Search

Background: Although the natural history of follicular lymphoma is indolent with a median overall survival of about 12-15 years, the disease is heterogeneous. The 5 and 10 year overall survival (OS) of low, intermediate and high risk FLIPI is 91%, 78% and 53% and 71%, 51% and 36% using standard rituximab-based treatment. 5-year progression-free survival (PFS) is 80%, 70% and 48% respectively. Methods: Based upon this we conducted an investigator-initiated single-centre Phase II trial of intensified therapy with CHOP-R followed by (90)Y ibritumomab tiuxetan consolidation and 24 months of rituximab maintenance as treatment for patients with intermediate and high-risk newly diagnosed symptomatic follicular lymphoma. 33 patients were enrolled. Results: The addition of (90)Y ibritumomab tiuxetan was well tolerated but resulted in asymptomatic grade 3 or 4 thrombocytopenia and neutropenia in11-36% and 10-24% of patients between weeks 2-8 post (90) Y. After 9 years of follow-up (median follow-up 61 months) the 0S for intermediate and high risk FLIPI was 95% and 78%. The 5 year PFS was 79% and 64% for intermediate and high risk FLIPI, respectively. Responses at three months post consolidation were as follows: 3/33 (9%) achieved CR, 25/33(76%), achieved CRU, 1/33(3%) had PR, and 1/33(3%) had PD. Three patients did not receive (90)Y ibritumomab tiuxetan due to disease progression 2/33(6%), or death 1/33(3%). Of 19 patients who had a molecular marker for their lymphoma, 18 (95%) achieved molecular remissions in peripheral blood with CHOP-R therapy. Nine (47%) of these patients have been recently assessed for MRD and remain in molecular remission. The therapy resulted in decreased levels of IgG, IgM and IgA below the lower normal level in 33%, 40% and 23% of patients respectively post therapy. These levels did not recover in most of these patients. B cells were depleted to undetectable levels during therapy including rituximab maintenance. In 18 evaluable patients only 11 recovered normal B cell counts post maintenance rituximab. There was no correlation between normal B-cell recovery and Ig levels. Many patients with low or no B cell counts had normal IgG levels, whereas some patients who regained normal B cell counts were still unable to reach normal Ig levels. No patient developed human anti-mouse antibody. Immunity to measles, mumps, or rubella was retained post therapy. Patients did not have significant infections or opportunistic infections (although 2 developed Grade 1 shingles post (90)Y ibritumomab tiuxetan) and none required IVIG. Conclusions: We conclude that this intensified regimen is highly active in cyto-reducing lymphoma in high and intermediate risk FLIPI follicular lymphoma patients. The toxicity is tolerable although a significant percentage of patients will end up with persistent asymptomatic reductions in B cells and serum Ig. Only randomized trials will determine whether this regimen enhances outcome over standard of care in this higher risk follicular lymphoma population. References: 1.Examination of the follicular lymphoma international prognostic index (FLIPI) in the National LymphoCare study (NLCS): a prospective US patient cohort treated predominantly in community practices. Nooka AK, Nabhan C, Zhou X, Taylor MD, Byrtek M, Miller TP, Friedberg JW, Zelenetz AD, Link BK, Cerhan JR, Dillon H, Sinha R, Shenoy PJ, Levy D, Dawson K, Hirata JH, Flowers CR. Ann Oncol. 2013 Feb;24(2):441-8. doi: 10.1093/annonc/mds429. Epub 2012 Oct 5 2.Validation, revision and extension of the Follicular Lymphoma International Prognostic Index (FLIPI) in a population-based setting. van de Schans SA, Steyerberg EW, Nijziel MR, Creemers GJ, Janssen-Heijnen ML, van Spronsen DJ. Ann Oncol. 2009 Oct;20(10):1697-702. doi: 10.1093/annonc/mdp053. Epub 2009 Jun 23. PMID: 19549712 Disclosures Buckstein: Novartis: Honoraria; Celgene: Honoraria, Research Funding.

We investigated the development of a positive direct antiglobulin test associated with the use of the nonsteroid anti-inflammatory drug sulindac. The drug was shown to be the cause of the positive direct antiglobulin test using red cells treated in vitro with solutions of native sulindac and its two major metabolites. Serum from the patient contained sulindac-dependent red cell antibodies which could not be demonstrated when red cells having the Rh phenotype D-- were employed in the test procedure. An eluate prepared from the patients' red cells reacted against untreated red cells having common Rh phenotypes, but not against target red cells with the Rh phenotypes D-- or Rh null. The eluate showed stronger reactivity against the cells having common Rh phenotypes when they were treated with solutions of a metabolite of sulindac, but failed to react against treated red cells having the Rh phenotype D-- or Rh null. The results of our investigations point to an interaction between sulindac and/or its metabolites and Rh structures on the red cell membrane as the initial step in the production of drug dependent and autoantibodies leading to the positive direct antiglobulin test.

We report a patient in whom a cell line of 47,XY,+X,t(1;14)(q21;q32) constitution was found in a lymph node excised from the neck. Histologic examination and immunophenotyping both in situ and by flow cytometry failed to confirm a diagnosis of B-cell lymphoma, but Southern analysis indicated the presence of B-cell clonal expansion. These observations support the concept that primary chromosomal abnormalities occur in clonal expansions in the very earliest stages of tumorigenesis.

