-hDEC205 also improved the cross-presentation of Gag to primed CD8+ T cells from HIV-infected individuals. In all tests, 3D6 and 3G9 targeting greatly enhanced immunization relative to nonbinding control mAb. These results provide preclinical evidence that in vivo hDEC205 targeting increases the efficiency with which proteins elicit specific immunity, setting the stage for proof-of-concept studies of these new protein vaccines in human subjects.

Description: Abstract 4013 Background: In recent years, azacytidine (AZA) has become the standard of treatment for high risk myelodysplasia (MDS). The approved schedule of AZA uses a 75mg/m2/d s.c. regimen for 7 days based on the CALGB-9221 (Silverman, JCO 2002) and the AZA-001 studies (Fenaux, Lancet Oncol 2009). The clinical response rates after AZA have been extensively presented but there is only limited data on the rates of cytogenetic response (CyR). Based on the review of the literature, there are no specific cytogenetic data published on prospective trials. Methods: We based our analysis on a randomized phase 2 study from the US Leukemia Intergroup (E1905 study, NCT00313586) testing 10 days of AZA (50mg/m2/d s.c.) vs 10 days of AZA+ the histone deacetylase inhibitor entinostat (4 mg/m2/d PO days 3 and day 10). MDS, CMML, and AML with myelodysplasia-related changes were included. This analysis includes all patients with cytogenetic abnormalities (at baseline or acquired following treatment) with available cytogenetic follow-up (cycle 6). Of 150 patients, 70 demonstrated baseline cytogenetic abnormalities. To date, forty patients (27 MDS and 13 AML) were evaluable for both time points. Karyotypes were performed at local laboratories, and reviewed centrally (RPK and GH). Cytogenetic response was assessed using IWG 2000 (Cheson et al, Blood 2000) criteria. Results: The clinical response rate (CR+PR+ trilineage HI) according to IPSS cytogenetic risk stratification were of 20%, 33%, and 35% for favorable, intermediate and poor cytogenetic risk groups respectively (p=NS). Patients with Chr 7 abnormalities (i.e. -7 or -7q, n=18) had a response rate of 28% including 17% CR. Of patients with complete cytogenetic data, the rate of overall CyR was 52% (n=21): 22% (n=9) complete CyR, 30% (n=12) partial CyR. This represents a complete CyR of 13% and a partial CyR of 23% as a proportion of all treated patients with initial cytogenetic abnormalities (including those who did not receive six cycles of therapy). To date, confirmatory FISH analyses were available for 4 patients with CyR (2 CCyR and 2 PCyR). All four had complete clearance of their cytogenetic clone. Among the cytogenetic responders, 15 had MDS and 6 had AML (p=NS). CyR did not differ between the two treatment arms. CyR and clinical response were highly correlated (p

Description: Single nucleotide polymorphism arrays (SNP-A) have recently been widely applied as a powerful karyotyping tool in numerous translational cancer studies. SNP-A complements traditional metaphase cytogenetics with the unique ability to delineate a previously hidden chromosomal defect, copy neutral loss of heterozygosity (CN-LOH). Emerging data demonstrate that selected hematologic malignancies exhibit abundant CN-LOH, often in the setting of a normal metaphase karyotype and no previously identified clonal marker. In this review, we explore emerging biologic and clinical features of CN-LOH relevant to hematologic malignancies. In myeloid malignancies, CN-LOH has been associated with the duplication of oncogenic mutations with concomitant loss of the normal allele. Examples include JAK2, MPL, c-KIT, and FLT3. More recent investigations have focused on evaluation of candidate genes contained in common CN-LOH and deletion regions and have led to the discovery of tumor suppressor genes, including c-CBL and family members, as well as TET2. Investigations into the underlying mechanisms generating CN-LOH have great promise for elucidating general cancer mechanisms. We anticipate that further detailed characterization of CN-LOH lesions will probably facilitate our discovery of a more complete set of pathogenic molecular lesions, disease and prognosis markers, and better understanding of the initiation and progression of hematologic malignancies.

Description: Abstract 3937 CHOP and CHOP-like chemotherapy programs remain the most commonly used regimens for the treatment of peripheral T-cell lymphomas (PTCLs) despite often sub-optimal results. Histone deacetylase inhibitors (HDACIs) are epigenetic agents known to be active in T-cell lymphoma. Recently romidepsin (R) was approved for patients with relapsed or refractory CTCL. Both R and belinostat (B) are being investigated in patients with relapsed or refractory PTCL. We have previously shown that hypomethylating agents as decitabine (D) produce synergistic interactions with HDACIs in B-cell lymphomas. We investigated the in vitro and in vivo activity of D, R and B alone or in combination in different T-cell lymphoma and leukemia cell lines including CTCL (H9, HH), and T- acute lymphoblastic leukemia (T-ALL) lines resistant to gamma-secretase inhibitors (GSI) (P12, PF-382). For all cytotoxicity assays, luminescent cell viability was performed using CellTiter-Glo™ followed by acquisition on a Biotek Synergy HT. The IC50s for D, B and R were calculated using the Calcusyn software (Biosoft). Drug: drug interactions were analyzed using the calculation of the relative risk ratios (RRR) based on the GraphPad software (RRR 20 uM. In the cytotoxicity assays, the combination of D and B or R at 72 hours showed synergism in all the cell lines studied. The most representative RRRs are showed in table 1. Table 1 D 0.5 uM 1uM B (nM) RRR H9 50 0.7 0.7 70 0.6 0.6 100 0.4 0.5 PF 382 150 0.8 0.7 0.5 uM 1 uM R (nM) RRR H9 0.5 0.9 0.9 1 0.8 0.8 2 0.3 0.3 PF 382 1 0.8 0.7 1.5 0.4 0.4 2 0.1 0.1 When H9, HH, P12 and PF382 cell lines were treated with D and B or R for 72 hours, all the combination groups showed significantly more apoptosis than the single drug exposures and controls. Table 2 displays the range of apoptosis induction for B, R and D or for them used in combination and the RRR value after the analysis for the most significant data. Table 2 B D B + D RRR (% Apoptotic + Dead Cells) H9 100nM (22.9%) 500nM (17.9%) 51.5% 0.7 HH 100nM (42.9%) 1uM (46.9%) 61.3% 0.8 P 12 150nM (16%) 1uM (42.7%) 80.1% 0.4 PF 382 100nM (8.3%) 1uM (27.9%) 40.1% 0.8 R D R + D H9 2nM (22.2%) 500nM (17.9%) 63.6% 0.5 HH 2nM (80%) 1uM (46.9%) 89.7% 0.6 P 12 2nM (9.9%) 10uM (58.7%) 98% 0.03 PF 382 2nM (54.5%) 500nM (17.9%) 88.7% 0.2 Increased acetylation of H3 was observed when H9 cells were treated with R alone and synergistically increased after exposing cells to the combination of D + B and D + R. The expression of phosphorylated Stat3 was decreased after exposure of H9 cells to the combination of D and R. Additional interrogation of the effects of this epigenetic therapy on the JAK-STAT signaling pathway are now underway. An in vivo xenograft study in six to eight weeks old female SCID beige mice injected subcutaneously with 2 × 107 HH cells has also begun and will be reported. Mice were separated into different cohorts and treated with intraperitoneal injections of D or B or their combination according to the following schedules: D alone at 1.5 mg/kg on days 1, 5; B alone at 35 mg/Kg/day for 7 days. Collectively, the data suggest that the combination of a hypomethylating agent like D and a HDACI (B and R) are synergistic in in vitro models of human T-cell lymphoma, and may lead to a new platform for the treatment of these diseases. Disclosures: O'Connor: Millennium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Abstract 285 Background: KW-0761 is a defucosylated, humanized, monoclonal antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC; Potelligent®) that binds to CC chemokine receptor 4 (CCR4). CCR4 is expressed on the surfaces of cells comprising several T-cell malignancies such as ATL, peripheral T-cell lymphoma (PTCL) and cutaneous T-cell lymphoma (CTCL). A phase I study of KW-0761 in patients with CCR4-positive ATL and PTCL demonstrated that 4 weekly intravenous infusions of KW-0761 were well tolerated up to 1.0 mg/kg and it showed encouraging clinical activity with an overall response rate (ORR) of 31.3% (4 of 13 ATL and 1 of 3 PTCL) in 16 patients (J Clin Oncol 2010;28:1591-8). Methods: A multicenter phase II study of KW-0761 has been conducted for relapsed patients with CCR4-positive ATL to evaluate its efficacy, pharmacokinetics (PK), safety and immunogenicity. Patients were planned to receive 8 weekly intravenous infusions of KW-0761 at 1.0 mg/kg. The primary endpoint was ORR. Objective responses were assessed after the 4th and 8th infusions of KW-0761 according to the response criteria for ATL (J Clin Oncol 2009;27:453-9) by each investigator and the independent efficacy assessment committee. The number of patients required was estimated to be 25, for 90% power to detect a lower limit of the 95% confidence interval (CI) exceeding the 5% threshold of ORR, based on the assumptions that the minimum required response ORR to a new drug for relapsed ATL is 5% and the expected ORR to KW-0761 is 30%. Results: Twenty-seven patients (12 males and 15 females) were enrolled and received KW-0761. The median age was 64 years (range: 49–83). The disease subtypes of ATL consisted of 14 acute-, 6 lymphoma-, and 7 chronic-types with unfavorable prognostic factors. Among the 27 patients enrolled, 14 patients (52%) completed the protocol treatment of 8 infusions. Eleven patients (41%) discontinued the protocol treatment because of progressive disease, and the remaining 2 discontinued because of skin rash or the concurrent colon tumor. The treatment-related grade (G) 2 or greater adverse events (AEs) were lymphopenia (96%), leukopenia (56%), skin rash (52%), neutropenia (33%), thrombocytopenia (26%), AST increase (26%), ALT increase (22%), hypoxemia (19%), anemia (15%), pruritus (15%), g-GTP increase (15%) and hypophosphatemia (15%). G2 or greater Infusion-related toxicities were observed in 22 of 27 patients (81%) including 1 G3, but immediately recovered after treatment with systemic steroids. Treatment-related severe AEs (SAEs) were observed in 5 patients, including a Stevens-Johnson syndrome (G3) and 4 skin rashes (each G3). All these AEs also improved by steroids. PK analysis demonstrated that Cmax and trough (C168h) after the 8th infusion was 38,853 ± 11,267 and 25,934 ± 10,193 ng/mL, respectively, and T1/2 after the 8th infusion were 457 ± 144 h. No anti-KW-0761 antibody has been detected. Among the 26 patients evaluable for efficacy, KW-0761 exhibited an ORR of 54% (14/26; 95% CI, 33 to 73) (acute: 6/14 patients, lymphoma: 3/6 patients, chronic: 5/6 patients) including 7 complete responses (CRs) (27%; 95% CI, 12 to 48) and 7 partial responses (PRs). These are remarkable results, considering that the ORR of relapsed or refractory patients with ATL to a single-agent chemotherapy has been reported to be low (7 to 39%). Response rates according to the affected disease lesion were 100% (13 patients, all CR), 71% (5 of 7 patients), and 38% (5 of 13 patients), respectively, for peripheral blood, skin, and lymph node disease. Conclusions: KW-0761 is a highly effective agent with acceptable toxicity profiles in relapsed patients with CCR4-positive ATL who have no standard therapies. A multicenter, randomized study for untreated ATL patients to compare mLSG15 (a dose-intensified multi-agent regimen, J Clin Oncol 2007;25:5458-64) + KW-0761 with mLSG15 alone has been initiated. Disclosures: Ogura: Kyowa Hakko Kirin Co Ltd: Consultancy. Akinaga:Kyowa Hakko Kirin Co Ltd: Employment.

Description: Abstract 3687 Background: Prednisolone at a dose of 1 mg/kg/day (PRD) for 2 to 4 weeks is administered as front-line therapy to most adult patients with immune thrombocytopenia (ITP) who need to be treated. Recent results from 2 large single arm studies suggest high dose dexamethasone (DEX) as first-line treatment may produce a high sustained response rate. Therefore, we conducted prospective randomized trial comparing DEX with PRD as initial treatment of adult patients with newly diagnosed ITP. Methods: We did a randomized multicentre trial based on a 1:1 design. We enrolled treatment-naïve patients, 16 years or older, with a diagnosis of ITP according to the practice guidelines of the American Society of Hematology and a platelet count of 30×109/L or less. Patients with ITP were randomly assigned to receive either DEX 40 mg/day on day 1–4 (If the platelet count dropped below 3×109/L within the first six months, another four-day course of DEX was given) or PRD 1mg/kg/day for 4 weeks. The primary object of this study was the sustained response (platelet count〉30×109/L after 6 months) rate. Second objectives are response rate at 4 weeks, predictors of steroid response, and toxicity. This study is registered at http://clinicaltrials.gov as NCT00451594. Results: From September 2005 to December 2009, 151 adults with ITP were randomly assigned (76 patients in the DEX arm, 75 patients in the PRD arm). Overall demographics were balanced between arms; Median age 44 years old (DEX:PRD 44 years old:44 years old); 69.5% female (DEX:PRD 73.7%:65.3%); median initial platelet count 17×109/L (DEX:PRD 16×109/L:17×109/L). Of 151 enrolled patients, 117 patients (57 patients in the DEX arm, 60 patients in the PRD arm) were evaluable in terms of sustained response. Sustained response rate by intention-to-treat (ITT) and per protocol were 25.0% and 33.3% in the DEX arm, 36.0% and 45.0% in the PRD arm, respectively (p=0.33 by ITT). Eight patients in the DEX arm and 17 patients in the PRD arm had a sustained platelet counts more than100×109/L. Fifteen patients had a sustained response after a single course of DEX and 6 patients among them required no further treatment during two to four year follow up. Median time to relapse in patients who had a sustained response was 1320 days and 1140 days, respectively. The response rate at day 28 was 68.2% in the DEX arm and 81.2% in the PRD arm. Pre-treatment platelet count was higher in patients who achieved sustained response (responder vs. non-responder: 17.1±7.8×109/L vs. 15.9±8.9×109/L). Eleven patients in the DEX arm and 8 patients in the PRD arm received splenectomy. Both treatments were well tolerated. Three patients represented side effects that were severe enough to discontinue the treatment in the PRD arm, including pneumonia (1), hyperglycemia (1), and myalgia (1). Six patients receiving DEX and 5 patients receiving PRD had G3 or more hyperglycemia. Conclusion: One or two courses of DEX were not more effective than PRD in adults with ITP. However, 19.7% of adult ITP had a sustained response with a single course of DEX. Initial treatment with DEX could be useful for identifying patients who may not require prolonged steroid treatment. Disclosures: No relevant conflicts of interest to declare.

Description: Oncogenic c-Myc is known to balance excessive proliferation by apoptosis that can be triggered by p53-dependent and p53-independent signaling networks. Here, we provide evidence that the BH3-only proapoptotic Bcl-2 family members Bcl-2 modifying factor (Bmf) and Bcl-2 antagonist of cell death (Bad) are potent antagonists of c-Myc–driven B-cell lymphomagenesis. Tumor formation was preceded by the accumulation of preneoplastic pre-B and immature immunoglobulin M–positive (IgM+) B cells in hematopoietic organs of Eμ-myc/bmf−/− mice, whereas Eμ-myc/bad−/− mice showed an increase of pre-B cells limited to the spleen. Although the loss of Bad had no impact on the tumor immunophenotype, Bmf deficiency favored the development of IgM+ B cell over pre-B cell tumors. This phenomenon was caused by a strong protection of immature IgM+ B cells from oncogene-driven apoptosis caused by loss of bmf and c-Myc–induced repression of Bmf expression in premalignant pre-B cells. Steady-state levels of B-cell apoptosis also were reduced in the absence of Bad, in support of its role as a sentinel for trophic factor-deprivation. Loss of Bmf reduced the pressure to inactivate p53, whereas Bad deficiency did not, identifying Bmf as a novel component of the p53-independent tumor suppressor pathway triggered by c-Myc.