Effective spleen-derived humoral responses are essential for protecting hosts from blood-borne pathogens. Increased susceptibility to infection is therefore a risk post-splenectomy but is also associated with smoking, chemotherapy and aging. In fact, decreased vaccine efficacy in the elderly may be due to splenic hypofunction. While investigating the role of sensory nerves in hematopoiesis, we discovered a lymphocytosis phenotype involving the splenic marginal zone (MZ). Dividing red and white pulp in the spleen, the MZ is a specialized micro-anatomic structure facilitating innate and adaptive crosstalk to incoming antigen. Sensory nerves were found to synapse at this site and in mice deficient in the major sensory neurotransmitter substance P (Tac-1-/-), MZs were expanded histologically and confirmed by flow cytometry as an increase involving the IgMhiIgDloCD21hiCD23lo MZ B cell population. Tac-1-/- mice also exhibited splenomegaly with overall Ki-67 proliferation indices of 40% vs. 5% in controls. In the peripheral blood, Tac-1-/- mice had persistent polyclonal pan-B cell lymphocytosis with an absolute increase of 200% relative to wildtype. Aberrant B cell homeostasis had functional consequences as demonstrated by significantly decreased total basal plasma IgG (P Disclosures No relevant conflicts of interest to declare.

The leukemia stem cell model suggests that elucidation of the genes that regulate growth ability within the leukemia cell hierarchy will have important clinical relevance. We showed that the expression of NR2F6 (EAR-2), is greater in clonogenic leukemia single cells than in leukemia cells that do not divide, and that this gene is over-expressed in patients with acute myeloid leukemia and myelodysplastic syndrome. In vivo, overexpression of EAR-2 using a retroviral vector in a chimeric mouse model leads to a condition that resembles myelodysplastic syndrome with hypercellular bone marrow, increased blasts, abnormal localization of immature progenitors, morphological dysplasia of the erythroid lineage and a competitive advantage over wild-type cells, that eventually leads to AML in a subset of the mice, or after secondary-transplantation. Interestingly, animals transplanted with bone marrow that over-expresses EAR-2 develop leukemia that is preceded by expansion of the stem cell compartment in the transplanted mice—suggesting that EAR-2 is an important regulator of hematopoietic stem cell differentiation. Here we report that over-expression of EAR-2 also has a profound effect on the differentiation of erythroid progenitor cells both in vitro and in vivo. Studies of the roles of EAR-2 in normal primary bone marrow cells in vitro showed that overexpression of EAR-2 profoundly impaired differentiation along the erythroid lineage. EAR-2 over-expressing bone marrow cells formed 40% fewer BFU-E colonies, but had greatly extended replating capacity in colony assays. While knockdown of EAR-2 increased the number of cells produced per BFU-E colony 300%. Normal mice transplanted with grafts of purified bone marrow cells that over-expressed EAR-2 developed a rapidly fatal leukemia characterized by pancytopenia, enlargement of the spleen, and infiltration of blasts into the spleen, liver and peripheral blood. Sick animals had profound reduction of peripheral blood cell counts, particularly anemia with a 55% reduction in hemoglobin levels. Anemia was evident even on gross inspection of the blood and the liver in EAR-2 overexpressing animals. Analysis of the leukemic cells revealed an erythroblastic morphology, with the immunophenotype lineageneg, CD71high, TER119med. Hence, we wondered weather EAR-2 caused leukemia by arresting erythroid progenitor cell differentiation. Examination of the bone marrow of pre-leukemic animals showed a four-fold increase in cells with a pro-erythroblastic immunophenotype (CD71highTER119med , region I), and a four-fold decrease in orthochromatophilic erythroblasts (CD71lowTER119high , region IV). We observed no change in the numbers of basophilic erythroblasts (CD71highTER119high , region II) or late basophilic and polychromatophilic erythroblasts (CD71medTER119high, region III). These data suggests that over-expression of EAR-2 blocks erythroid cell differentiation at the pro-erythroblastic stage. Since EAR-2 over-expressing recipients died within 4 week, we wanted to definitively test whether animals had compromised radioprotection. We showed that decreasing the size of the bone marrow graft, reduced survival of the EAR-2 over-expressing cohort by a week, but had no effect on control animals proving that EAR-2 over-expression has a profound effect on erythropoietic reconstitution in vivo. Mechanistically, we show that DNA binding is necessary for EAR-2 function, and that EAR-2 functions in an HDAC-dependent manner, regulating expression of several genes. Pre-leukemic pro-erythroblastic cells (CD71highTER119med) that over-expressed EAR-2 had lower expression of genes involved in erythroid differentiation such as GATA1, EBF1, inhibitor of NFKB (NFKBia), ETV6, CEBP/a, LMO2, and Nfe2, and increased expression of GATA2, GLI1, ID1 and PU.1 than GFP control pro-erythroblasts. These data establish that EAR-2 is a novel oncogene whose cellular function is to regulate terminal differentiation of erythroid cells at the proerythroblastic (CD71highTER119med) stage by deregulating gene expression necessary for erythroid differentiation. Disclosures Ichim: Entest BioMedical: Employment, Equity Ownership, Patents & Royalties, Research Funding. Koos:Entest BioMedical: Employment, Equity Ownership, Patents & Royalties, Research Funding.

A permanent cell line, HSC-M1, was established from a child with advanced CD30 (Ki-1)+ anaplastic large-cell lymphoma (ALCL). Clinical features included irritability, fever, weight loss, tender lymphadenopathy, pneumonitis, neutrophilia, and bone marrow erythrophagocytosis. While HSC-M1 cells exhibited an immunophenotype characteristic of ALCL of T-cell lineage, the cell line also demonstrated features of monocyte-macrophage lineage. Cytogenetic and polymerase chain reaction (PCR) analysis of the HSC-M1 cell line and involved bone marrow demonstrated the characteristic non-random chromosomal translocation t(2:5)(p23:q35). Reverse transcriptase PCR for mRNA expression of cytokines and cytokine receptors showed that HSC-M1 cells expressed the message for multiple cytokines and their receptors. Measurement of cytokine levels in serum samples using enzyme-linked immunosorbent assays showed increased concentrations of several cytokines. The increased levels of some cytokines correlated with disease activity and clinical symptoms. Although spontaneous production by HSC-M1 cells of some of these cytokines was demonstrated, the production of others was only detectable after stimulation with exogenous CD30 ligand. With few exceptions, there was good correlation between serum cytokine levels and cytokines produced by HSC-M1 cells. These findings indicate that cytokine production is a feature of ALCL cells and that some of the clinical manifestations in ALCL may result from cytokines produced by either the malignant or accessory cells.