Description: The long-term prognosis of indolent adult T-cell leukemia-lymphoma (ATL) is not clearly elucidated. From 1974 to 2003, newly diagnosed indolent ATL in 90 patients (65 chronic type and 25 smoldering type) was analyzed. The median survival time was 4.1 years; 12 patients remained alive for more than 10 years, 44 progressed to acute ATL, and 63 patients died. The estimated 5-, 10-, and 15-year survival rates were 47.2%, 25.4%, and 14.1%, respectively, with no plateau in the survival curve. Although most patients were treated with watchful waiting, 12 patients were treated with chemotherapy. Kaplan-Meier analyses showed that advanced performance status (PS), neutrophilia, high concentration of lactate dehydrogenase, more than 3 extranodal lesions, more than 4 total involved lesions, and receiving chemotherapy were unfavorable prognostic factors for survival. Multivariate Cox analysis showed that advanced PS was a borderline significant independent factor in poor survival (hazard ratio, 2.1, 95% confidence interval, 1.0-4.6; P = .06), but it was not a factor when analysis was limited to patients who had not received chemotherapy. The prognosis of indolent ATL in this study was poorer than expected. These findings suggest that even patients with indolent ATL should be carefully observed in clinical practice. Further studies are required to develop treatments for indolent ATL.

Description: Long-lived plasma cells in the bone marrow produce memory antibodies that provide immune protection persisting for decades after infection or vaccination but can also contribute to autoimmune and allergic diseases. However, the composition of the microenvironmental niches that are important for the generation and maintenance of these cells is only poorly understood. Here, we demonstrate that, within the bone marrow, plasma cells interact with the platelet precursors (megakaryocytes), which produce the prominent plasma cell survival factors APRIL (a proliferation-inducing ligand) and IL-6 (interleukin-6). Accordingly, reduced numbers of immature and mature plasma cells are found in the bone marrow of mice deficient for the thrombopoietin receptor (c-mpl) that show impaired megakaryopoiesis. After immunization, accumulation of antigen-specific plasma cells in the bone marrow is disturbed in these mice. Vice versa, injection of thrombopoietin allows the accumulation and persistence of a larger number of plasma cells generated in the course of a specific immune response in wild-type mice. These results demonstrate that megakaryocytes constitute an important component of the niche for long-lived plasma cells in the bone marrow.

Description: Abstract 2191 Background: Treatment protocols for newly diagnosed AML typically use age (often 60 years) alone to restrict eligibility to either younger or older patients. Implied in this practice is the assumption that age is the principal predictor of therapeutic failure in AML due to either early treatment-related mortality (TRM) or resistance to therapy in patients who do not incur TRM. Yet, clinical observation and previous studies indicate that other prognostic factors modulate the effect of age on TRM and resistance, suggesting that age as sole or primary criterion for treatment allocation may be suboptimal. Methods: To test this hypothesis in newly-diagnosed non-APL AML, we quantified the relative effects of age and other covariates using 1,127 patients (median age: 57 years) treated on Southwest Oncology Group (SWOG) trials from 1986–2009 and 1,604 patients (median age: 61 years) treated on various protocols at M.D. Anderson Cancer Center (MDA) from 2000–2008. We calculated weekly hazard rates (the probability of death in week × given that the patient was alive at the beginning of the week) for both cohorts overall and in various age subgroups. We used the area under the receiver operator characteristic curve (AUC) to quantify the effects of covariates for prediction of TRM and resistance (no TRM but patient does not enter CR or relapses within 1 year of CR), where an AUC of 1 indicates that a covariate is perfect at prediction while an AUC of 0.5 indicates no prediction (i.e. it is no better than flipping a coin). Results: Despite the use of different treatment protocols, survival in the SWOG and MDA cohorts was virtually superimposable. In both cohorts, the maximum weekly hazard occurred at weeks 3 and 4 from start of treatment, after which it decreased. The maximum hazard was relatively independent of age and remained between weeks 3 and 5 in patients age 70 years, respectively. The existence of such a discrete cut-point suggested that patients who die early are qualitatively distinct and prompted us to examine the relative effect of age and other covariates in patients who (a) died within the first 30 days of treatment (our empirically-based definition of TRM, 9% of MDA and 12 % of SWOG patients, respectively) and (b) survived at least 30 days but did not enter complete remission or relapsed within 1 year (“resistant”, 43% of MDA and 59% of SWOG patients, respectively). A model including age alone to predict early mortality had an AUC of 0.67, while a model including performance status (PS) alone had an AUC of 0.72. By comparison, a more refined model hat included PS, age, platelet count, cytogenetics, secondary AML, albumin, white blood cell count, peripheral blood blast count, and LDH yielded an AUC of 0.86. Elimination of age resulted in a model with an AUC that was only minimally lower (0.85). Prediction of resistance was more difficult with a model including age, secondary AML, cytogenetics, peripheral blood blasts, race, hemoglobin, and marrow neutrophils giving an AUC of only 0.70. Elimination of age had little effect (AUC 0.67) while age alone gave an AUC of 0.64. Conclusion: Age alone appears inadequate in predicting resistance and particularly TRM. The use of models based on several covariates improves predictive ability, but the ability to predict resistance is still limited, suggesting the value of randomized trials to assess treatment designed to reduce resistance. The observation that elimination of age has little effect on the predictive ability of such models, suggests that age is primarily a surrogate for other covariates. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2793 Background: DLBCL is the most frequent aggressive non Hodgkin's lymphoma in adults. The International Prognostic Index (IPI) is currently the most used tool to identify different risk patients. The role of an interim PET to early identify patients with bad response to chemotherapy is still controversial. Aims: The aim of this analysis is to evaluate the predictive value for event-free survival of an interim PET (after 2 chemotherapy cycles) and at the end of therapy (after 6 chemotherapy cycles) of dose-dense R-CHOP in patients with DLBCL. Methods: This is a prospective clinical trial for patients with DLBCL older than 65 with IPI 0–5 or younger than 65 with IPI 0–2. Treatment consists on 6 cycles of R-CHOP administered every 14 days followed by pegfilgrastim (6 mg per cycle) on day 2. In this sub-analysis we have included 69 patients who completed the 6 cycles of chemotherapy and who have PET evaluation at diagnosis, after 2 cycles of R-CHOP and at the end of treatment. All evaluations were made by combined PET/CT, except for 14 patients who were evaluated by PET alone. PET was determined to be positive or negative based on the local radiology reports. Interim PET results did not change the planned treatment. Results: 124 patients were included in the trial, 19 (15.3%) did not finish 6 cycles of treatment: 2 due to serious adverse events, 2 due to progression disease/stabilization, 7 due to investigators decision and 8 due to death. Over 105 patients who completed 6 cycles of treatment, 69 patients with complete PET evaluation (at diagnosis, after 2 cycles and at the end of treatment) were included in the analysis. Median age was 61.6 years old (range 18.2–82.8), 34 (49.3%) were older than 65 yo, 37 (53.6%) were male, 59 (85.5%) had ECOG 0–1. Characteristics of the disease at diagnosis were as follows: stage III-IV: 45 (65.2%), bulky disease: 17 (24.64%), 〉2 extra-nodal sites involvement: 4 (5.8%), B symptoms: 18 (26.1%), elevated LDH: 36/67 (53.7%), elevated beta-2-microglobulin: 23/62 (37.1%), IPI 3–5: 23 (33.3%). The median relative dose intensity (RDI) for cyclophosphamide and adriamycin calculated according to 3-week interval as a reference was 139.8% (≥130% in 75.2% of patients): 141.6% in younger patients and 138.6% in the elderly. Thirty-five (50.7%) patients achieved a negative PET evaluation after 2 R-CHOP cycles and 57 (82.6%) at the end of therapy. Patients with bulky disease had a positive interim PET more frequently (64.7% vs 44.2%), but most of them turned negative at the end of treatment (positive end-of-treatment PET for bulky disease: 11.7%). With a median follow-up of 28 months (limits 2.3–49), event-free survival (EFS) was 70.6% for patients with positive interim PET and 92.9% for patients with negative interim PET (p=0.14). The negative predictive value (NPV) of a negative interim PET was 94.3% and the positive predictive value (PPV) of a positive interim PET was 14.7%. EFS was 47.6% for patients with positive end-of-treatment PET and 95.6% for patients with negative end-of-treatment PET (p

Description: Minimal residual disease (MRD) at the end of remission-induction therapy predicts relapse in acute lymphoblastic leukemia (ALL). We examined the clinical significance of levels below the usual threshold value for MRD positivity (0.01%) in 455 children with B-lineage ALL, using polymerase chain reaction amplification of antigen-receptor genes capable of detecting at least 1 leukemic cell per 100 000 normal mononucleated cells (0.001%). Of the 455 clinical samples studied on day 46 of therapy, 139 (30.5%) had MRD 0.001% or more with 63 of these (45.3%) showing levels of 0.001% to less than 0.01%, whereas 316 (69.5%) had levels that were either less than 0.001% or undetectable. MRD measurements of 0.001% to less than 0.01% were not significantly related to presenting characteristics but were associated with a poorer leukemia cell clearance on day 19 of remission induction therapy. Patients with this low level of MRD had a 12.7% (± 5.1%; SE) cumulative risk of relapse at 5 years, compared with 5.0% (± 1.5%) for those with lower or undetectable MRD (P 〈 .047). Thus, low levels of MRD (0.001%-〈 0.01%) at the end of remission induction therapy have prognostic significance in childhood ALL, suggesting that patients with this finding should be monitored closely for adverse events.

Description: Abstract 364 Patients (pts) with advanced stage CTCL have a poor prognosis with standard therapy whereas AHCT possibly is curative. Published experience with AHCT for CTCL is modest. We report the outcomes of a large cohort of CTCL pts receiving AHCT (N=133) reported to the CIBMTR from 2000–2009. The majority of pts 129 (97%) were diagnosed with Mycosis Fungoides or Sezary Syndrome. Only 8 (6%) received AHCT in a complete remission (CR) while 46 pts (35%) never achieved CR pre-AHCT. Most pts (83%) were transplanted beyond 12 months from diagnosis. Median (range) time from diagnosis to transplant is 29 (4-206) months. Peripheral blood was the main graft source (83% vs. 14% marrow and 3% cord blood). Donor types included: 66 HLA-identical siblings (50%), 56 unrelated donors (42%), 11 from other related donors (8%). Reduced intensity conditioning (RIC) was used in 64%. Recipients of RIC were on average older (median age 51 vs. 44 yr for myeloablative) and transplanted in later study years. Among the myeloablative regimens, cyclophosphamide with total body irradiation was the most common (38%) whereas among RIC, fludarabine in combination with busulfan, cyclophosphamide or melphalan was used in 50%. GVHD prophylaxis regimens varied. Overall mortality at 30 and 100 days was 7% and 16%, respectively. Overall survival at 6 mo and 2 yr was 75% (95% CI 67–82%) and 44% (95% CI 34–53%), respectively. Conditioning intensity was not different statistically for the overall survival of these pts (figure 1). Treatment related mortality (TRM) at 6 mo and 2 yr was 10% (95% CI 5–17%). Progression/relapse of CTCL was 44% (95% CI 34–54%) at 6 mo and 58% (95% CI 47–68%) at 2 yr. Progression free survival at 6 mo was 46% (95% CI 36–57%) and at 2 yr 32% (95% CI 23–42%). The incidence of grade II-IV acute GVHD was 36% (95% CI 22–50%) and of chronic GVHD 31% (95% CI 19–45%) in a subset of reported pts. Infection as a cause of death does not seem to be increased in CTCL transplanted pts. Conditioning intensity did not impact TRM or the risk of progression. This very large series of allogeneic HCT demonstrates feasibility in pts with advanced CTCL with a low TRM and long term progression free survival in approximately one third of pts. Progressive disease was the primary cause of treatment failure in this cohort of pts with advanced disease. Figure 1. Figure 1. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2472 Background: As a result of the previously demonstrated efficacy of the cyclin dependent kinase inhibitor, flavopiridol, and the immunomodulatory agent, lenalidomide, in heavily pre-treated patients (pts) with bulky adenopathy and adverse cytogenetics, we conducted a phase I trial to determine the dose limiting toxicity (DLT) and maximum tolerated dose (MTD) of combined therapy. Both agents are active in pts with relapsed/refractory cytogenetically high risk CLL, utilize different novel mechanisms of action, and do not deplete T-cells, leading to the potential development of a well tolerated regimen for pts with del (17p13.1) and del (11q22.3) with greater efficacy and less infectious toxicity than observed with currently accepted fludarabine or alemtuzumab combinations. Methods: Pts with histologically confirmed CLL relapsed after at least 1 prior therapy with a WBC ≤ 150,000/mm3, ANC ≥ 1000/mm3, platelets ≥ 30,000/mm3, and creatinine ≤ 1.5 mg/dL were enrolled. Treatment consisted of flavopiridol alone (30 mg/m2 bolus + 50 mg/m2 4-hour continuous IV infusion (CIVI)) days 1, 8, and 15 of cycle 1 to decrease the pre-treatment disease burden and minimize the potential risks of simultaneous tumor flare and tumor lysis syndrome (TLS) with concurrent lenalidomide and flavopiridol. Starting in cycle 2, flavopiridol 30 mg/m2 bolus + 30 mg/m2 4-hour CIVI days 3, 10, and 17 was combined with lenalidomide 2.5, 5.0, 7.5, 10, 15, or 25 mg days 1–21 every 28 days. All pts received 20 mg of dexamethasone 30 minutes prior to flavopiridol dosing and 4 mg of dexamethasone 24 hours after flavopiridol to minimize cytokine release symptoms. Results: Twenty-one pts (12 males) with a median age of 59 (range 42–71) previously treated with a median of 4 prior therapies (range 1–10) were enrolled. All pts received prior fludarabine, 9 pts were fludarabine refractory, no pts previously received flavopiridol, and 1 pt was previously treated with lenalidomide. Pts had Rai stage I/II (n=6) or IV (n=15) disease and 13 pts had bulky adenopathy ≥ 5 cm. Thirteen pts had del (17p13.1) and 8 pts had del (11q22.3). Seventeen pts completed two or more cycles of therapy (median 3, range 2–8), receiving 2.5 mg (n=6), 5.0 mg (n=7), and 7.5 mg (n=4) of lenalidomide. Four pts completed only one cycle of therapy and were removed prior to receiving lenalidomide due to progressive disease (n=2), TLS requiring dialysis (n=1), and grade 4 thrombocytopenia (n=1). DLT consisting of grade 3–4 transaminitis persisting 〉 7 days occurred in 2 pts treated with 2.5 mg (n=1) and 5.0 mg of lenalidomide (n=1), respectively. Grade 3–4 toxicities consisted of neutropenia (86%), diarrhea (62%), transaminitis reversible in ≤ 7 days (57%), anemia (38%), thrombocytopenia (38%), hyperglycemia (38%), infection without neutropenia (24%, cellulitis in 1 pt, pneumonia in 3 pts, and catheter-associated in 1 pt), febrile neutropenia (14%, unknown source in 2 pts and pneumonia in 1 pt), TLS not requiring dialysis (14%), and fatigue (14%). Grade 1 tumor flare occurred in 1 pt and did not require additional steroids or cessation of lenalidomide. In 15 evaluable pts who completed 1 or more cycles of combined lenalidomide and flavopiridol, partial responses were observed in 7 pts, including 4 pts with del (17p13.1), 3 pts with del (11q22.3), 5 fludarabine-refractory pts, 3 pts with bulky lymphadenopathy ≥ 5 cm, and 1 pt previously treated with lenalidomide. Conclusions: Combined flavopiridol and lenalidomide is well tolerated, with activity in pts with previously treated, cytogenetically high-risk CLL. The MTD has not been reached and dose escalation continues. This trial is supported by NCI R21 CA133875, NCI K23 CA109004, NCI U01 CA076576, LLS SCOR 7080-06, and the D. Warren Brown Foundation. Disclosures: Blum: Celgene: Research Funding. Off Label Use: The efficacy of the cyclin dependent kinase inhibitor, flavopiridol, and the immunomodulatory agent, lenalidomide, are under investigation in CLL.

Description: Abstract 2461 Background: Fludarabine, rituximab, and cyclophosphamide combinations have shown high CR rates in CLL, but concerns remain about both short and long term toxicity. Lenalidomide has emerged as a potential alternative addition to the fludarabine/rituximab backbone that may increase anti-tumor activity. The purpose of this Phase I/II study was to determine optimal lenalidomide dosing and to evaluate its potential benefits when used in combination with fludarabine/rituximab. Methods: Eligible patients (pts) had untreated Rai stage III/IV or symptomatic stage 0-II B cell CLL with no CNS involvement, ECOG PS 0–2, and adequate organ function. The Phase I portion of this trial (n=19) explored fixed doses of fludarabine (25mg/m2/IV on days 1, 2, and 3 every 28-day cycle) and rituximab (375mg/m2/IV in divided dose days 1 and 2 cycle 1, followed by 500mg/m2/IV day 1 cycles 2–6) while receiving one of two dose levels (DLs) of lenalidomide. Pts on DL1 received 2.5mg PO on days 8–28 of cycles 1–6; those on DL2 received 2.5mg PO on days 8–28 of cycle 1 and 5.0mg on days 8–28 of cycles 2–6. Tumor lysis syndrome prophylaxis was administered to all pts throughout the first 2 cycles (allopurinol, 300mg PO daily). Disease assessment (NCI WG Guidelines) occurred post-cycle 3 during active treatment, 2 months after completion of the last treatment cycle (≤6 cycles), and every 6 months in follow-up until disease progression. Results: Between 2/2008 and 5/2010, 28 pts were enrolled; the first 26 are included in this analysis. Pts were all untreated, and 64% male with median age 66.5 yrs (range: 48–82 yrs) and Rai stage 0/I/II/III/IV of 2/10/7/4/3. For the Phase I portion of the trial, the original lenalidomide dosing schedule specified that treatment begin on day 1 concurrently with fludarabine/rituximab, continuing for 21 days. Of the first 4 pts enrolled, 2 experienced persistent grade (g) 3 rash and/or dose-limiting toxicity (DLT), including g4 febrile neutropenia. Only 1 pt completed all 6 cycles of therapy but still 3 achieved PR; 1 came off study prior to disease evaluation (DLT). Due to concerns about toxicity in these first 4 pts, the protocol was amended to delay lenalidomide until days 8–28. Results for DL1 pts (n=6) from the Phase I portion of the amended protocol are as follows: 5 pts completed 6 cycles of therapy and achieved an objective response rate (ORR) of 83% (CR, 4; PR, 1); the remaining pt had SD. Median time-to-CR was 34.0 weeks (range 32.4–35.4). Of these 6 pts, the most common DL1 g3/4 toxicities included neutropenia (4), leukopenia (4), and rash (1); 1 pt was hospitalized for g3 upper respiratory infection (possibly-related). The maximum tolerated dose of lenalidomide (5.0mg, DL2) is currently being studied within the Phase II portion of this study. Currently, the findings for DL2 (Phases I/II pooled, n=16) are as follows: 5 pts were unevaluable (2 too early for assessment; 3 due to toxicity). Two pts achieved CR; 5 pts are PR, 2 of which are still currently receiving treatment. Four pts are stable, including 2 who are still on-study. In DL2 pts the most common g3/4 toxicities were neutropenia (10), leukopenia (5), fatigue (3), anorexia (2) and rash (2). Five pts on DL2 were hospitalized, including 1 with g3 tumor lysis syndrome while receiving prophylaxis (related) and another with g3 allergic reaction/hypersensitivity (possibly-related); 1 pt later expired from g5 diarrhea (possibly-related). Conclusion: The activity of lenalidomide, when added to fludarabine/rituximab, appears promising, despite notable toxicity. The dosing schedule of lenalidomide in combination with fludarabine/rituximab appears to influence the overall toxicity of the three-drug regimen, as evidenced by the improvement in tolerability with sequential dosing. Phase II study is ongoing which will further characterize the toxicity and efficacy of this regimen in pts with CLL. Disclosures: Flinn: Celgene: Research Funding; Genentech: Research Funding. Off Label Use: This study uses lenalidomide off-label, in combination with fludarabine/rituximab, as an investigational treatment for patients with chronic lymphocytic leukemia. Cooper: Bristol-Myers Squibb: Consultancy.

Description: Abstract 4279 Background: A fraction of patients (up to 30%) with transfusional iron overload on deferasirox (DFX, Exjade®) have less-than-adequate chelation responses at doses above 30 mg/kg/day. Both transfusion burden and adherence play significant roles in observed chelation effectiveness, but a biological difference between good and poor DFX responders is detectable by pharmacokinetic (PK) assessment. Indeed, we have reported that patients with poor responses at doses〉30 mg/kg/d have decreased exposure (area under the concentration:time curve) after a single oral dose of 35 mg/kg (Chirnomas et al, Blood, 2009, 114:4009). In principle, iron status in these individuals could be improved by strategies to reduce iron input or intensify chelation. Here we report results of these strategies for the poor responders of the published PK trial. Patients and Methods: We retrospectively assessed response of ferritin levels and hepatic iron measurements by R2 Ferriscan® MRI to various strategies in the 9 thalassemia (thal) patients (pts) with poor DFX response from the phase 4 PK trial cited above. One sickle cell pt from the trial with documented poor adherence is not included. Strategies were individualized, based on prior dose of DFX, comorbidity (e.g. liver disease or dose-limiting toxicity), transfusion burden, and estimates of pt adherence. Strategies included: splenectomy to reduce transfusion burden (n=2); dose increase (n=5, including 1 with rapid elimination half-life who also received b.i.d. dosing) and combination (“combo”) therapy with DFX and deferoxamine (DFO, n=2). As ferritin values are variable and not normally distributed, log-transformed ferritin values in three successive months from the time of study eligibility determination and after intervention strategies were compared by two-way ANOVA. Liver iron content pre- and post-interventions, was compared by paired t-test. Results: As shown in the figure, ferritin improved in 8/9 pts (p

Description: Abstract 1407 The development of factor VIII (FVIII) inhibitors is usually considered uncommon among patients with mild and moderate hemophilia A (HA) and less frequent than in patients with severe HA. We report here the prevalence of FVIII inhibitors and their caracteristics in 167 patients with mild and moderate HA followed in Caen Hemophilia Treatment Centre (Table). FVIII molecular defects were identified by direct sequencing in 167 patients including 30 and 137 with mild and moderate HA, respectively. Following FVIII concentrates infusions, FVIII inhibitors occured in 7.8% of patients (13/167). Fifteen percent (2/13) were low-responding inhibitors. The risk of inhibitor development appeared to be associated with high-risk FVIII genotypes clustered in the A2 and C2 domains, especially p.Arg2150His (50%) and p.Arg593Cys mutations. Interestingly, we described inhibitor development associated with novel missense-mutations (p.Tyr1786Ser, p.Asp115Tyr and -219C〉T substitutions in FVIII gene promoter). In addition, high regimen infusion of FVIII concentrates appeared as risk factor for FVIII inhibitors development. Indeed, 60% (8/13) developped FVIII inhibitors following massive infusion of FVIII concentrates associated with FVIII:C levels above 1.2 UI/dL. Inhibitors in mild HA usually cross-react with endogenous factor VIII reducing the circulating basal FVIII:C level and are associated with more bleeding events. Similarly, we observed the evolution of bleeding patterns in our cohort to severe phenotypes. Bleedings were treated with FVIII concentrates and bypassing therapies (activated FVII and activated-prothrombin complex). About 25% (3/13) of these inhibitors disappeared spontaneously. Induction of Immune Tolerance (ITI) protocoles with high doses of FVIII were initiated for 7 high-responding patients with a success rate of 85 % (6/7). However, inhibitors persisted long-term and remained troublesome in 1 of these patients despite of ITI protocole. For two patients, immunosuppressive treatment with corticosteroids was started. Inhibitors disappeared and the levels of FVIII:C became detectable within 6 months. Development of FVIII inhibitors, their disappearance and the efficacy of ITI regimen seem to be different from our experience between patients with mild or moderate HA and severe HA. Disclosures: No relevant conflicts of interest to declare.

Description: A pilot study was undertaken to assess the safety, activity, and immunogenicity of a polyvalent Wilms tumor gene 1 (WT1) peptide vaccine in patients with acute myeloid leukemia in complete remission but with molecular evidence of WT1 transcript. Patients received 6 vaccinations with 4 WT1 peptides (200 μg each) plus immune adjuvants over 12 weeks. Immune responses were evaluated by delayed-type hypersensitivity, CD4+ T-cell proliferation, CD3+ T-cell interferon-γ release, and WT1 peptide tetramer staining. Of the 9 evaluable patients, 7 completed 6 vaccinations and WT1-specific T-cell responses were noted in 7 of 8 patients. Three patients who were HLA-A0201-positive showed significant increase in interferon-γ–secreting cells and frequency of WT1 tetramer-positive CD8+ T cells. Three patients developed a delayed hypersensitivity reaction after vaccination. Definite related toxicities were minimal. With a mean follow-up of 30 plus or minus 8 months after diagnosis, median disease-free survival has not been reached. These preliminary data suggest that this polyvalent WT1 peptide vaccine can be administered safely to patients with a resulting immune response. Further studies are needed to establish the role of vaccination as viable postremission therapy for acute myeloid leukemia. This study was registered at www.clinicaltrials.gov as #NCT00398138.

Description: Abstract 439 Background: TET genes have been implicated in DNA demethylation in mammals (Thalihani, Science, 2009; Ito, Nature 2010) and the TET2 gene is mutated in 15–22% of MDS and AML (Kosmider et al. Blood 2009, Nibourel et al. Blood 2010). Azacitidine (AZA) is a hypomethylating agent providing about 50% of responses in MDS and AML with low blast count (Lancet Oncol 2009, JCO 2010). Routine clinical and biological variables can predict OS with AZA, but are poor predictors of response to AZA (Itzykson et al. ASH 2009, and other abstract submitted to ASH 2010), while no consensus genetic predictor of response has been reported so far. Methods: In consecutive MDS (including RAEB-t and CMML) and AML post MDS (with 〉30% blasts) (AML/MDS) patients treated by AZA in 6 centers, we prospectively sequenced the TET2 gene from PBMC or BMNC DNA samples stored prior to AZA onset. Standard PCR and sequencing procedures were performed as previously described (Delhommeau et al, NEJM 2009), allowing detection of mutations in the entire coding sequence of the TET2 gene (exons 3 to 11). Patients were to receive AZA at the FDA/EMEA approved schedule (75mg/m2/d, 7d/4 weeks). Patients (pts) having received ≥ 1 cycle of AZA and who had bone marrow evaluation after ≥ 4 cycles, or who died or progressed before completion of 4 cycles were considered evaluable (the last 2 groups were considered as treatment failures). Responses were scored according to IWG 2006 criteria for MDS and to Cheson et al. (JCO 2003) for AML. Results: The study population included 103 pts: F/M: 36/67; median age 72 (range 43–91). Diagnosis at AZA onset was MDS in 89 (RAEB-1 n=20, RAEB-2 n=43, RAEB-t n=23, CMML-1 n=2, CMML-2 n=1; IPSS int-1 in 13, int-2 in 38, high in 36, undetermined in 2) and AML/MDS in 14 pts; 29 pts had previously been treated by LD AraC or intensive chemotherapy (IC). Cytogenetics according to IPSS was favorable in 48, intermediate in 20, unfavorable in 31, unknown in 4. 78 pts received the approved and 25 (24%) a reduced AZA schedule (mostly 75 mg/m2 for 5 days every 4 weeks). Median number of cycles was 7 [range 1–39] and median follow-up was 18.2 months. 21 TET2 mutations were found in 17 (17%) pts, including 12 frameshift (all inducing a premature STOP codon), 6 non sense and 3 missense mutations. Predicted TET2 protein length was preserved in 3, and truncated in 14 pts, respectively. Patients with mutated (MUT) TET2 had similar age, diagnosis, BM blast %, neutrophil and platelet counts as those with wild type (WT) TET2 (all p〉0.3), but less frequent unfavorable cytogenetics (6% vs 37%, p=0.02) and a trend for higher WBC (p=0.12), lower Hb (p=0.11) and more pretreatment by LD AraC or IC (47% vs 24%, p=0.08) than WT TET2 pts. The 2 groups received AZA at similar schedules (p=0.4) for a median of 6 [1-39] cycles (TET2 WT) or 11 [4-34] cycles (TET2 MUT, p=0.01). Ten WT pts (vs 0 MUT) received

Description: Abstract 123 Background: Lenalidomide is an effective agent in the treatment of MDS particularily in those possesing the deletion 5q (del5q) abnormality. The disease phenotype is partly related to loss of genetic material found in the commonly deleted region (CDR) on the long arm of chromosome 5. Recently implicated genes include miRNA-143, 145 and 146 all of which appear to play a role in innate immune signalling. In this study we examined whether upregulation of miRNAs in pretreatment CD34+ marrow cells from patients with MDS (both del5q and non-del5q) exposed to lenalidomide in vitro predicts for clinical response to the drug. Patients and Methods: In total 31 pre-treatment patient samples were obtained from three North American centres. 11 patients' samples were positive for del5q. All patients had received lenalidomide as per local protocols. Clinical response was defined as per the International Working Group 2000 criteria. Clinical data were available for 26 patients. Hematologic improvement (HI), specifically the eythroid response (HI-E), was examined as the main clinical endpoint. Changes in miR-143, miR-145, miR-146a and miR-146b expression in CD34+ cells after in vitro lenalidomide exposure (10uM for 48 hours) were examined. The CD34- fraction was also analyzed. dCT values were normalized to 5S. A ≥ 1.5 fold change in miRNA expression was considered significant. Results: For the entire cohort mean fold changes in miRNA-143 and 145 post lenalidomide exposure were 1.6 and 1.7, respectively. Little change was seen in miR-146a and miR-146b expression (mean fold change 1.1 and 1.2, respectively). No significant increase was seen in any of the examined miRNA in the CD34 - fraction (Figure 1a). When separated based on del5q status both del5q and non-del5q groups showed a 〉 1.5 fold increase in expression of miR-143 and miR-145. No significant change in either miR-146a or miR-146b expression was seen (Figure 1b). In the CD34- fraction no significant change was seen in any of the examined miRNAs, irrespective of del5q status (Figure 1c). Examining HI-E, major responses (MR) were seen in 42% of patients (87.5% in the the del 5q cohort (7/8 had a MR) and 22.2% for the non-del5q cohort (4/18 had a MR)). Correlation with clinical response and miRNA expression in the CD34+ fraction is shown in Table I. In the del5q cohort MR was associated with an increase in both miR-143 and miR-145. No correlation with miRNA expression and clinical response was seen in the non del5q cohort. Conclusion: Exposure of CD34+ cells from patients with MDS to lenalidomide results in up regulation of miR-143 and miR-145, irrespective of del5q status. Clinically, upregulation of miR-143 and miR-145 correlates with a HI-E in patients with del5q and may be predictive of response to therapy. Interestingly, the non-responder in the del5q group failed to upregulate miR-145. A similar gene induction pattern was seen in the non-del5q cohort, yet no correlation was seen with clinical outcome. Given that non-del5q patients do not harbor any abnormalities within the CDR of chromosome 5 these miRNAs are unlikely to play a role in their disease phenotype, nor in their response to lenalidomide. Disclosures: Nevill: Celgene: Honoraria. Karsan:Celgene: Research Funding.