Abstract 1855 Introduction: Solid tumors commonly recruit and condition immature myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMΦs), which impair immune-surveillance and facilitate growth. Whether they play a pathogenic role in hematological malignancies is not known. Our previous studies in an MDS/CMML model (SHIP-/- mice) revealed MΦs that are skewed to an alternatively activated (M2) phenotype (reminiscent of TAMΦs), characterized by arginase 1 (Arg1)-mediated redirection of L-arginine metabolism away from cytotoxic NO and towards the production of healing intermediates, as well as over-expression of the Th2-skewing and anti-apoptotic chitinase-like protein, Ym1. Moreover, MDSCs are also increased in SHIP-/- mice, leading to T-cell suppression. We therefore hypothesized that MDSCs/M2-MΦs may be expanded in the BM of MDS and CMML patients and/or their properties hijacked by the malignant clones. Methods: With informed consent, BM mononuclear cells (MNCs) and plasma were obtained from 40 specimens in the Sunnybrook MDS Biobank (6 normals, 11 CMML-1, 1 CMML-2, 1 RARS, 2 del(5q), 6 RCMD, 2 RCMD-RS, 8 RAEB-1, and 3 RAEB-2) (20 at presentation; 20 at follow-up). BM plasma was subjected to YKL-40 ELISA and Quantibody (RayBiotech) cytokine array (bFGF, BMP6, IL-1α, IL-1β, IL-6, PDGF-BB, TGFβ1, TNFα, VEGF, and VEGF-R2). BM-MNC protein lysates were prepared and subjected to arginase enzymatic assay, and Arg1/YKL-40 Westerns. Micro-vessel density (MVD) was recorded as the concordant counts of two observers per mm2 of H&E BM biopsies. Surrogate MVD analysis was performed by automated CD31 immunohistochemistry (IHC) analysis (Vias, Ventana). Linear regression and unpaired, two-tailed t tests were performed with Prism (GraphPad) software. Results: Mean BM-MNC arginase activity (μg urea/h/μg protein, recorded as % of control mean) and BM plasma YKL-40 levels were significantly higher in CMML patients, as compared to controls (350 ± 69 % vs 100 ± 5 %, p = 0.022, and 300 ± 32 μg/L vs 150 ± 22 μg/L, p = 0.0033, respectively). Westerns confirmed intracellular Arg1/YKL-40 over-expression in CMML BM-MNCs. Heterogeneous Arg1/YKL-40 over-expression was observed in MDS patients, and was strongly associated with the lowest risk IPSS and WPSS MDS scoring categories (Figure 1) (CMML not included). Moreover, high Arg1, but not YKL-40, was able to further discriminate IPSS Low/Int1-risk patients with poor prognosis using the low-risk MD Anderson score (Figure 1). This low-risk MDS/high-Arg1/YKL-40 profile was confirmed by negative correlations with (poor-prognostic) MVD (Arg1: p = 0.049, YKL-40: p = 0.089) (similar trends with CD31), albeit in a seemingly paradoxical pro-angiogenic BM plasma milieu (trends to increased bFGF, PDGF-BB, VEGF, and VEGF-R2). These results were unexpected, given that Arg1/YKL-40 are normally thought to be pro-angiogenic, and were reminiscent of recent studies suggesting VEGF-mediated recruitment of suppressive myeloid cells may inhibit angiogenesis and tumor progression in some situations (Stockmann et al., Nature 456:814-8). Accordingly, MDS BM arginase activity was associated with a larger BM myeloid compartment (using > 60% as a cutoff; p = 0.042) and a striking linear correlation with peripheral blood (PB) neutrophil counts on the day of BM collection (p = 0.0003), likely reflecting BM and systemic expansion of suppressive myeloid populations. MDS BM-MNC Arg1/YKL-40 Westerns, stratified by PB neutrophil counts, confirmed these associations. Conclusions: 1) Arg1/YKL-40, effectors of MDSCs/M2-MΦs, are over-expressed in BM MNCs and plasma in most CMML patients, as predicted from SHIP-/- mouse studies. Perturbations in SHIP and associated pathways may warrant investigation in CMML. 2) Arg1/YKL-40 are over-expressed in low-risk MDS patients, as assessed using 3 prognostic scoring systems. Their immune-suppressive nature and pro-proliferative/anti-apoptotic effects may create a permissive environment for low-risk MDS clones. 3) The unexpected association of high Arg1/YKL-40 with a pro-angiogenic BM milieu and paradoxically low MVD, may be in keeping with the ability of MDSCs/M2-MΦs to inhibit angiogenesis under certain conditions. Understanding what regulates the “immune/angiogenic switch” to low suppressive myeloid/high MVD/high-risk MDS may have important future treatment implications. Disclosures: Krystal: Aquinox Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Abstract 438 Type II Abl kinase inhibitors such as imatinib achieve high selectivity by specifically targeting the inactive Abl/c-Kit conformation. However, these inhibitors are prone to inactivation by resistance mutations that cause conformational changes. A better understanding of mutant Abl conformations would provide information to design inhibitors that recognize multiple states of Abl with lower rates of resistance. To more easily examine Abl kinase conformations, conformer specific reagents are needed that are (i) easily generated against a given Abl conformation, (ii) compatible with genetic selections, in vivo and in vitro assays, and structural studies, and (iii) amenable to chemical synthesis. Recently, we developed a novel genetic screen for isolating “lariat” peptide inhibitors of protein function. Lariats are lactone-cyclized peptides that possess the above-mentioned characteristics and can be used to evaluate the function and therapeutic potential of proteins. Previously, we isolated specific lariat inhibitors against the inactive conformation of the bacterial repressor LexA. To obtain conformer specific lariats against Abl, we screened a combinatorial seven amino acid lariat library for interactions with the Abl SH1 domain using the yeast two-hybrid (Y2H) assay. We obtained two lariat peptides named A1 (SGWQRLPFEY) and A2 (SGWHRLSEEY) that interacted with the Abl SH1 domain. In vitro studies with purified Abl kinase demonstrated that the A1 lariat competitively inhibited ATP binding with an inhibitory constant of 5.95 μM. By performing site-saturation mutagenesis, we defined acceptable and tolerable substitutions at each position of the A1 lariat. To obtain tighter binding variants of the A1 lariat, we rationally designed mutations and constructed a second-generation lariat (GWQTLDWNY) with | 10 times higher affinity for Abl. We compared the affinity of the lariats for imatinib resistant Abl kinase mutants using the lariat Y2H binding assay. Mutations that (i) promote conformational dynamics of the kinase T315I), (ii) destabilize the Abl/c-Kit like inactive state (H396R), (iii) disrupt the flexibility or hydrophobicity of the lariat-binding pocket (E355G), and (iv) distort the ATP phosphate binding loop (Y253F), have a marked decrease in lariat affinity. Together, these results show that the lariat preferentially bound to the Abl/c-Kit like inactive conformation and act similarly to type II kinase inhibitors. A specificity screen against a panel of related and distant kinases also demonstrated the conformer specific nature of the lariats. We used lariats that recognized the Abl/c-Kit like inactive state to probe various conformations of Abl His396 mutants. His396 is located in the activation loop and does not alter the Abl/c-Kit like inactive form. His396Pro and His396Arg substitutions are clinically relevant imatinib resistant mutations that alter the conformation of Abl. Previous structural studies showed that the His396Pro mutation causes Abl to adopt the active conformation. It is hypothesized that the His396Arg mutation favors the inactive Src/Cdk like state. Using inactive conformation specific Abl lariats as probes, we showed that these lariats interact weakly with the His396Arg relative to His396, suggesting that this mutation destabilized the Abl/c-Kit like inactive state. We also showed that the lariats interacted stronger with a previously unreported His396Ile mutation than His396, indicating that His396Ile shifts Abl to a highly inactive conformation. Preliminary studies suggest that this residue plays a key role in determining the flexibility of the activation loop and hence the conformational state of Abl. These results provide information on the structure and sequence requirements for maintaining an auto-inhibited activation loop conformation. In summary, we isolated lariat peptide inhibitors against the Abl SH1 domain, characterized their mechanism of action, and improved their affinity for wild type and mutant Abl SH1domains. Additionally, we demonstrated that lariat peptides can be used as affinity reagents to probe conformational states of Abl. Disclosures: No relevant conflicts of interest to declare.