Description: The type-III interferon (IFN) family is composed of 3 molecules in humans: IFN-λ1 (interleukin-29 [IL-29]), IFN-λ2 (IL-28A), and IFN-λ3 (IL-28B), each of which signals through the same receptor complex. Plasmacytoid dendritic cells (pDCs) are major IFN-λ producers among peripheral lymphocytes. Recently, it has been shown that IFN-λ1 exerts a powerful inhibitory effect over the T-helper 2 (Th2) response by antagonizing the effect of IL-4 on CD4+ T cells and inhibiting the production of Th2-associated cytokines. Here, we asked whether Th2 cytokines exert reciprocal control over IFN-λ production. IL-4 treatment during stimulation of human peripheral lymphocytes significantly elevated IFN-λ1 transcription and secretion. However, pDCs were not directly responsive to IL-4. Using depletion and reconstitution experiments, we showed that IL-4–responsive monocytes are an intermediary cell, responding to IL-4 by elevating their secretion of IL-1 receptor antagonist (IL-Ra); this IL-1Ra acts on pDCs to elevate their IFN-λ1 output. Thus, our experiments revealed a novel mechanism for regulation of both IFN-λ1 production and pDC function, and suggests an expanded immunomodulatory role for Th2-associated cytokines.

Description: Abstract 4147 Whole-body diffusion-weighted magnetic resonance imaging (DWI-MRI) provides functional information and is able to highlight oncological lesions, but the usefulness has not been established in malignant lymphoma especially for monitoring therapeutic response. Positron emission tomography with fluorine-18 fluorodeoxyglucose (FDG-PET) is a useful imaging producer for tumor staging and therapy monitoring that can visualize active tumor tissue including malignant lymphoma. The spatial resolution of FDG-PET is limited, and low accuracy rates in diabetic patients and those with low grade lymphoma have been reported. We prospectively studied the utility of DWI-MRI with T2 imaging and apparent diffusion coefficient (ADC) for staging and monitoring therapeutic responses in patients with malignant lymphoma compared with FDG-PET/CT. Twenty-eight patients with malignant lymphoma (16 patients with diffuse large B cell lymphoma: DLBCL, 7 with follicular Lymphoma: FL, 3 with aggressive T cell lymphoma: TCL and 2 with Hodgkin lymphoma: HL, including one diabetic patient) received both MRI and FDG-PET examination before (n=28), after 2 courses of chemotherapy (n=25) and one month after the end of chemotherapy (n=9). MRI examination was performed with a 3-Tesla MR system (Signa Excite, Generel Electrics). Whole-body DWI-MRI was performed with echo planar imaging sequence with short T1 inversion recovery (STIR) fat suppression. ADC measurement was performed based on the region of interest (ROI) method. ROI was placed on the lesion showing the highest standardized uptake value (SUV) on FDG-PET/CT scanner (Discovery LS, General Electrics) in each patient, and crucial parameters of the ADC and SUV were compared. Based on staging by PET/CT, 4 patients were clinical stage I, 8 were stage II, 7 were stage III and 9 were stage IV. DWI-MRI findings alone matched PET/CT in 22 patients (79%), whereas these findings combined with T2 imaging increased match in 26 patients (93%). Regarding the early response to chemotherapy, 19 of 25 patients (76%) were considered to show CR on PET/CT and the DWI findings matched PET/CT 23 patients (92%). To evaluate the final response after chemotherapy, 7 of 9 patients (78%) were considered to show CR on PET/CT and the DWI findings matched PET/CT in 8 of 9 patients (89%). Of these nine, one patient with DLBCL who did not show a match was a false positive on PET/CT. In all patients with TCL and HL, the DWI-MRI findings combined with T2 imaging matched PET/CT findings for staging and therapeutic response. Interestingly, the ADC values on DWI-MRI did not differ between DLBCL and FL (0.77 +/− 0.23 and 0.70 +/− 0.08, p=0.99, mean +/− SD respectively), whereas the SUV values of DLBCL on PET/CT were higher than those of FL (14.5 +/− 6.97 and 6.09 +/− 2.54, p 〈 0.0005, mean +/− SD respectively), suggesting the DWI-MRI could detect the lymphoma lesion more accurately than PET/CT in patients with indolent lymphoma such as FL. We conclude that whole-body DWI-MRI combined with T2 imaging and ADC analysis could be promising sensitive method for staging and therapeutic response evaluation in patients with malignant lymphoma. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2967 Activin-A is a member of the transforming-growth factor-beta superfamily most commonly associated with embryogenesis and gonadal hormone signaling. Activin-A is also involved in bone homeostasis having growth stimulatory effects on osteoclasts, but unclear effects on osteoblast function. A recent study showed that, in multiple myeloma (MM) microenvironment, activin-A is a stromally derived osteoblast inhibitor induced by myeloma cells and thus it seems to be implicated into the pathogenesis of myeloma bone disease (Vallet et al, PNAS 2010;107:5124). The aim of this study was to evaluate the circulating levels of activin-A in newly-diagnosed and relapsed patients with MM and explore possible correlations with clinical and laboratory data, including lytic bone disease and survival. We studied 113 MM patients (63M/50F, median age 69 years, range 31–92 years): 98 newly-diagnosed patients (13 with asymptomatic and 85 with symptomatic MM) and 15 patients with relapsed MM after previous response to front-line therapy, who received the combination of lenalidomide and high-dose dexamethasone (RD). All symptomatic patients received frontline treatment with novel agent-based regimens. For newly-diagnosed patients, serum was stored at -80°C at the time of diagnosis, while for relapsed patients serum was stored on day 1 of the first RD cycle and then on day 28 of the 4th cycle. Serum activin-A was measured using ELISA methodology (R&D Systems, Minneapolis, MN, USA) along with a series of markers of bone resorption (C-terminal cross-linking telopeptide of collagen type-1, CTX and tartate-resistant acid phosphatase isoform-5b, TRACP-5b) and bone formation (bone-specific alkaline phosphatase and osteocalcin). Evidence of bone involvement was documented using plain radiographs in patients at diagnosis and at the time of relapse. Activin-A and the above bone markers were also measured in 10 MGUS patients and in 17, gender- and age-matched, healthy controls. Circulating levels of activin-A of newly-diagnosed patients with symptomatic MM (median: 555 pg/mL, range: 129–2336 pg/mL) and of relapsed patients (677 pg/mL, 272–2088 pg/mL) were increased compared to controls (393 pg/mL, 204–899 pg/mL; p

Description: Abstract 1791 Background Advanced stage T-cell or NK/T-cell lymphomas usually show aggressive clinical course and their treatment outcomes are worse than B-cell non-Hodgkin lymphoma. Furthermore, the optimal treatment regimen is not still established for these disease entities. At present, cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP) regimen is still used as a primary treatment for advanced stage T or NK/T cell lymphomas although its efficacy is not satisfactory. Thus, more effective treatment regimen is required to improve treatment outcome. The incorporation of new targeted agents into CHOP regimen has been a widely used strategy to develop new regimen for the treatment of lymphoma. Bortezomib, a proteasome inhibitor approved for the use of treatment of multiple myeloma has been tried in many B-cell non-Hodgkin lymphomas. A recent in vitro study results showed that proteasome inhibitor could inhibit the growth of NK/T lymphoma cells. Based on these results, we designed a regimen combining CHOP with. Our previous phase I study determined the maximum tolerated dose of bortezomib as 1.6mg/m2 for combination with CHOP. Thus, we performed the phase II study to evaluate the efficacy of bortezomib plus CHOP chemotherapy. Methods We enrolled patients with newly diagnosed T or NK/T cell lymphoma. All patients were Ann Arbor stage III/IV and had adequate organ function. Patients received bortezomib on days 1 and 8 (weekly schedule, 1.6 mg/m2 per dose) in addition to 750 mg/m2 cyclophosphamide, 50 mg/m2 doxorubicin, 1.4 mg/m2 vincristine on day 1 and 100 mg/day prednisolone on days 1 to 5, every 3 weeks. Six cycles of therapy administered every 21 days were planned. All patients provided written informed consents and this trial was registered at www.ClinicalTrials.gov (NCT00374699). Results 46 patients were enrolled between April 2007 and August 2009. Peripheral T-cell lymphoma, unspecified (n=16) and extranodal NK/T cell lymphoma (n=10) were dominant subtypes while angioimmunoblastic T-cell lymphoma (n=8) and ALK-negative anaplastic large cell lymphoma (n=6) account for 30.4% of all patients. Five patients with cutaneous T-cell lymphoma and one hepatosplenic T-cell lymphoma were also recruited. The median age at diagnosis was 52 years (range 21 – 66 years). Serum LDH elevation (n = 28, 60.9%) and stage IV patients were dominant (n = 32, 69.6%). Thus, the International Prognostic Index risk was dominantly high or high-intermediate (n = 26, 56.5%). Complete response was achieved in 30 patients (65.2%) and partial response was 5 patients (10.9%). As a result, the overall response rate was 76.1%. The comparison of complete response rate based on the subtype demonstrated that the complete response rate of peripheral T-cell lymphoma, unspecified (12/19, 63.2%), angioimmunoblastic T-cell lymphoma (6/8, 75%), anaplastic large cell lymphoma (4/6, 66.7%) and cutaneous T-cell lymphoma (5/5, 100.0%) was better than extranodal NK/T cell lymphoma (3/10, 30.0%). Five patients with extranodal NK/T cell lymphoma progressed during the treatment with bortezomib and CHOP. The hematologic toxicity was the major toxicity of this regimen, thus, grade 3/4 leucopenia and febrile neutropenia were the most frequent toxicity. However, there was no treatment-related mortality. In addition, neurotoxicity was tolerable, so the majority of peripheral neurotoxicity was grade 1 or 2. Conclusion The combined treatment of bortezomib with CHOP is an effective regimen for advanced stage T-cell lymphomas with acceptable toxicity. However, it may not be efficient for advanced stage extranodal NK/T-cell lymphomas. Disclosures: No relevant conflicts of interest to declare.

Description: The shear stress–induced transcription factor Krüppel-like factor 2 (KLF2) confers antiinflammatory properties to endothelial cells through the inhibition of activator protein 1, presumably by interfering with mitogen-activated protein kinase (MAPK) cascades. To gain insight into the regulation of these cascades by KLF2, we used antibody arrays in combination with time-course mRNA microarray analysis. No gross changes in MAPKs were detected; rather, phosphorylation of actin cytoskeleton-associated proteins, including focal adhesion kinase, was markedly repressed by KLF2. Furthermore, we demonstrate that KLF2-mediated inhibition of Jun NH2-terminal kinase (JNK) and its downstream targets ATF2/c-Jun is dependent on the cytoskeleton. Specifically, KLF2 directs the formation of typical short basal actin filaments, termed shear fibers by us, which are distinct from thrombin- or tumor necrosis factor-α–induced stress fibers. KLF2 is shown to be essential for shear stress–induced cell alignment, concomitant shear fiber assembly, and inhibition of JNK signaling. These findings link the specific effects of shear-induced KLF2 on endothelial morphology to the suppression of JNK MAPK signaling in vascular homeostasis via novel actin shear fibers.

Description: Intravenous immunoglobulin (IVIg) is an effective treatment against immune thrombocytopenia (ITP). Previous studies suggested that IVIg exerts this ameliorative role through 2 different leukocyte subsets. Dendritic cells (DCs) modulate the immunosuppression in an adoptive cell transfer model, and phagocytes up-regulate their inhibitory IgG Fc receptors (FcγR)IIB expression and thereby ameliorate the inflammatory response and platelet clearance. However, whether or not regulatory mechanisms exist among DCs, phagocytes, and platelets is still largely unknown. In this study we present findings that IVIg-primed splenic CD11c+ DCs (IVIg-DCs) primarily mediate their anti-inflammatory effects at the level of the platelet rather than the phagocyte. IVIg-DCs did not ameliorate ITP in Fcgr2b−/−, Fcgr3−/−, nor P-Selp−/− mice, implicating the potential involvement of these pathways in IVIg action. As platelets are a component of DC regulatory circuits, these findings may suggest an alternative perspective for the use of IVIg treatment.

Description: Extracellular factors control the angiogenic switch in endothelial cells (ECs) via competing survival and apoptotic pathways. Previously, we showed that proangiogenic and antiangiogenic factors target the same signaling molecules, which thereby become pivots of angiogenic balance. Here we show that in remodeling endothelium (ECs and EC precursors) natural angiogenic inhibitors enhance nuclear factor-κB (NF-κB) DNA binding, which is critical for antiangiogenesis, and that blocking the NF-κB pathway abolishes multiple antiangiogenic events in vitro and in vivo. NF-κB induction by antiangiogenic molecules has a dual effect on transcription. NF-κB acts as an activator of proapoptotic FasL and as a repressor of prosurvival cFLIP. On the FasL promoter, NF-κB increases the recruitment of HAT p300 and acetylated histones H3 and H4. Conversely, on cFLIP promoter, NF-κB increases histone deacetylase 1 (HDAC1), decreases p300 and histone acetylation, and reduces the recruitment of NFAT, a transcription factor critical for cFLIP expression. Finally, we found a biphasic effect, when HDAC inhibitors (HDACi) were used to test the dependence of pigment epithelial-derived factor activity on histone acetylation. The cooperative effect seen at low doses switches to antagonistic as the concentrations increase. Our study defines an interactive transcriptional network underlying angiogenic balance and points to HDACi as tools to manipulate the angiogenic switch.

Description: Mutations within the FMS-like tyrosine kinase 3 (FLT3) gene on chromosome 13q12 have been detected in up to 35% of acute myeloid leukemia (AML) patients and represent one of the most frequently identified genetic alterations in AML. Over the last years, FLT3 has emerged as a promising molecular target in therapy of AML. Here, we review results of clinical trials and of correlative laboratory studies using small molecule FLT3 tyrosine kinase inhibitors (TKIs) in AML patients. We also review mechanisms of primary and secondary drug resistance to FLT3-TKI, and from the data currently available we summarize lessons learned from FLT3-TKI monotherapy. Finally, for using FLT3 as a molecular target, we discuss novel strategies to overcome treatment failure and to improve FLT3 inhibitor therapy.

Description: Induced pluripotent stem cells (iPSCs) can be generated from various differentiated cell types by the expression of a set of defined transcription factors. So far, iPSCs have been generated from primary cells, but it is unclear whether human cancer cell lines can be reprogrammed. Here we describe the generation and characterization of iPSCs derived from human chronic myeloid leukemia cells. We show that, despite the presence of oncogenic mutations, these cells acquired pluripotency by the expression of 4 transcription factors and underwent differentiation into cell types derived of all 3 germ layers during teratoma formation. Interestingly, although the parental cell line was strictly dependent on continuous signaling of the BCR-ABL oncogene, also termed oncogene addiction, reprogrammed cells lost this dependency and became resistant to the BCR-ABL inhibitor imatinib. This finding indicates that the therapeutic agent imatinib targets cells in a specific epigenetic differentiated cell state, and this may contribute to its inability to fully eradicate disease in chronic myeloid leukemia patients.