Abstract 1357 Introduction: High dose chemotherapy (HDT) with autologous stem cell transplantation (ASCT) is historically effective treatment for patients with relapsed follicular lymphoma (FL) but relapse is common possibly due to contaminated stem cell grafts or inadequate eradication of primary disease. The addition of immunotherapy to HDT/ASCT may augment “in vivo” graft purging and achieve more effective eradication of minimal residual disease (MRD) post ASCT. This may lead to improved PFS and OS. Methods: We conducted 3 sequential prospective phase 2 trials of HDT/ASCT combined with immunotherapy in patients with relapsed FL from 1997–2010. Protocol 1 (n=13) used a-interferon 3 MU/m2 SC TIW for 2 years post ASCT. Protocol 2 (n=23) used a single infusion of rituximab (R) 375 mg/m2 for ‘in vivo’ purging 3–5 days pre-stem cell collection and 2 sets of 4 weekly R at 2 and 6 months post ASCT respectively. Protocol 3 (n=32) used 3 infusions of R 375 mg/m2 pre stem cell collection, followed by a-interferon 3 MU/m2 SC TIW × 2 years + R 375mg/m2 × 6 weekly infusions post ASCT. Eligibility included adult patients age 18–65 with stage III or IV follicular lymphoma grades 1–3a in first or second relapse. Salvage chemotherapy was either CHOP or DHAP depending on prior anthracycline exposure. High dose therapy was cyclophosphamide, carmustine, and etoposide (CBV). Minimal residual disease (MRD) assessment of t(14; 18) by quantitative PCR was serially performed in blood, PBSC and marrow. Studies were REB approved and all patients gave informed consent. Results: Sixty-eight patients from the 3 trials are included. Median age at study enrolment was 47 (30-71) and patients were a median of 31 mo (9-197) from diagnosis. 54% were male. Median FLIPI score was 2. Median # of prior chemotherapies was 1. Median number of cycles of salvage chemotherapy was 4 (2 - 10). 63 patients proceeded to ASCT. 5 patients were not transplanted either do to disease progression (2), or failed stem cell mobilization (3). Baseline characteristics of the patients enrolled in the three trials were similar, with the exception of rituximab(R) exposure in 22% of the patients in Protocol 3. 4 (6%) patients were lost to follow up. Median time to death or last follow was 84.5 mo (7-166). 32 patients (47%) relapsed and 20 (29%) have died. Median follow up times for each trial were 116, 99 and 57 mo. Actuarial median OS for Protocol 1 was 136 mo from enrolment and has not yet been reached for Protocols 2 and 3. Actuarial median PFS for Protocols 1 and 3 are 49 and 78 mo respectively and has not yet been reached for Protocol 2. MRD assessment by PCR was collected on 48 patients. Compared with study 1, the increased use of R pre SC collection improved in vivo purging by 2 logs. In Protocols 1 and 2, most still had detectable disease in the graft to a sensitivity of 1 cell/100,000. In Protocol 3, 56% had MRD negative apheresis products. 91% of 46 evaluable patients achieved molecular remission post stem cell transplant. Thirteen patients (19%) developed secondary malignancies post ASCT with a median time to malignancy of 88.5 mo (39-144). 1 patient developed 2 distinct malignancies. Secondary malignancies present were: MDS (4), AML (1), ALL (1), basal cell carcinoma (2), squamous cell carcinoma (3), thyroid cancer (1), non small cell lung cancer (1) and adenocarcinoma of unknown origin (1). Five (7%) patients transformed to DLBCL, with a median time to transformation post ASCT of 43.5 mo (17-142). The majority of patients who received rituximab immunotherapy pre and post ASCT developed persistent hypogammaglobulinemia. 17 patients (26%) developed interstitial pneumonitis or >2 respiratory complications post rituximab. 4 patients (6%) require monthly IVIG to treat recurrent respiratory infections. 10 patients (15%) developed herpes zoster 15% and 30% of patients in Protocols 1 and 3 did not complete the 2 years of alpha-IFN post ASCT secondary to one or more of depression, fatigue or cytopenias. Conclusion: HDT + ASCT combined with R +/− a-IFN produces durable PFS and molecular remissions but is associated with prolonged hypogammaglobulinemia and higher rates of interstitial pneumonitis. Three infusions of R pre SC collection achieve higher rates of molecular remission. Compliance with 2 years of a-IFN added to R post ASCT is poor and may not add clinical benefits to R alone. The additional role of HDT/ASCT in the era of R-chemotherapy for FL needs to be explored. Disclosures: No relevant conflicts of interest to declare.