Description: Abstract 535 PCL is associated with a failure of the plasma cells to retain their normal homing patterns to the bone marrow, is difficult to treat and developing targeted treatments based on its pathogenesis would be a step forward. PCL lies at the end of the multistep pathway from normal to malignant plasma cells and little is known about the genetic mechanisms mediating the final stages of this pathway. The methylation status of genes in myeloma can change as the malignancy progresses and as such identifying genes deregulated by methylation that mediate the progression of MM to PCL may offer epigenetically relevant therapeutic targets. We have previously analyzed 181 samples including B cell, normal plasma cells, MGUS and MM samples for methylation differences using the Illumina Infinium humanmethylation27 array, which interrogates 27,578 highly informative CpG sites per sample at the single-nucleotide resolution using bisulfite converted DNA. Here we have added 32 PCL samples to our dataset in order to further investigate the methylation changes that occur on the transition from MM to PCL. Data are presented as an average beta-score where 1.0 is fully methylated and 0 is fully unmethylated. Data were analyzed in GenomeStudio using the methylation module (Illumina). Further analyses were performed using R and the LIMMA package. Differential methylation between samples was identified using an empirical Bayes moderated t-test and the resulting p-values were adjusted using the Benjamini and Hochberg method. P-values below 0.05 were considered significant. We have shown genome-wide hypomethylation at the transition from MGUS to MM with gene-specific hypermethylation of tumor suppressor genes. The methylation patterns within myeloma samples correlate with translocations or hyperdiploidy, where the latter are split into 2 distinct clusters with a significant difference in overall survival, p=0.03. However, the t(4;14) subgroup had the most distinct methylation profile with hypermethylation of genes specific to the t(4;14) subgroup including CD79A, FAM49A and SOCS2 which correlate with loss of expression in this subgroup. Herein, we analyzed presentation MM (n=161) and PCL (n=32) samples for methylation changes which may contribute to expression and phenotypic changes. When comparing all MM and PCL samples, irrespective of cytogenetic subgroup, we find hypermethylation of the genome in PCL samples. Upon clustering we find that PCL samples segregate within the translocation groups with which they belong i.e. t(11;14) PCL samples cluster with t(11;14) myeloma samples. However, when MM and PCL samples from the same cytogenetic group are compared there is exclusive hypermethylation of genes occurring as the disease progresses. Although t(4;14) myeloma samples have considerable more methylation than t(11;14) samples, the increase in methylation from MM to PCL is consistent in both groups, resulting in t(4;14) PCL samples with increased methylation over t(11;14) PCL samples. The genes which are hypermethylated in the transition of MM to PCL can be classified as either tumor suppressor genes, genes involved in cell-cell signaling, or as cell adhesion molecules. The further analysis of these genes will allow us to identify genes which are down-regulated through methylation and mediate the progression of MM to PCL allowing the clone to become independent of the bone marrow microenvironment. In addition, these analyses will enable us to identify targets which may be sensitive to modulation by epigenetic therapies in vivo. In conclusion, we have demonstrated that methylation changes play a significant part in differentiating the various cytogenetic subgroups of MM and in mediating the transition to PCL. Hypermethylation affects genes and pathways important in retaining plasma cells in the bone marrow as well as in their growth factor independent growth in the absence of stromal cell support. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4558 Myeloid Sarcoma (MS) is a rare malignant disorder usually presenting as an extramedullary myeloid cell tumor proliferation. MS can be observed in 3 to 7% of AML cases (called “leukemic” MS, LMS hereinafter). MS prognosis is rather poor with overall survival (OS) not exceeding 2 years. We have previously shown, that allo-SCT following an AML-like chemotherapy is likely the best available therapeutic option for patients with LMS (J Clin Oncol 2008). However, it is still unclear whether allo-SCT is a valid therapeutic option for patients with isolated MS (IMS). The aim of this analysis was to assess the role of allo-SCT in patients with IMS as compared to patients with LMS. We retrospectively analysed the outcome of 99 MS patients with a median age of 40 years (range: 18–70), including 30 cases of IMS and 69 cases of LMS, reported to the EBMT registry between January 1991 and June 2009. The majority of patients were in complete remission (CR) at time of transplant (IMS: first CR n=14, second CR or beyond n=7 vs. LMS: first CR n=38, second CR or beyond n=21, P=0.20). In this series, patients received either a standard (IMS, n=24 vs. LMS, n=55, P=NS) or a reduced-intensity conditioning regimen (IMS, n=6 vs. LMS, n=14, P=NS) prior to allo-SCT from a matched-related (IMS n=21 vs. LMS, n=48, P=NS) or unrelated donor (IMS, n=9 vs. LMS, n=21, P=NS). After allo-SCT, engraftment was observed in all patients. The incidences of grade 2–4 and grade 3–4 acute GVHD were 32% and 9%, respectively. The incidence of chronic GVHD at 2 years was 45%. With a median follow-up of 48 (range: 6–213) months, the 5-years OS, and leukaemia-free survival (LFS), and the cumulative incidences of relapse (RI), and non-relapse mortality (NRM) were 48%, 36%, 40% and 19%, respectively. When comparing the two sub-groups (IMS vs. LMS), there were no significant differences in term of outcomes: neutrophils recovery (15 vs 18.5 days), grade 2–4 acute GVHD (27% vs 35%), chronic GVHD (54% vs 41% at 2 years), 5-year OS (33% vs. 48%), 5-year LFS (30% vs. 38%), 5-year RI (45% vs. 38%) and 5-year NRM (19% vs. 17%). In all, we conclude that allo-SCT is an effective treatment for patients with MS. The current data suggest that patients with isolated or leukemic MS have similar outcome after allo-SCT. Therefore, allo-SCT is likely to be considered as the optimal therapy for both isolated and leukemic MS. Disclosures: Tilly: Amgen: Honoraria.

Description: Abstract 4560 Introduction: About 15% of acute myelogenous leukemia (AML) patients will have disruption of the core binding factor (CBF) transcription factor as indicated by the detection of t(8;21) or inv(16) on metaphase cytogenetic analysis. In general, patients with CBF-AML are younger and regarded as having a favorable outcome when treated with anthracycline-based induction chemotherapy and multiple cycles of high-dose cytarabine consolidation. Autologous and allogeneic hematopoietic stem cell transplantation (HSCT) are usually perceived as unnecessary in the management of CBF-AMLs. However, most data indicating the favorable prognosis of CBF-AML with non-HSCT management originates from young selected patients participating in clinical trials and information on relapse treatments is often lacking. We performed a retrospective analysis of consecutive patients diagnosed with CBF-AML at a regional leukemia center over the last twelve years to assess long term survival of unselected patients and the likelihood of therapeutic success without ever requiring HSCT. Methods: The study cohort was identified through a search of all AML reports issued by the institutional clinical cytogenetics laboratory since the introduction of electronic records (1998-2010). We subsequently reviewed the charts of all patients with CBF-AML (irrespective of other cytogenetic abnormalities) to extract demographic, treatment, and outcome information. Survival status and subsequent treatments in other institutions were determined by contacting the patient, attending physician or review of public death records. Data collection and analysis were approved by the Institutional Review Board. Results: Thirty patients with CBF-AML were identified, 14 with t(8;21) and 16 with inv(16) AML. Median follow up for survivors in this series is 35.3 months (range 6.4–117.4) with all survivors currently in remission. Median age at diagnosis was 43 years (range 18–69) with 10 (33%) patients older than 55. All patients initially received therapy with curative intent with anthracycline-based induction chemotherapy. Only 47% of the patients were treated on clinical trials. There was 1 death during induction therapy and 22/29 patients (73.3%) achieved a complete remission with initial induction therapy. Overall 13 patients (43.3%) have required some modality of HSCT. Six patients received HSCT in CR1 due to perceived high risk of relapse (5 autologous, 1 allogeneic). None of the 6 patients who underwent HSCT in CR1 (5 autologous, 1 allogeneic) have relapsed. Among the 16 patients receiving non-transplant consolidation in CR1, 6 have subsequently relapsed and required a HSCT (3 autologous, 3 allogeneic) and 3 of these patients are alive 20.9–117.4 months from the initial diagnosis. Seven patients received HSCT in CR2 or in relapse (4 autologous, 3 allogeneic) and 6 of these patients are long-term survivors. Estimated 5 year overall survival for the entire cohort is 58.8 +/− 10.8%, comparable to what has been described for younger patients entering clinical trials (Grimwade et al, 2010). However, the likelihood of survival at 5 years without requiring HSCT was only 28.1+/− 8.8% (Figure). Conclusions: The favorable outcome previously reported for CBF-AMLs was reproducible in unselected patients. However, only a minority of patients were long term survivors relying exclusively on conventional chemotherapy. In the near future, strategies for molecular risk stratification of CBF-AML patients need to be coupled with risk-adapted therapy, likely including early use of HSCT for high-risk patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4546 Hemorrhagic cystitis (HC), occurring mostly within first month after allogeneic hematopoietic stem cell transplantation (allo-HSCT), is a common complication which often requires prolonged hospitalization, considerable expense, and occasionally causes significant morbidity. The data from different transplantation centers about this complication are different in the incidence, risk factors, therapeatic strategies and outcome. In this study, we retrospectively analyzed the incidence, risk factors, prophylaxis and treatment regimens of HC after unrelated donor HSCT(URD-HSCT)in our center From October 2000 to February 2008, 168 consecutive patients underwent URD-HSCT were enrolled,including 113 male patients and 55 female patients. The median age was 26 years (range,8-52 years). The main myeloablative conditioning regimens used were busulfan/cyclophosphamide (BuCy) without total body irradiation (TBI); Reduced-intensity conditioning regimens (RIC) were predominantly fludarabine-based combinations without irradiation. Anti-thymocyte globulin(ATG) were added to the conditioning regimen in the patients receiving HLA-mismatched URD-HSCT. The prophylaxis regimen for HC included intravenous mesna,hyperhydration and intermittent diuretic. The diagnosis of HC was based on the presence of sustained microscopic or macroscopic hematuria in the absence of other clinical conditions such as urinary bacteria and fungi infection. It could be clinically graded to I to IV according to hematuria and divided into early onset (EOHC) and delayed (LOHC) according to the time of onset. In total, 27 of 168 patients (16.1%) developed HC at a median interval of 40 days post engraftment (range, 8–89 days). Among them,11 patients developed grade I (6.5%), 10 patients developed grade II (6.0%), 6 patients developed grade III (3.6%), while no patients with grade IV. Five patients(2.98%) presented EOHC and 22 patients (13.17%) presented LOHC. We discovered that in 27 patients with HC only 3 were female (11.1%), however in 141 patients without HC, 52(36.9%) were female (P