Abstract 204 Treatment of chronic myeloid leukemia (CML) patients with tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, and dasatinib, results in a dramatic reduction in proliferating BCR-ABL expressing leukemia cells. However, these agents do not eliminate the CML stem cell population, indicating that inhibiting BCR-ABL kinase activity alone is not sufficient to eradicate the disease. In vitro studies of human CML cell lines and CD34+ cells isolated from CML patients, have shown that bone marrow stromal cell factor (BMSF) conditioned media can maintain important pro-survival and self-renewal activities downstream of BCR-ABL in the presence of TKIs, suggesting a role for secreted BMSFs in innate resistance to BCR-ABL kinase inhibition. However, the ability of BMSFs to maintain the leukemic potential of CML stem cells upon exposure to TKIs has not been reported. We used a standard murine retroviral transduction system to model CML blast crisis (BC-CML) and obtain cells highly enriched for leukemia initiating potential. Purified LIN-, Sca-1+, CD117+ cells (LSKs) were isolated from the bone marrow of C57BL6/J mice and retrovirally-transduced with BCR-ABL-GFP and Nup98/HoxA9-YFP then injected intravenously into recipient C57BL6/J mice. All animals developed leukemia within 21 days characterized by leukocytosis and extensive infiltration of bone marrow and spleen with leukemic blasts. LSKs expressing both BCR-ABL-GFP and Nup98/HoxA9-YFP (GFP+/YFP+ LSKs) were purified from the spleens or bone marrows of leukemic mice and cultured for 72 hrs in BMSF conditioned media across a range of concentrations (0% - 50%) in the presence and absence of imatinib (0 - 1000 nM). BMSF conditioned media reduced the cytotoxic effects of imatinib on GFP+/YFP+ LSKs as assessed by cell counts, trypan blue viability assays, and Annexin V expression by flow cytometry. Furthermore, BMSF conditioned media reduced the inhibitory effects of imatinib on GFP+/YFP+ LSK colony formation in methylcellulose, and beta-catenin expression as assessed by flow cytometry. These observations strongly suggest that signaling by stromal cell-derived soluble factors protects BC-CML stem cells from imatinib therapy by re-activating pro-survival and self-renewal pathways. The ability of BMSFs to reduce the inhibitory effect of imatinib on BC-CML stem cell self-renewal in vivo was assessed by performing secondary transplantation assays. GFP+/YFP+ LSKs were purified from primary CML mice and transplanted into secondary recipients following in vitro exposure to BMSF conditioned media in the presence and absence of 1000 nM imatinib. Survival after transplantation was compared in cohorts of 5 mice per experimental condition: Group 1 (0% BMSF, 0 nM imatinib), Group 2 (50% BMSF, 0 nM imatinib), Group 3 (50% BMSF, 1000 nM imatinib) and Group 4 (0% BMSF, 1000 nM imatinib). Survival was significantly prolonged in Group 4 mice treated with 1000 nM imatinib and this effect was abrogated by treatment with 50% BMSF conditioned media, indicating that cell-derived soluble factors contribute to maintaining BC-CML stem cell function in the presence of imatinib. Our findings strongly suggest that signaling by soluble BMSFs plays an important role in the innate imatinib resistance of CML stem cells, implicating these factors in disease relapse. Genetically defined murine models of CML provide a powerful in vivo system to identify and target soluble factors that contribute to stromal-mediated cytoprotection of CML stem cells from TKIs. Disclosures: No relevant conflicts of interest to declare.

Abstract 2571 Background: Myelodysplastic syndromes (MDS) are clonal hematopoetic stem cell disorders characterized by ineffective hematopoiesis and a propensity to develop AML predicted by conventional scoring systems such as the International Prognostic Scoring System (IPSS). Azacitidine (AZA), a hypomethylating agent is indicated for high and high intermediate IPSS scores based on survival and leukemia-free survival benefits demonstrated in randomized trials (Silverman 2002, 2006, Fenaux 2009). Additionally, improvements in fatigue, dyspnea, physical functioning, affect and psychological distress were demonstrated in the CALGB study (Kornblith 2002). We previously showed that most symptom and functional domains of quality of life (QOL) are impaired in MDS patients measured by several instruments and are primarily determined by Hb and transfusion dependence (Buckstein 2009). With the exception of the CALGB paper, there is a paucity of data assessing the ‘real world' QOL in MDS patients treated with AZA longitudinally. All consented patients with MDS followed at our center have QOL assessed every 3–4 months as part of routine care. We present the QOL scores of patients on AZA as assessed by the EORTC QLQ-C30, EQ-5D and a global fatigue scale. Methods: Clinically significant score differences were considered to be 10 points for the EORTC, and 0.05 for the EQ-5D. Linear regression analysis was used to detect each QOL change over time. Log-transformation was applied for all QOL scores to normalize the distribution. To search for significant predictive factors of each QOL, linear regression analysis (for continuous predictive factors) or Analysis of Variance (for binary predictive factors) was conducted at baseline. A two-sided p-value less than 0.05 was considered statistically significant. Results: 30 patients in our database were/are currently treated with AZA. The median age was 73 years, with 63% being male. Of the 26 patients with measureable IPSS scores, 54% were high/high intermediate risk. Seventy percent had a Hb Of the 19 TD patients only 3 became transfusion independent (TI) on AZA and 3 patients who were TI at baseline became TD. Of the 30 patients, 20 have QOL data available for analysis with a median follow up time of 10 weeks (range 0–80) and an interval duration between QOL assessments of 15.5 weeks. Fourteen out of twenty patients have serial QOL assessments, 5 with two, 9 with three or more. The only clinically significant improvements were observed with the EORTC physical functioning and fatigue subscales but constipation scores were higher and global health status/QOL deteriorated over time (Figure 1). At baseline assessment ferritin ≥ 1000 ug/L was negatively associated with physical functioning (p=.0007), cognitive functioning (p=.0012), global QOL (p=.0048) and global fatigue (p=.0003) while transfusion dependence was not predictive of QOL scores. No significant clinical improvements were detected by linear regression or ANOVA over time, but constipation worsened using both models. The health utilities (determined by the summary score of the EQ-5D) are seen in table 1. Conclusion: Many clinically important function and symptom domains of 3 different QOL instruments have not changed significantly over time in our patients receiving AZA. This is likely explainable by the limited sample size and serial number of assessments in our patients. Our present patient population is higher risk than that tested in the CALGB study and our previous report on the MDS patients in our database. Furthermore, we have yet to see the rates of transfusion independence that might be associated with improved QOL. The negative association of increased ferritin with numerous symptom and function scores may simply be a surrogate for the extent of transfusion dependence. We hope that with longer follow up and larger sample size, we will be able to reproduce the QOL benefits observed in the pivotal CALGB trial (Kornblith 2002). Disclosure: No relevant conflicts of interest to declare.