Description: Abstract 4556 A 37 year old male with T cell acute lymphoblastic leukemia with normal cytogenetics 46XY in second complete remission (CR2) after 8 cycles of HyperCVAD and 4 cycles of salvage nelarabine underwent myeloablative conditioning with cyclophosphamide TBI for a matched related donor allogeneic stem cell transplant from his brother. His day+100 bone marrow biopsy showed CR2 and was 100% donor. His post transplant course was unremarkable until he relapsed d+162. The immunosuppression was tapered off rapidly and 2 cycles of nelarabine and a donor lymphocyte infusion (1×107 CD3 cells/kg) were completed by d+254 and CR3 was achieved. He did well until d+608 when he presented with a month long history of dyspnea and recurrent bilateral large pleural effusions (despite multiple large volume thoracenteses) and anasarca including facial edema. He had gained 15 kilograms in 2 weeks. He had no fever, skin rash, sicca syndrome or lichen planus. Chest and abdomen CT showed massive bilateral pleural effusions, but no lung infiltrate, pericardial effusion or ascites or lymphadenopathy. The complete blood count, renal function and liver function tests were normal and there was normal hepatopedal flow in the main portal vein and its branches. The echocardiogram showed a left ventricular ejection fraction of 60% (normal) and there were no vegetations. The serum ANA, anti-Ro and anti-La were negative. The bone marrow biopsy showed no evidence of relapse of T cell ALL. The peripheral blood STR was 100% donor. The pleural fluid was transudative and cytology showed no malignancy. Pleural fluid flow cytometry showed no CD20+ B cells but did show polyclonal T cells with a CD4:CD8 ratio of 0.22. The pleural biopsy showed reactive mesothelial cells with mixed acute and chronic inflammation and no malignancy. Ebstein Barr virus in-situ hybridization and HHV-8 immunohistochemistry was negative on the pleural biopsy. All bacterial, fungal and viral (including CMV, EBV, HSV, adenovirus, VZV, HIV, BK) of the blood, urine and pleural fluid were unremarkable. A random skin biopsy of the forearm showed scleroderma. Methylprednisolone 2 mg/kg iv daily and therapeutic tacrolimus with extracorporeal photoporesis was begun with resolution of the pleural effusions and anasarca two months later. On d+903 he remains in CR3 and is on tapering courses of prednisone and tacrolimus without recurrence of the pleural effusions or anasarca. Conclusion: Isolated sterile pleural effusions with anasarca should be recognized as a rare feature of late onset chronic graft versus host disease so as to allow prompt initiation of immunosuppression. The patient shared the HLA antigens HLA DR15 and HLA DQW6 which are associated with autoimmune disease such as systemic lupus erythematosus suggesting that patients with this HLA combination may be more prone to this presentation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4542 Immunosuppressive treatment is widely used, especially after allogeneic stem cell transplantation (HSCT) to prevent or treat graft versus host disease (GvHD). Common drugs are Ciclosporin A or Tacrolimus in combination with steroids. However, immunosuppressive treatment and the underlying conditions are associated with an increased risk of viral reactivations with persistent pathogens like cytomegalovirus or adenovirus. In the absence of a protective immune response, virus infection remains a life-threatening complication after HSCT. Here we investigated the antiviral T-cell response (n=12) after ex vivo exposition with Ciclosporin A, Tacrolimus, Prednisolone or Mycophenolate at time of immunosuppression, 24 hours and 72 hours later. Analysis has been done with IFNgamma Elispot assays, confirmed by intracellular cytokine staining in flow cytometry and analysis of T-cell proliferation detected by CFSE. The antiviral T-cell response is suppressed after 24 hours using normal serum concentrations (100-200ng/ml) of cyclosporine A. T-cell annergy, induced by cyclosporine, could be reversible, after 72 without immunosuppression. Tacrolimus has a stronger immunosuppressive effect on T-cell activation within the same time and even low levels of 1ng/ml induce T-cell suppression after 72 hours. Peak levels of calcineurin inhibitors even suppressed the T-cell response to superantigens like staphylococcal antigen B or PHA. As expected, Prednisolone had a short and dose dependend effect on T-cell activation. Mycophenolate has a mild effect on the activation of virus-specific T-cells. However, all three drugs induced a significant reduction in Ag-specific T-cell proliferation. In conclusion, Interferonγ detection in virus-specific T-cells is a good diagnostic tool for clinicians to monitor the risk of viral complications in immunosuppressed patients. Tacrolimus, Cyclosporin A, Prednisolone and Mycophenolate induce an activation defect in Ag-specific T-cells with decreasing severity. The effect is reversible and corresponds to high or low serum levels. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4530 We present the case of a 34 year old African American male with T-cell non-Hodgkin's Lymphoma who received a matched related allogeneic transplant using a preparative regimen of cyclophosphamide and TBI from his brother on 11/4/09. The patient's blood type was O+ and the donor's blood type was B+. The cell dose was 10.3 × 106 CD34/kg. His post transplant course was generally uncomplicated. Although he recovered his ANC on D+18 and he was platelet transfusion independent by D+28, he never became red cell transfusion independent. A bone marrow biopsy on day 33 showed no evidence of disease with minimal erythroid recovery. His engraftment studies were all 100% donor cells. His reticulocyte counts are detailed in the table below. On day 126 he had a positive qualitative PCR for parvovirus. He received IVIG (400 mg/kg) for 5 consecutive days beginning 3/15/10. Weekly surveillance for CMV by PCR remained non-detectable. Repeat bone marrow biopsies on day 63 and 88 continued to show neutrophil engraftment, hypoplastic megakaryocytes and aplasia of red cells. His immune suppression was discontinued on day 99. A fourth bone marrow biopsy on day 155 again showed erythroid hypoplasia with neutrophil and megakaryocyte hypoplasia. He received a second infusion of PBSC without a conditioning regimen. He received 2.5 × 106 G-CSF mobilized HPC-A from the original donor on day 205 post transplant. Tacrolimus was reinitiated on day 203. As noted in the table below, his reticulocyte count normalized three weeks post infusion. His last RBC transfusion was given two days prior to the stem cell infusion and he remains transfusion independent today. He continues on tapering doses of tacrolimus and has not experienced any graft versus disease.DateT+WBCHgbPLTANCreticParvo PCR12/7/09336.68.8632.412/9/09356.17.8692.50.68%negative2/25/101131.98.8691.10.10%3/10/101262.18.50.4positive3/25/101423.59.1732.20.10%negative4/1/101492.48.0491.2negative5/25/102032.77.0871.50.10%6/18/10227/233.18.31131.96.10%6/25/10234/303.68.41172.25.20% Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4551 The diagnosis of intrapulmonary nodules in patients with haematological malignancies is problematic as such lesions are often small and impalpable and “non-targeted” surgical biopsy is difficult. The aim was to evaluate the utility of image-guided “targeting” of small pulmonary nodules with methylene-blue before video-assisted thoracoscopic (VATS) biopsy. Eleven patients (8M:3F) with median (range) age of 48 (27-62) years with haematological malignancies (lymphoma, n=6, AML/MDS, n=3, ALL, n=1,Castleman's disease, n=1) were referred for VATS biopsy. Equal volume of iodinated contrast) was injected in the vicinity of the target lesion and along a track (including the pleural surface and the overlying chest wall), using a 20G needle. The platelet count, diameter of targeted nodules, “perpendicular” distance from the pleural surface and complications were recorded. Patients were transferred to surgery from the CT suite. The median (range) platelet count was 256×109/L (54-453). The mean (range) diameter of targeted nodules was 12.5(5-22) mm and these were at a mean distance of 14.3±8.3mm from the pleural surface. Complications included small pneumothoracis in 4/11 (36%) patients and pain in 1/11 (9%). A definitive histopathological/microbiological diagnosis was achieved in 10/11 (91%) patients and included: organising pneumonia (n=4), respiratory bronchiolitis (n=2), Kaposi's sarcoma (n=1), mycobacterium fortuitum infection (n=1) and chronic GVHD (n=1). There were no cases of angioinvasive aspergillosis. This preliminary data suggest that pre-biopsy methylene-blue targeting of intrapulmonary lesions is a safe and promising technique for the diagnosis of indeterminate lung nodules in patients with haematological malignancy. Disclosures: Mufti: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Description: Abstract 4525 High dose therapy and autologous transplantation is considered a standard of care procedure in symptomatic, transplant eligible multiple myeloma patients. The chemotherapy of choice in this situation is high dose melphalan. The later has marginal solubility and limited chemical stability upon reconstitution and dilution. The marginal stability of this compound has limited the use of regimens utilizing higher absolute dosages and/or longer infusions (〉 60 minutes) that could potentially lead to improved outcomes. Accordingly, co-solvents are used in the marketed formulation, which are believed to contribute to the side effects of the therapy. A co-solvent used in Alkeran for Injection is propylene glycol, which has been reported to cause renal dysfunction, arrhythmias, hyperosmolality, increased anion gap metabolic acidosis, and sepsis-like syndrome. On the other hand, Melphalan HCl for Injection (Propylene Glycol-Free), a reformulation of Alkeran for Injection, incorporates the Captisol® brand of β-cyclodextrin sulfobutyl ethers, sodium salts (also known as [SBE]7m-β-CD) into a freeze-dried product developed by CyDex Pharmaceuticals, Inc. (CyDex). Captisol improves stability allowing for potentially longer infusion times. Study Design/Goals: In this phase IIa, open-label, randomized, cross-over design, the PK of Melphalan HCl for Injection (Propylene Glycol-Free) and Alkeran for Injection are assessed in the same MM patients undergoing transplantation. Furthermore, the rates of myeloablation and subsequent engraftment are determined, and any difference in expected safety and tolerability due to high-dose Melphalan HCl for Injection (Propylene Glycol-Free) is assessed in transplanted patients. Results: Seven patients of the planned 24 patients have already enrolled in the study at the University of Kansas Medical Center. All patients achieved myeloablation followed by successful engraftment. Median time to myeloablation, defined as number of days from the start of chemotherapy until absolute neutrophil count dropped below 500/Ul, was 6.4 days (range 5–7 days). Median time to neutrophil engraftment, defined as first day of three consecutive days where ANC was higher than 500/Ul following their nadir, was day +9. Beside expected grade 2–3 toxicities related to high dose melphalan, no additional toxicities were reported. In specific, no renal insufficiency was noted. Preliminary PK analysis has been performed on five patients. Preliminiary parameter estimates (Mean± SEM) are presented in the Table. The mean bioavailability was 113%. The mean half-lifes for the Melphalan HCl for Injection (Propylene Glycol-Free) and Alkeran for Injection were 70 and 75 min. Following Melphalan HCL for Injection (PropyleneGlycol-Free) and Alkeran for Injection, the distribution volumes were 0.23 and 0.35 L/kg and clearances were 0.29 and 0.34 L/hr per kg, respectively. Conclusion: Melphalan HCl for Injection (Propylene Glycol-Free), administered as half of a high-dose conditioning regimen, appears to result in successful myeloablation and subsequent engraftment at no extra-toxicity, and trending toward higher bio-availability compared to Alkeran. Future studies that are designed to expose patients exclusively to the propylene glycol-free formulation will delineate potential safety and efficacy advantages over the current therapy.ProductN=Cmax (μ g/mL)t½α(min)t½ β(min)Vc (L)Vdss (L)AUC (μ g* min/mL)CL (mL/min)Melphalan HCl for Injection55.5 ± 2.112.9 ± 2.769.6 ± 2.825.5 ± 11.042.0 ± 15.4424 ± 146529 ± 187Alkeran54.4 ± 2.624.2 ± 19.774.8 ± 14.039.3 ± 25.752.3 ± 22.1375 ± 167631 ± 209 Disclosures: Aljitawi: CyDex Pharmaceuticals, Inc.: Research Funding. Off Label Use: Melphalan use in high dose therapy and autologous transplantation. Robinson:CyDex Pharmaceuticals, Inc.: Consultancy. Pipkin:CyDex Pharmaceuticals, Inc.: Employment.

Description: Abstract 4508 Long-term follow-up data of the patients who underwent HSCT more than 2 years ago at a single institution in Japan was presented. The patients who received first allogeneic hematopoietic stem cell transplantation (HSCT) at Tokyo Metropolitan Komagome Hospital between January, 1990 and December, 2007 under hematology division were included. Follow-up data were obtained annually from the medical records if the patients visited our institution regularly. If the patients were not followed in our institution, follow-up data were requested to the hospitals where the patients had been followed or questionnaires were sent directly to the patients unless their addresses are not known. These letters were sent annually. The most recent laboratory data and any kind of complications under treatment including hypertension, hyperlipidemia, diabetes including usage of insulin, renal failure, dialysis, and any kind of malignancies were requested to be reported. Survival was the most primitive data and the reason of death was defined by one of the authors, HA. Chronic GVHD was excluded from the independent etiology of death. Progression of the disease or complications of the treatment for the progressive disease were considered to be due to relapse. The incidence of each complication was calculated using the number of the patients whose data are available. In total, 622 patients had received transplantation and 370 patients survived more than 2 years. During last two years, 211 patients had been followed in our institution while 74 patients by the institutions outside. Letters were sent directly to rest of surviving patients. As the result, 6 patients could not be reached by any method. Letters had been received without response in 15 patients. 72 patients died later than 2 years after transplantation. Relapse was the most important reason of death even more than 2 years after the transplantation. Although the incidence declined annually, the latest relapse was observed in the patient with CML almost 15 years after the transplantation. Pulmonary complications including bronchiolitis obliterans and infections followed. Secondary malignancy was the reason of death in 7 patients. Chronic kidney disease was already observed in 27 % of the patients who survived 2 years after transplantation and one of the devastating complications. In total of 7 patients needed to start regular dialysis or kidney transplantation and another 2 patients showed eGFR level of less than 15 ml/min/m2. Nephrotic syndrome was another renal complication observed in 4 patients. Hypertension was reported on 46/244 (19%) of the patients. Diabetes was reported on 27/241 (11.2%) and 13 of them were on regular insulin treatment. Definitive diagnostic criteria of diabetes were not indicated on this analysis suggesting higher incidence of glucose intolerance in these patients. There were 14 patients developed secondary malignancies diagnosed later than 2 years after transplantation and oral mucosa, tongue, esophagus and colon were the main organs involved. Physicians taking care of those patients were recommended to check kidney function and gastrointestinal tract including head/neck, esophagus and colon for secondary malignancy, as well as hematological status. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4522 Background: Conditioning regimens utilized to prepare patients for stem cell transplantation uniformly result in suppression of marrow function, leading to prolonged recovery of neutrophils and platelets. Neutropenia increases the risk of infection while thrombocytopenia and prolonged platelet engraftment time can result in increased risk of bleeding as well as morbidities associated with subsequent transfusions. The variations in duration of cytopenias are affected by multiple factors including regimen intensity and other clinical factors. Methods: In this retrospective study of consecutive patients undergoing stem cell transplantation at the University of Rochester between 2005 and 2009 for radiation—containing regimens, and between 2007 and 2009 for non-radiation containing regimens, we assessed the relationship between platelet engraftment times and source of stem cells, type of graft, and the effect of conditioning regimen intensity. Platelet engraftment was defined per standard CIBMTR definitions – platelet engraftment occurred at the first of 3 days of platelet count 〉20,000/μ L without platelet transfusions for 7 days, and/or the first day of platelet count 〉100,000/μ L. Guidelines for transfusion support stipulated a 10,000/μ L threshold for platelets in the absence of bleeding and a hemoglobin of 8.0 g/dL for red cells in the absence of other symptoms. The time to platelet engraftment and the number of platelet transfusions required prior to engraftment were analyzed in those patients with platelet nadir

Description: Abstract 4510 Recently the increasing number of cases of solid malignancies developed in recipients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) has attracted more attention while less donor-derived solid malignancies after allo-HSCT have been found. We describe here a case of donor-derived tongue squamous cell carcinoma (SCC) in a young adult patient who had undergone allogeneic peripheral stem cell transplantation for acute leukemia 2 years ago. The female patient had a history of acute lymphoblastic leukemia (ALL-L2) at 20 years of age in July 2004 and received a successful busulfan and cyclophosphamide (BU/CY2) myeloablative allografting (allogeneic peripheral blood stem cells) from a HLA-matched unrelated male donor in August 2006. Acute graft versus host disease (GVHD) prophylaxis consisted of cyclosporine A 2.5 mg/kg/day, methotrexate 10 mg on day 1,3,6, and mycophenolate mofetil 0.5 g/day. However, on day 12 the patient developed skin grade II acute GVHD. Subsequently, she complicated with intestinal grade II acute GVHD characterized by recurrent diarrhea on day 49. 2 mg/kg/day of methylprednisolone and 4 mg/kg/day of cyclosporine A were given. 7 days later acute GVHD was controlled gradually. After that, grade II chronic GVHD with skin rash and oral mucositis accompanied by leukoplakia occurred after 5 months post engraftment. Prolonged immunosuppressive agents containing prednisolone and cyclosporine A were administered. In April 2008 (20 months post engraftment), the patient presented with symptoms of pain in the tongue which worsened in the following 2 months. A firm mass was seen in the tongue which was highly suspicious for malignancy. The histology of the puncture biopsy showed a well-differentiated SCC. The neoplastic cells were pleomorphic and prominent nucleoli. Cell nests and keratinous pearls could be seen clearly. Then she was treated with mass excision in addition to supraomohyoid neck dissection immediately. At the same time peripheral blood STR analysis showed full donor's chimerism, and bone marrow aspiration as well as minimal residual disease with FACS indicated complete remission of the primary disease. The patient is now under follow-up with no recurrence or metastasis. Further investigation of the tissue specimens by fluorescent in situ hybridization (FISH) (X,Y probes) combined with double-SP immunohistochemical staining (CD45, Pan-cytokerin) revealed the presence of one Y chromosome-specific signal and one × signal within most of the neoplastic cells which was identified as pan-cytokerin (+), CD45(-) cells and this suggested that donor-derived stem cells might transform into malignancies in the recipient by several potential mechanisms (Hu YX et al, Onkologie 2010). Till now about 13 cases developing donor-derived solid malignancies including oral SCC (Janin A et al, Blood 2009; Tomihara K et al, Head Neck 2009), colonic adenoma (Cogle CR et al, Stem cells 2007), breast adenoma (Golfinopoulos V et al, Breast Cancer Res Treat 2009), gastric adenoma (Arai Y et al, AM J Transplant 2006), larynx squamous cell carcinoma (Avital I et al, Stem cells 2007), Kaposi's sarcoma (Avital I et al, Stem cells 2007), lung adenoma (Avital I et al, Stem cells 2007) and glioblastoma multiforme (Avital I et al, Stem cells 2007) have been reported post-HSCT. The presence of donor-derived solid malignancies suggests that human bone marrow-derived stem cells have a role in the carcinogenesis of solid organ cancer especially in the organ with inflammation. But which kind of bone marrow derived stem cells transformed into solid malignancies and its mechanisms need further investigation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4503 Background&Objective: The management of lymphoma which has relapsed after allogeneic hematopoietic cell transplantation (HCT) is difficult. Therapeutic options may include donor lymphocyte infusion (DLI), withdrawal of immunosuppression, rituximab, chemotherapy, radiation, immunotherapy and experimental treatments, but response and survival are uncertain. Result: We analyzed 72 patients with relapsed or progressive lymphoma after allogeneic HCT (1991-2007); 44 non-Hodgkin's lymphoma (7 mantle cell, 5 indolent, 19 diffuse large B [DLBCL], and 13 T/NK cell) and 28 Hodgkin's lymphoma (HL). At HCT, 62 patients (86%) were in remission (22 CR, 40 PR); nine (13%) had progressive disease and one had stable disease. Conditioning was myeloablative (n=17) or reduced intensity conditioning (RIC) (n=55). Relapse or progression occurred at a median of 99 days (0-1898 days; 25–75% 43–194 days). At relapse, 41 (57%) had extensive nodal disease and 56 (78%) had extranodal organ involvement. Management of relapse included: no therapy (n=5, 7%), reduction of immunosuppression (RIS) (n=58, 81%), chemotherapy alone (n=14, 19%), immunotherapy alone (n=20, 28%), combined chemo-immunotherapy (n=7, 10%) donor lymphocyte infusion (DLI) (n=7, 10%), second allogeneic HCT (n=2, 3%), radiation (n=23, 32%) and other therapy (n=7, 8%). Twenty-four patients achieved a remission (15 CR, 9 PR) to the initial salvage therapy: RIS (n=13), combined chemo-immunotherapy(n=5), chemotherapy alone (n=2), radiation (n=3) or DLI (n=1). Overall, 40 (56%) patients responded (30 CR, 8 PR, 2 stable disease) with a median post-relapse overall survival of 28 months (range 0–147 months). At 3 years following relapse, 44% (95% CI, 32–56%) survive. Favorable prognostic factors for improved 3-year survival after relapse include stage I-III at relapse, single site of relapse and most importantly, later relapse (〉100 days after HCT [3 year survival 53% vs. 36%, p=0.02]). Conclusion: The overall prognosis of relapsed lymphoma after allogeneic HCT differs based upon extent of disease at relapse and time of relapse. Appropriate therapeutic approaches (RIS, combined chemo-immunotherapy, radiation or DLI) in these high risk, post transplant relapsed patients can yield promising outcome. Prospective study of post-relapse therapy to determine the most effective management strategies are warranted. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4498 Background: Chronic Myeloid Leukemia(CML) is an myloproliferative disorder characterized by the expansion of clone of hematopoietic cells that carries the Philadelphia chromosome(Ph).The Ph chromosome results from a reciprocal translocation between the long arms of chromosome 9 and 22,t(9;22) (q34;q11).The molecular consequence of this translocation is a fusion gene. Imatinib inhibits constitutively active BCR-ABL tyrosine kinase of Chronic Myeloid Leukemia (CML).This is a novel molecule, which inhibits the protein product of this fusion gene and hence has been used in the treatment of CML. We are now report 51 months of median follow-up data and focus on 645 number of patients who received Imatinib as a primary treatment from 2002 to 2010. Materials and Methods: We included total 645 patients of CML during the period of January 2002 to June 2010.The age range was 02–87 years with median age being 36±11.6 years. Among 645 patient 366 was male and 280 female. A total 645 patients 608was in chronic phase, 31 in accelerated phase and 5 patients had blast crisis at the time of presentation. At present the molecular status of the disease has been detected by RT PCR, Flowcytometry (By Fluorescence Activated Cell sorter, FACS), Karyotyping and FISH.As a runtime protocol we have used Imatinib as 400 mg/day as standard dose at chronic phase, 600 mg/day in accelerated phase and 800 mg/day in blast crisis.We have studied a comparative analysis of Hematological parameter values. Result: The best observed average rate of complete hematological response was 97%, complete cytogenetic response was 50%, complete molecular response 35% with partial response in 45% and poor response in 15%.After 1 year levels of BCR-ABL transcripts had fallen in 52% patients and after 4 years levels had fallen in 79%. Conclusion: At 51 months of median follow-up in our study, comparative analysis of haematological parameters like haemoglobin, Total Count, Platelet Count between patient and normal population revels that pre-treatment value against normal value is very significant. The post treatment value of the haematological parameters is very close to the normal values and hence it can be proved that Imatinib therapy is effective. We observed that Imatinib drug is tolarence in case of 90% patients and resistance in about 10 % patient. So, we can conclude that Imatinib is an effective and safe drug for CML patient. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4494 Background: Although imatinib could induce efficacious and stable responses in patients with chronic myeloid leukemia (CML), resistance is increasingly problematic. Dasatinib, a BCR-ABL inhibitor with 325-fold greater potency in vitro than imatinib, induced MCyR 59%, CCyR 48%, PFS 90% and OS 96% for patients who can not tolerate or are resistant to imatinib in START-C. Method: We assessed retrospectively the efficacy and safety of dasatinib in patients with chronic phase (CP)-CML, resistant or intolerant to imatinib who received dasatinib 70 mg to 140 mg per day. Dose was adjusted according to toxicity. Result: Between 21 June 2005 and 31 March 2010, medical records of 47 patients from 6 centers in Korea were reviewed: 22 with imatinib-resistant and 1 with imatinib-intolerant CP-CML. 8 Imatinib–resistant ABL kinase domain mutations were found including E255K, T315I, F317L. Median duration of dasatinib therapy was 20.6 months. CHR, MCyR, CCyR and MMR was attained in 91%, 79%, 56% and 44% of patients, respectively. 18 month MMR rate was 71%, 67% in imatinib-intolerant and 53% in imatinib-resistant group. There was no difference in PFS according to the Sokal score, to the best response of imatinib to the type of mutation. 3-year PFS was 71% and OS was 79% with a median follow up of 20.6 months. There was no disease progression or death in imatinib intolerant group. Grade 3/4 anemia, neutropenia and thrombocytopenia were reported in 36%, 49% and 45% of patients, respectively. Non-hematologic toxicity (grade 3/4) consisted of infection(15%), dyspnea(4%), pleural effusion(6%), abdominal pain(2%) and skin rash(2%). Conclusion: Dasatinib showed promising efficacy and tolerability in imatinib-resistant or -intolerant CP-CML in Korean patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4575 Introduction: Anti-HLA antibodies constitute potentially important factor that may influence outcomes of HLA-mismatched allogeneic hematopoietic stem cell transplantation (allo-HSCT). The rationale of this study was to detect presence of anti-HLA antibodies in recipients of allo-HSCT from HLA-mismatched unrelated donors. Patients and Methods: Anti-HLA-A,B,C,DR,DQ,DP antibodies were identified in sera collected from 46 recipients of allo-HSCT from HLA-mismatched unrelated donors. Sera were collected between 1 month and 5.5 years after allo-HSCT, and additionally before allo-HSCT in 17 pts. We have used microchips spotted with purified HLA class I and HLA class II antigens to allow binding of anti-HLA antibodies present in tested sera to the surface of the microchip, pre-optimised reagents and DynaChip Processor (Dynal Invitrogen Corporation) for assay processing, data acquisition and analysis. Results: Antibodies against HLA class I, II or I and II were detected in 15%, 11% and 35% of pts whereas no antibodies were detected in 39% of patients. Antibodies were directed against HLA-A, B, C, DR and DQ in 37%, 46%, 35%, 48% and 35% of pts, respectively. Pre-transplant anti-HLA antibodies have been detected in 7 pts (41%) out of 17 tested before allo-HSCT. In this group percent of Panel Reactive Antibodies (% PRA) increased following allo-HSCT in 3 pts and decreased in 4. In 5 out of 10 remaining pts without pre-transplant antibodies, %PRA increased post-transplant. DynaChip software allowed to define specificities of HLA-A,B,C,DR and DQ antibodies on low and high resolution levels. The specificity of antigens that masked results of antibody identification has been also defined in 2 pts. At this stage we did not define exactly whether detected anti-HLA antibodies were donor-specific. Cross-reactive groups (CREG's) analysis has been also used to compare antibodies’ reactivity. Anti-HLA-DP antibodies were not detected in the examined group of transplanted patients. Conclusions: Presented preliminary study results indicate, that anti-HLA antibodies can appear post-transplant in mismatched allo-HSCT recipients. Further analysis aiming to evaluate their influence on transplant outcomes is ongoing. We intend to extend the search for anti-HLA antibodies with use of Luminex LabScreen method. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4496 The multikinase BCR/ABL inhibitor dasatinib exerts major growth-inhibitory effects in imatinib-resistant patients with chronic myeloid leukemia (CML). Although previously reported to be a well-tolerated drug, several side effects such as pleural and pericardial effusion have been reported at a dose of 140 mg daily. Therefore, the approved standard dose of dasatinib was reduced to 100 mg daily. More recent data suggest that freshly diagnosed patients with CML treated with dasatinib at 100 mg once daily may still develop pleural effusions, but the frequency of this side effect was reported to be lower compared to patients receiving 140 mg daily dasatinib. Moreover, effusion formation was reported to be less severe, with almost no grade 3/4 events, in patients receiving 100 mg daily dasatinib. We examined a cohort of CML patients (n=13) treated with dasatinib at 100 mg or 50 mg daily, for development of severe non-hematologic side effects. Patients received dasatinib for at least 3 months (range: 3–38 months; observation period: March 2005 to June 2010). Of these 13 patients, 4 had previously received 140 mg dasatinib daily. In all 4 patients, the dose of dasatinib was reduced to 100 mg or 50 mg daily because of effusion formation. In the other 9 patients, the initial dose of dasatinib was 100 mg daily. Of these 9 patients, 2 patients in chronic phase (CP) received dasatinib as frontline therapy. Four of our 13 patients developed severe effusions (grade 3/4) while on dasatinib at 100 mg or 50 mg daily, including one CP patient receiving dasatinib as frontline therapy. In two patients, massive pleural effusions were recorded, and two developed pericardial effusion together with pleural effusions. In two patients, effusion formation required paracentesis and surgical intervention. No pre-existing cardiac or pulmonary diseases were known in these 4 patients. In 3 patients, dasatinib had to be discontinued despite treatment with low dose glucocorticosteroids and diuretics. After switching to an alternative BCR/ABL inhibitor, no further effusion formation occurred. In summary, dasatinib-treated patients are at risk for the development of pleural and pericardial effusions even when the drug is administered at a dose of 100 mg or 50 mg daily. Therefore, all patients should be examined carefully for pre-existing relevant comorbidities and cardiovascular risk factors before dasatinib is started, and all patients should have repeated chest × ray controls during long-term treatment with dasatinib. Disclosures: Valent: Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding.

Description: Abstract 4490 Background: Imatinib is currently standard therapy for patients with success in Chronic Myeloid Leukemia (CML). Several instances of cardiac adverse events have been reported for patients while on therapy with imatinib. In many instances, these events occur in patients with pre-existing cardiac conditions. The aim of our study was to determine the occurrence of cardiac events in patients with CML treated with Imatinib, and the impact that cardiac risk factors and pre-existing cardiac conditions had on the risk of developing cardiac adverse effects. Methods: We reviewed the medical records of 51 patients with chronic phase CML who were treated with imatinib after failing prior therapies. For each patient we collected cardiac risk factors, pre-existing cardiac disease, pre-treatment EKG and echocardiogram (ECHO) readings, as well as post-treatment changes in EKG and ECHO findings. Results: Pre-existing cardiac conditions were found in 14 (27%) patients, including congestive heart failure in 2 (4%), myocardial infarction in 4 (8%), atrial fibrillation in 1 (2%), benign arrhythmias in 1(2%), aortic regurgitation in 1(2%), mitral valve prolapse in 1 (2%), mitral regurgitation in 1(2%), pericarditis in 1(2%), bradycardia in1(2%) and benign arrhythmia in 1(2%). Cardiac risk factors were present in 26 patients (51%), including smoking in 10 patients (20%), hypertension in 17 (33%), diabetes mellitus in 9 (18%), obesity in 2 (4%), hyperlipidemia in 5 (10%), stress (self-reported by patient or on anxiolytic therapy) in 5 (10%), alcohol in 11 (22%), atherosclerosis in 3 (6%), and positive family history for cardiac disease in 5 patients (9.8%). Cardiac events were noted in 19 patients (37%) of whom 11 (58%) had pre-existing cardiac conditions prior to initiating imatinib treatment and 14 patients (27%) had at least one cardiac risk factor. Congestive heart failure with clinical manifestations was seen in 9 patients (18%) with documentation of decreased ejection fraction on echocardiogram seen in 3 patients (6%) who had a decrease in LVEF from a median of 55% (range 50% to 72%) to a median of 45% (range 25% to 60%). Out of the patients who developed CHF while on treatment with imitanib, 3 patients (6%) had history of cardiac conditions (atrial fibrillation in 1 (2%), congestive heart failure in 2 (4%)). Myocardial infarction was documented in 3 patients (6%), one of which had prior myocardial infarction and pacemaker, another had history of mitral valve prolapse and hypertension; one patient had hypertension, diabetes mellitus and positive family history but no prior history of heart disease. Arrhythmia was seen in 3 patients (6%). Post-treatment EKG changes occurred in 14 patients (27%) including bradycardia, PAC's, PVC's, ST-T wave changes, tachycardia and other rhythm abnormalities. These changes were usually asymptomatic. Gated cardiac study done after a median of 63 months (range 29 to 83 months) after initiation of imatinib treatment showed EF below 60% in 9 patients (18%) with a median of 55% (range 36% to 59%). None of the patients died of cardiac conditions and none discontinued imatinib therapy because of cardiac events. Conclusion: Although cardiac events occur in some patients treated with imatinib, these are much more common among patients with pre-existing cardiac conditions and/or cardiac risk factors. These patients need to be monitored closely to minimize their risk and intervene early when new cardiac events arise. Disclosures: Cortes: novartis: Research Funding; BMS: Research Funding; Pfizer: Consultancy, Research Funding. Kantarjian:novartis: Research Funding; BMS: Research Funding. O'Brien:Novartis: Research Funding.

Description: Abstract 4472 Chronic myeloid leukaemia (CML) is a clonal disorder of haematopoietic stem cells, characterized by constitutively active BCR-ABL kinase. It was previously shown, that transient inhibition of BCR-ABL by tyrosin kinase inhibitor (TKI) dasatinib induces apoptosis in CML cells (Shah 2008). Dasatinib mediated apoptosis is accompanied by increasing expression level of proapoptotic protein BIM and inhibition of its degradation in proteasome. Our aim is to find alterations in BCR-ABL downstream pathways and changes in BIM expression after pulse (wash-out after 20min) or continuous treatment with TKI dasatinib alone or in combination with proteasome inhibitor bortezomib in K562 cells. Higher mRNA levels of BIM were confirmed in both transiently and continuously treated (100nM dasatinib) K562 cells compared to low dose continuous (1nM dasatinib) treatment. Western blot analysis of BCR-ABL induced signalling pathways activity revealed, among others, sustained inhibition of MEK/ERK pathway even in the case of re-activation of BCR-ABL after drug wash-out. MEK/ERK inhibition leads to activation of proapoptotic BIM expression and inhibition of its degradation. We used bortezomib, a reversible inhibitor of proteasome, in order to test the block of BIM degradation in dasatinib treated cells. However, we have not found any difference in cell viability caused by adding of 30nM bortezomib to dasatinib treated cells neither in continuous or transient exposure. These findings suggest that equal increase of BIM in the case of pulse and continuous dasatinib treatment is caused by MEK/ERK inhibition in cells with restored BCR-ABL activity. This project is supported by grants from the Ministry of Education of Czech Republic NPVII (2B06052) and MSM-0021622430. The substance (dasatinib) for this project was supplied by BMS company. Disclosures: Mayer: BMS: Consultancy, Research Funding.

Description: Abstract 4531 Introduction: Cytomegalovirus (CMV) is one of the most common viral pathogens post-allogeneic stem cell transplantation (SCT). However, CMV reactivation and disease are relatively uncommon events after autologous stem cell transplantation (ASCT), with a reported incidence of 2–9%. There are few studies describing risk factors for development of CMV reactivation post-ASCT. Objective: To describe the incidence and risk factors for CMV reactivation post-ASCT in our institution and its impact on survival. Methods: The charts of patients who underwent ASCT at our institution from January 2005 until July 2009 were manually reviewed. CMV reactivation was detected by antigenemia assay and/or real-time PCR. Variables associated with CMV in a univariate analysis with a p

Description: Abstract 2828 The immunomodulatory compound lenalidomide induces apoptosis in tumor cells, exerts antiangiogenic effects and activates immune effector cells. Apoptosis resistance, increased neoangiogenesis and impaired immunity critically contribute to Hodgkin‘s Lymphoma (HL). However, the activity of lenalidomide in HL is unknown so far. 42 patients with multiple relapsed or refractory HL were treated with 25 mg oral lenalidomide for 21 days of a 28-day cycle in a named patient program in 18 different german hospitals. Participants gave written informed consent, had no curative treatment options, an ECOG performance status ≤ 2, and normal organ functions including peripheral blood counts within the normal range. Patients were staged and restaged with computed tomography (CT) of the initially involved sites; the use of PET was optional. Response was defined according to the “Cheson-criteria” from 1999 and evaluated after two to three cycles and every other cycle thereafter. Treatment was continued until disease progression or in case of intolerable toxicity occurred. Concomitant anti-thrombotic prophylaxis with 100 mg daily oral acetylsalicylic acid was mandatory. Patients with a history of thrombosis or thrombembolic events received low molecular heparin. To date, in the 24 patients who are eligible for analysis, treatment was well tolerated. No hematological or other toxicities above CTC grade-2 were reported; adverse events included diarrhea, constipation, neuropathy, and mild dyspnea. At restaging, eight of the analyzed patients had stable disease and twelve patients achieved clinical responses (11× PR, 1× CR) as measured by CT-scan. However, five of the heavily pretreated patients had progressive disease. Results on the remaining patients are currently being analyzed and will be presented at the ASH meeting. In conclusion, these first clinical observations indicate that lenalidomide is well tolerated and effective in patients with relapsed or refractory HL. Consequently, international clinical trials evaluating the role of lenalidomide in HL are in preparation and a phase I/II trial evaluating lenalidomide with AVD (Adriamycine, Vinblastine and Dacarbacine) in elderly HL patients is currently recruiting. Disclosures: Off Label Use: Lenalidomide approved for Multiple Myeloma and MDS (5q−).