Abstract 1761 Poster Board I-787 Introduction Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis culminating in peripheral blood cytopenias and a propensity to Acute Myelogenous Leukemia (AML). The gold standard diagnostic test is the bone marrow aspirate and biopsy but clinicians are often hesitant to refer patients with unexplained cytopenias or macrocytosis for this test. Consequently undiagnosed patients are deprived access to effective treatments. Previously (Buckstein et al., Leukemia Research 33 (2009) 1313–1318) we identified factors which were independently predictive of diagnosing MDS at the time of bone marrow, including; age, mean corpuscular volume (MCV), red cell distribution (RDW), and lactate dehydrogenase (LDH). In this current study, we set out to validate our findings and determine the sensitivity and specificity of our scoring system for routine clinical practise. Methods We reviewed all bone marrow reports conducted at a tertiary care center for the years (January through December) 2006–2008 inclusive. Our inclusion criteria included bone marrows done for unexplained cytopenias and/or macrocytosis. We excluded all outside consultative referrals and bone marrows done for staging or remission assessment in patients with pre-existing or strongly suspected hemato-lymphoid diagnoses. In cases where the patient had more than one bone marrow, only the most recent marrow was used. The bone marrows were reviewed by two experienced hematopathologists, and the diagnosis of MDS was made using the WHO and/or FAB classification systems. Marrows were classified as ‘confirmed MDS’, ‘suspected MDS’ or ‘Not MDS’. Electronic patient charts were reviewed to determine the age, MCV, LDH and RDW at the time of diagnostic bone marrow. A factor was considered positive if the age was > 65 or if its value exceeded the upper range of normal for our laboratory standards (MCV > 96 fl, LDH > 250 IU/L, RDW >14.5%). All patients for whom we had 4 recorded factors were then assigned a score that ranged from 0 to 4. We determined the distribution of scores within this population, and the sensitivity and specificity of this scoring method in predicting a histopathological diagnosis of MDS. Results Three-hundred and forty bone marrows met our inclusion criteria, and 289 (85%) had all four factors recorded at the time of diagnosis. The predictive ability of the MDS score is summarized in Table 1. The probability of diagnosing MDS increased from 8% with a score of 0 to 46% with a score of 3 or 4. A similar trend was seen when all 344 marrows were analysed. Our scoring system had high sensitivity (> 96%), when only one predictive factor was present and high specificity (96%) when 4 predictive factors were present at the time of diagnostic marrow. (Table 2) Conclusions In patients with unexplained cytopenias, or macrocytosis undergoing a diagnostic BM test, the pre-test probability of a MDS diagnosis increases as the number of predictive factors increases. The specificity of our MDS score also follows a similar trend. The sensitivity is high with only one positive factor indicating a lower score can be used as a screening tool. We have effectively validated a scoring system that may help clinicians decide on the utility of arranging diagnostic bone marrow examinations for patients with undiagnosed cytopenias and macrocytosis. Disclosures Buckstein: Celgene : Honoraria; Novartis: Honoraria.

This abstract was removed at the request of the author.

Introduction: The biology of myelodysplastic syndrome (MDS) is poorly understood, and treatment options are limited. Thus, most MDS patients require chronic red blood cell transfusion, and many develop secondary iron overload. Although the pathophysiological consequences of iron overload to the heart, liver, and endocrine organs have been well characterized, its effects on haematopoiesis have not been studied. However, it has been observed that chelation therapy in iron-overloaded MDS patients may result in reduction of transfusion requirements, and recent studies have suggested a correlation between the use of iron chelation therapy and improvement in leukaemia-free survival in MDS. At the cellular level, iron toxicity is mediated in large part via the generation of reactive oxygen species (ROS). It has been shown in animal models that accumulation of ROS leads to senescence of haematopoietic stem cells, and that ROS cause DNA damage and promote the development of malignancy. These effects of ROS may be particularly important in MDS, in which haematopoiesis is already severely compromised and genetic instability is a striking feature. Hypothesis: We hypothesize that iron overload secondary to transfusion leads to increased levels of intracellular ROS in early haematopoeitic cells in MDS. The increase in intracellular ROS in MDS would be predicted to lead further impairment of haematopoiesis via stem cell exhaustion and while promoting accumulation of DNA damage by myelodysplastic stem cells and early progenitors, thus accelerating progression of MDS to acute leukaemia. Results: To test this hypothesis, we examined the relationship between transfusion-related iron overload and ROS content of CD34+ bone marrow cells in MDS. ROS content was measured in CD34+ cells by flow cytometry in bone marrow aspirates from 34 consecutive MDS patients (CMML=4, MDS/MPD=2, RA=4, RARS=3, RCMD=2, RAEB 1=6, RAEB 2=12, RAEB-t/AML=1). The patients represented a wide range of prior transfusion burden (0->300 units PRBC) and serum ferritin levels (11->10000 μg/L). ROS was strongly correlated with serum ferritin concentration for patients with iron overload (serum ferritin >1000 μg/L; n=14, R=0.733, p To assess the effect of iron overload on normal stem cell and progenitor function, we established a mouse model of subacute bone marrow iron overload. B6D2F1 mice were loaded with iron dextran by intraperitoneal injection (150mg total iron load over 21 days), and sacrificed three days after the end of iron loading. Iron staining of tissue sections confirmed iron deposition in the bone marrow, liver, and myocardium. The development of splenomegaly was noted in iron-loaded animals. Flow cytometric analysis revealed increased apoptosis of bone marrow cells in iron loaded mice based on annexin V+/7 AAD-staining (6.26±0.96% versus 3.54±0.99% for control mice, paired student’s t-Test p Conclusions: These results establish a relationship between CD34+ cell ROS content and serum ferritin concentration in MDS patients with iron overload, and indicate that iron chelation therapy in this patient population reverses this ROS accumulation. The physiological consequences of this relationship are currently being investigated in this patient set by haematopoietic colony assays and assessment of DNA damage in CD34+ cells. Nonethelesss, these data may have key implications for the deployment of iron chelation therapy in MDS patients, and may explain the association between the use of iron chelation and improved leukaemia-free survival in MDS.