Description: Abstract 4281 Introduction: Quantification of HbA2 is a well established screening test for beta-thalassemia trait (BTT). However, there is a small subset of individuals heterozygous for beta-thalassemia mutations who have HbA2 levels less than or equal to 3.5%. Some reports have suggested that iron deficiency in BTT patients causes HbA2 to be lower than expected, while others have found no significant relationship between iron deficiency and the level of HbA2. These conflicting reports have led to confusion amongst clinicians as to the reliability of HbA2 measurement when screening for BTT in iron deficient individuals. Methods: From a database of 444 individuals with heterozygous beta-thalassemia, confirmed by molecular testing, we assessed the variability in HbA2. Individuals were classified as “iron deficient” or “non-iron deficient” based on their serum ferritin, using two definitions of iron deficiency (serum ferritin

Description: Abstract 4941 Since the multidrug resistance phenotype was first observed 30 years ago, other 48 proteins, belonging to an evolutionary highly conserved family of transport proteins, named ABC proteins, has been identified and grouped in eight subclasses, according to divergent evolution. In eukaryotes, ABC proteins are involved in transport across the membrane to extracellular space or into intracellular organelles playing an important role in cell physiology. Almost three distinct subfamilies (B, C and G) have been recognized to transport a wide variety of amphipatic or hydrophobic molecules, including most of anticancer drugs compounds, affecting drug absorption, disposition, metabolism, excretion and toxicity (ADMEtox) and has been associated to chemotherapy failure in solid and hematologic neoplasia. In particular the role of ABCG2 has been pinpointed over the past years for its high expression on primitive hematopoietic and on progenitors cells and for its ability to confer to the cancer cell the properties of cancer stem cell, with increased survival capacity. At present over 40 single nucleotide polymorphisms of ABCG2 protein have been identified. Among them the 421C〉A (Q141K) variant has been shown able to alter protein function and to modify in vitro sensitivity to many anticancer drugs, compared to the wild type protein. In the present paper we have evaluated the incidence and the impact on therapy outcome in a series of 100 caucasic patients with acute myeloid leukemia, receiving the same chemotherapy program including idarubicin. Q141K polymorphism was detected in 19/100 (19%) patients, without correlation with any clinical-biological characteristic at diagnosis (such as sex, age, peripheral blast count, FAB subtype, karyotytpe, CD34 expression). ABCG2 protein was overexpressed in 10/19 (52.6%) of mutated patients and in 37/83 (44.5%) of patients expressing the wt form of ABCG2 and no difference in intensity of ABCG2 expression was observed in the two groups. Again no difference in ABCG2 mRNA transcription was detected between patients carrying wt or polymorphic protein. Complete remission rate was comparable in wt and mutated patients (38/81, 45,7% vs 10/19, 52,6%). However patients with Q141K polymorphism (irrespective to the intensity of expression of ABCG2) protein) had a leukemia free survival comparable to patients overxpressing wt protein, and significantly shorter than patients without wt ABCG2 overepxression (chi square 12,2, p=0,002, logrank test). In conclusion our data demonstrated Q141K polymorphism of ABCG2 transporter protein identified a subset of patients with high probability of relapse when treated with idarubicin suggesting that other drugs should be considered in consolidation/maintenance treatment to improve patients outcome. Finally the impact of Q141K polymorphism on treatment toxicity is under investigation. Work supported by PRIN 2007WEYB34-004 Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4891 Background CD20 is a non-glycosylated phosphoprotein expressed on the surface of all mature B-cells. CD20 is expressed on all stages of B cell development except the first and last. However, complete lack of CD20 expression occurred in a few cases without previous rituximab (R-) treatment. The immunohistochemostry (IHC) studies which we used were not perfect for confirmation of expression. However, we intended to investigate characteristics and clinical outcome of CD20-negative diffuse large B-cell lymphoma (DLBCL), not detected with usual method, and to compare with CD20-positive. Methods The records of Non-Hodgkin's Lymphoma patient registry were reviewed in Asan Medical Center. Between September 2003 and February 2009, a total of 407 patients were diagnosed DLBCL and 16 patients (3.9%) out of 407 confirmed CD20-negative DLBCL by IHC. The rest of patients (n=391) were CD20-positive and unconfirmed cases were excluded. Retrospective analysis of complete response (CR), disease-free survival (DFS), and overall survival (OS) was performed. Results The median age was 60.5 years old (range 31–81) in CD20-negative patients. Ten patients were males. The Ann Arbor stage was I in 3 patients, II in 3 patients, and III or IV in 10. Six patients were low risk group, 7 patients in intermediate, and 3 in high risk group according to international prognostic index (IPI). Most of patients (62.5%) received cyclophosphamide, doxorubicin, vincristin, and prednisone (CHOP) chemotherapy in CD20-negative and 295 patients (75.4%) with R-CHOP in CD20-positive DLBCL. The Baseline characteristics was not different in both groups except Hans classifier (p=0.02). With a median follow-up time of 32.3 months (range 0.5–83.4), the CR rate was 73.5%, the 3-year OS was 69.5%, and 3-year DFS 74.2% in all patients. CD20-positive and CD20 negative groups had a CR rate of 73.7%, 68.8% (p=0.146), respectively, a 3-year OS of 70.7%, 40.0% (p=0.003), a 3-year DFS of 75.4%, 44.6% (p〈 0.001), respectively. The 3-year OS and DFS also had significant difference with adjusted IPI (p

Description: Abstract 491 CD200 is a type-1 transmembrane glycoprotein which suppresses inflammatory and autoimmune responses by signalling through its cognate transmembrane receptor homologue (CD200R). Normally, CD200 expression is restricted to immune privileged sites where it enhances immune tolerance through mechanisms that include modulating the expansion of FOXP3+ regulatory T-lymphocytes (T-regs) and suppressing macrophage cytolytic activity. Furthermore, leukocyte associated CD200 has been reported to suppress Natural Killer (NK) cell activity in vivo. Pathologically, we have previously shown that CD200 over-expression on leukemic blasts in around 50% of acute myeloid leukemia (AML) patients is significantly associated with a poor overall survival (Tonks et al, Leukemia, 2007). Given the existing evidence that T-reg frequency and NK cell function influence blast clearance and long-term survival in AML, we investigated the possibility that CD200 expression in AML may be directly suppressing anti-tumor immunity in this disease. Here we present evidence that CD200+ AML can suppress host anti-tumor responses by augmenting the frequency of AML patient T-regs and by direct inhibition of NK cell anti-tumor activity. We also show that targeting the interaction between CD200 and its receptor might provide a new strategy for the treatment of AML. Bone marrow aspirates from 91 diagnostic AML patients were analysed by multiparameter flow cytometry for blast CD200 protein expression. We found that the level of blast CD200 expression directly correlated with an increased frequency of T-regs (CD4+CD25++FoxP3+; R=0.78, p=0.0008). Measuring 3H-thymidine incorporation, we show that T-regs isolated from AML patients by MACS® separation inhibited T-cell proliferation (induced by CD3 and CD28 stimulation) at ratios

Description: Abstract 4899 Treatment of central nervous system (CNS) lymphoma remains challenging, and balance between therapeutic effectiveness and toxicity is difficult to find. The addition of systemic chemotherapy to whole brain radiotherapy has significantly improved the outcome of these patients. However, this combined strategy is burdened with possible acute and/or delayed severe neurotoxicity, particularly in elderly patients. Chemotherapy-only approaches, with or without concomitant intrathecal (IT) therapy, for CNS lymphoma have been explored. Moreover, deferring radiotherapy to the time of first or subsequent relapse may reduce the risk of severe neurotoxicity, without altering outcome. Recently, high-dose methotrexate and temozolomide (HD-MTX-TMZ) without IT CNS prophylaxis have been studied in PCNSL elderly patients. This regimen appears effective, while substantially decreasing acute and/or delayed neurotoxicity. Conflicting data exist regarding the safety of combining IT liposomal cytarabine (LC) and systemic CNS-penetrating therapy. We report our preliminary experience with HD-MTX-TMZ plus IT LC used upfront or as salvage in 4 primary or secondary CNS lymphoma patients, only one of which under the age of 60 (56). Treatment consisted of induction: MTX 3g/ms d 1, 10, 20, TMZ 100 mg/ms d 1–5; and maintenance for ≥SD patients, and for up to 5 additional cycles: MTX 3g/ms d 1, TMZ 100 mg/ms d 1–5, every month. Fifty mg IT LC were given concomitantly for up to 6 doses planned. LC doses were separated by at least 14 days from one another and at least 7 days from HD-MTX. Pt n. 1 (56 year-old male) had a history of testicular diffuse large B cell lymphoma (DLBCL), stage IVA. He received chemo-radiotherapy with 4 concomitant IT LC injections as CNS prophylaxis. Complete response (CR) was achieved. Two months later, he presented with headache and dizziness. MRI and PET revealed metabolically active righ cerebellar lesion. Karnofsky performance status (KPS) was 50%. Rituximab plus TMZ and IT MTX were initiated but soon discontinued due to a G4 CMV-related pneumonia. After complete recovery, HD-MTX-TMZ was started with concomitant 4 IT LC injections, and precautionary stem cell harvest. No significant G3-4 toxicities were observed during treatment. At 9 months of follow-up post-treatment, the lesion is stable with no contrast enhancement at MRI and PET is negative, suggesting a CRu. Pt 2 (76 year-old male) was referred in May 2009 with a diagnosis of CNS peripheral T-cell lymphoma not otherwise specified with MRI- and PET-documented multiple brain localizations (i.e. hypophyseal infundibulum, cavernous sinus, cerebellum). KPS was 60%. He was treated with systemic steroids and TMZ 150 mg/ms d 1–5/28. In August 2009 disease progressed after two cycles, and HD-MTX-TMZ was started. IT LC was added as CNS prophylaxis with 4 drug injections. Toxicity consisted of G2 renal insufficiency and G3 steroid-induced diabetes mellitus, both resolved. Treatment resulted in marked shrinkage of all lesions, with no contrast enhancement and PET is negative, indicating CRu, which is stable at 5 months of follow-up. Pt 3 (68 year-old female) was diagnosed with PCNSL, DLBCL, with left cerebellar localization. KPS was 50%. HD-MTX-TMZ and concomitant IT LC injections were initiated as first-line therapy. Four maintenance cycles and 5 IT LC injections have been performed so far. Toxicities included G3 atrial fibrillation. Interim MRIs documented very good partial remission of the disease. Pt 4 (71 year-old female) was diagnosed with DLBCL stage IIIE (i.e. subcutaneous facial localization) in July 2009, and treated with R-COMP (liposomal doxorubicine in lieu of the free formulation) for 8 cycles. She obtained CR, but in July 2010 presented with aphasia and facial hemiparesis. Brain MRI showed multiple subcortical lesions in both hemispheres, biopsy-confirmed to be lymphoma. The first IT LC injection and HD-MTX dose were given without significant toxicity. All patients completed induction and 5 maintenance cycles. HD-MTX-TMZ and concomitant IT LC therapy appeared feasible and overall well tolerated. No acute neurologic complications were observed. Follow-up is too short to make comments on delayed neurotoxicity. Increasing the number of LC injections might be reasonable and at least 7 days should be interposed between systemic and IT therapy. HD-MTX-TMZ plus IT LC deserves further evaluation in a larger prospective setting. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 490 T lymphocytes play central roles in cellular immunity, exerting their proliferative and effector activities when they recognize antigens, in HLA-restricted and antigen-specific manner, via T-cell receptors (TCRs). Successful treatment of leukemias/cancers with T-lymphocytes infusions is a direct proof that human immunity has the potential to eradicate cancers. However, continuous exposure to tumor/self antigens drives T lymphocytes into a highly exhausted state, with loss of potentials for long-term survival, proliferation, and effector functions that can end up with deletion of antigen-responding T-lymphocyte pools. Several workers have endeavored to develop clinical protocols for expanding antigen-responding T cells from the few naïve T-cell pools remaining in the patient. However, highly expanded T cells in such protocols have not proved fully effective so far, because functional losses like those in the patient occur during ex vivo manipulation. To overcome this obstacle to T-lymphocyte based immunotherapy, we endeavored to induce antigen-specific TCR-expressing T lymphocytes from induced pluripotent stem (iPS) cells, which were derived from antigen-reactive single T lymphocytes. iPS cells have a capacity for unlimited self-renewal while maintaining pluripotency. These features enabled us to induce an unlimited number of T lymphocytes, especially naïve T lymphocytes, showing reactivity to specific antigens. If they retain properties of naïve T lymphocytes, they may proliferate for a longer period and achieve better therapeutic effects than their peripheral blood counterparts expanded in vitro. Peripheral T lymphocytes were isolated from healthy volunteers. Then three reprogramming factors (OCT4, SOX2, and KLF4) and additional factors (c-MYC and/or NANOG) were transduced into fresh or frozen/thawed T lymphocytes using a retrovirus. The virus-infected T lymphocytes were then transferred onto mouse embryonic fibroblasts (MEFs) in the presence of cytokines and chemicals favorable for T-lymphocyte survival/proliferation. iPS-like colonies were observed within 3 weeks after infections. Single T lymphocyte-derived colonies were isolated and clonally expanded. They exhibited standard ES-like morphology, cell surface markers and alkaline phosphatase activity, as well as differentiation potential into various tissues related to all three germ layers. Human TCRs are encoded in four genes (TCRA, TCRB, TCRG, TCRD), which should be genetically assembled in an irreversible manner during T-lymphocyte development. This feature allowed us to retrospectively confirm the iPS cells were generated from T lymphocyte. The TCR genes rearrangement encoded in an iPS colony was single in all iPS lines, indicating that the iPS colony was derived from single T lymphocyte. Sequence analyses of TCR genes revealed whether the rearrangements were productive, and the productivity might promise the conservation of TCR genes rearrangement during the reprogramming process. Next, we tried to re-differentiate T-lymphocyte derived-iPS (T-iPS) cells into T-lineage cells by co-culturing them with murine stromal cell layers (OP9 and OP9-DL1). These T-lineage committed cells were expressed TCRab heterodimer and T-cell surface markers such as CD3. They could activate via TCR stimulation, and produce IL-2 and IFN-g as maturing T lymphocytes. The re-differentiation efficiency of T-iPS cells was higher than those of embryonic stem cells, fibroblasts derived-iPS cells, or cord blood derived-iPS cells. mRNA sequence of TCRs transcribed in re-differentiated T-lineage cells was identical to that engraved in the pre-differentiated T-iPS cells genome. The invariance of the sequence, especially antigen-recognition site sequence, indicated that the antigen-specificity in original T lymphocyte was conserved during re-differentiation process. Here we show that the conservation of the antigen-specificity encoded in TCR genes throughout induction of T-iPS cells and re-differentiation into T-lineage cells. These data suggest that further optimization of these processes for clinical application could open the door to the development of novel T-lymphocyte therapy, repeatedly supplying patient-compatible and disease-specific naïve T lymphocytes. Disclosures: No relevant conflicts of interest to declare.