High-dose therapy and autologous stem cell transplantation (HDT/ASCT) and rituximab immunotherapy have been increasingly applied in the management of non-Hodgkin’s lymphomas (NHL). Although both approaches have been individually associated with B-cell depletion and hypogammaglobulinemia, the incidence, time course, and predictors of prolonged deficiencies following a combined treatment approach is unknown. Methods: We completed a series of prospective phase II studies of HDT/ASCT combined with rituximab for patients with relapsed follicular lymphoma (FL) or diffuse large B cell lymphoma (DLBCL). In two phase II trials of patients with FL (R/Tx and R-IFN/Tx), patients received 9 infusions of rituximab 375mg/m2 as both an in vivo purge and as maintenance post HDT/ASCT. In a trial with relapsed DLBCL or transformed lymphoma, patients received 8 infusions with rituximab 375mg/m2 only as part of the salvage chemotherapy regimen (R-ESHAP/Tx). Immunoglobulin levels were expressed as percentages with 100% representing the lower limit of normal at the institutional lab. Hypogammaglobulinemia was defined as Results: Fifty-one patients with FL were transplanted and were subsequently treated with maintenance rituximab (R/Tx study) or with maintenance rituximab (MR) and interferon-alpha (R-IFN/Tx). Twenty-six patients with relapsed aggressive-histology lymphoma were transplanted with the R-ESHAP salvage regimen. The mean age of the FL patients was 45 and the mean age of the aggressive-histology lymphoma patients was 49. The median number of prior therapies was 1 (range 1–3) for the FL patients and bone marrow involvement at study entry was present in 63%. The median number of prior therapies was 1 (range 1–5) for the aggressive-histology lymphoma patients and bone marrow involvement was present in 15%. Baseline IgG hypogammaglobulinemia was seen in 18% of patients with relapsed FL and 15% of patients with relapsed DLBCL/transformed lymphoma. Median baseline Ig levels in each study are displayed in Figure 1. Patients with FL undergoing HDT/ASCT and MR (combined R/Tx and R-IFN/Tx studies) had prolonged IgG hypogammaglobulinemia compared to patients with aggressive-histology lymphoma (log-rank p=0.0047; see Figure 2). The median time-to-recovery from hypogammaglobulinemia had not been reached after a median follow-up of 42 months. Similarly, IgM level recovery was delayed in the FL group compared to the aggressive-histology patients (p=.0001), although late recovery was noted for this Ig subtype (median time-to-recovery 36 months post-ASCT). Univariate analyses revealed that persistent hypogammaglobulinemia (at 24 months) was associated with FL histology (vs. DLBCL/transformed; p=0.006), bone marrow involvement at study entry (p=0.008), and hypogammaglobulinemia at study entry (p=0.026). Factors not associated with persistent hypogammaglobulinemia included: number of prior therapies, age, and administration of maintenance interferon-alpha. Post-transplant (>3 months) infections included herpes zoster reactivation (n=7) and pneumonia (n=8). One individual in the R-ESHAP/Tx study died of PCP pneumonia 4 months post HDT/ASCT. A relationship between grade III-IV infections and prolonged hypogammaglobulinemia was not evident on univariate analysis (data not shown). Conclusions: Patients with follicular lymphoma undergoing high dose therapy and stem cell transplantation together with rituximab maintenance are likely to experience a prolonged hypogammaglobulinemia whereas this is less likely with patients with aggressive-histology lymphoma undergoing similar doses of rituximab as part of their salvage therapy. Further research is required to elucidate the relative contributions of disease histology, bone marrow involvement, baseline hypogammaglobulinemia, timing of rituximab infusions or other factors not yet identified. Figure Figure Figure Figure

A central conundrum in the biology of myelodysplastic syndrome (MDS) is the observation that haematopoietic cells derived from the MDS clone have an increased disposition to apoptosis, yet gain clonal dominance in the bone marrow (BM). One explanation for this apparent paradox could be that MDS stem cells attain clonal dominance by acquiring an augmented probability of self-renewal. We therefore sought to identify novel genes associated with self-renewal that are overexpressed in both MDS and AML. By analyzing the growth of individual clonal siblings derived from low passage cultures of OCI-AML4 we found the expression of the orphan nuclear receptor NR2F6 to be four-fold higher in leukaemia cells with high proliferative capacity, compared to those which spontaneously lose proliferative ability. NR2F6 mRNA transcripts were more abundant in patients (n=37) with MDS, CMML, and AML compared to normal BM (n=16; p