ALBERT

Description: Abstract 99 Whole genome sequencing with next generation technologies represents a new, unbiased approach for discovering somatic variations in cancer genomes. Our group recently reported the DNA sequence and analysis of the genomes of two patients with normal karyotype acute myeloid leukemia (AML). Improvements in next generation sequencing technologies (principally, paired-end sequencing) led us to reevaluate the first case (Ley et al, Nature 456:66–72, 2008) with deeper sequence coverage. We discovered a novel frameshift mutation in DNMT3A, one of the three genes in humans (DNMT1, DNMT3A, and DNMT3B) that encodes a DNA methyltransferase that catalyzes the addition of methyl groups to cytosine within CpG dinucleotides. We then sequenced all the coding exons of this gene in 280 additional de novo cases of AML to define recurring mutations. 62/281 de novo AML cases (22%) had mutations with translational effects in the DNMT3A gene. 18 different missense mutations were identified, the most common of which was at amino acid R882 (37 cases). Frameshifts (n=6), nonsense mutations (n=6), splice site mutations (n=3), and a 1.5 Mbp deletion that included the DNMT3A gene were also identified. DNMT3A mutations were highly enriched in cases with intermediate risk cytogenetics (56/166=33.7%; p

Description: Abstract 3204 A high pressure circulatory system has two diametrically opposed requirements for its function: it must be able to rapidly gel to prevent blood loss when the integrity of the vasculature is compromised while simultaneously maintaining fluidity when the vasculature is intact. The endothelium is primarily responsible for maintaining blood fluidity, producing rapidly acting labile substances that inhibit both the clotting of blood and the adhesion and aggregation of platelets. Among these substances are the prostaglandins (PGE1, PGI2, PGD2), which bind platelet membrane receptors, raise concentrations of intracellular cyclic adenosine monophosphate (cAMP), and inhibit platelet functions. The major effector of increased cAMP is the serine/threonine kinase protein kinase A (PKA). Of the numerous targets for PKA, one of the most highly phosphorylated upon cAMP increase is glycoprotein (GP) Ibβ, a component of the GPIb-IX-V complex, the platelet receptor for VWF that mediates the initial adhesion of platelet to the vessel wall at sites of injury. The GPIb-IX-V complex consists of 4 type I transmembrane polypeptides, GPIbα, GPIbβ, GPV and GPIX. GPIbα and GPIbβ are disulfide linked in a 1:2 ratio, and the resulting GPIb is non-covalently associated with GPIX and GPV in a 2:2:1 ratio. The VWF-binding site resides within the N-terminal 300 amino acids of GPIbα 500 Å above the platelet surface. Although current data indicate that PKA phosphorylation of the GPIbβ cytoplasmic domain (at Ser166) inhibits the ability of GPIbα to bind VWF, the molecular mechanism(s) have yet to be elucidated. The cytoplasmic domain of GPIbβ associates with calmodulin (in the juxtamembrane 20 amino acids) in resting platelets; calmodulin dissociates upon platelet activation. With elevated cytosolic cAMP, GPIbβ Ser166 becomes phosphorylated and associates with 14-3-3ζ. An interesting feature of the cytoplasmic sequence N-terminal to Ser166 is its extreme cationic nature, containing 8 Arg residues in a stretch of 17 amino acids. Other cytosolic proteins with similar polybasic sequence (MARCKS, GAP43) function as organizers of the signaling phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), and promote the formation of lipid rafts; we reasoned that the polybasic region of GPIbβ might function similarly, organizing rafts when unbound by protein, but not when occupied by calmodulin or 14-3-3ζ. Platelet activation increases raft-associated GPIb-IX-V two fold, with concomitant dissociation of calmodulin from GPIbβ. Here we present evidence that the cytoplasmic domain of GPIbβ plays a role in the localization of the GPIb-IX-V complex to lipid rafts. Treatment of platelets with agents that increase cAMP (PGI2 or forskolin) inhibited GPIb-IX-V-dependent platelet functions, including ristocetin-induced aggregation, shear-induced aggregation and adhesion to VWF under flow. This effect was prevented by the cell-permeable PKA-specific inhibitor H-89. Consistent with the functional importance of GPIb-IX-V localization to lipid rafts, PGI2 and forskolin reduced the raft content of GPIb-IX-V by 35%, and this effect was reversed by H-89. We have thus uncovered a mechanism for long-observed inhibition of platelet adhesion by agents that elevate cytosolic cAMP concentrations, which depends on modulating the quantity of GPIb-IX-V complexes associated with lipid rafts. “Resting” platelets ex vivo are relatively quiescent because calmodulin occupies the GPIbβ polybasic region. The situation changes rapidly when platelets are activated, with more of the complex assuming a ligand-competent state as calmodulin dissociates and the complex organizes rafts. Elevations of cAMP promote phosphorylation of GPIbβ, enabling 14-3-3ζ association, which also displaces the GPIbβ tail from the membrane, disrupting raft association and adhesive function. Disclosures: No relevant conflicts of interest to declare.

Description: The recent shift to the use of stem cells mobilized by granulocyte colony-stimulating factor (G-CSF) for hematopoietic transplantation has increased chronic graftversus-host disease (GVHD), although the mechanisms of this are unclear. We have found that G-CSF invokes potent type 17 rather than type 1 or type 2 differentiation. The amplification of interleukin-17 (IL-17) production by G-CSF occurs in both CD4 and CD8 conventional T cells and is dependent on, and downstream of, G-CSF–induced IL-21 signaling. Importantly, donor IL-17A controls the infiltration of macrophages into skin and cutaneous fibrosis, manifesting late after transplantation as scleroderma. Interestingly, donor CD8 T cells were the predominant source of IL-17A after transplantation and could mediate scleroderma independently of CD4 T cells. This study provides a logical explanation for the propensity of allogeneic stem cell transplantation to invoke sclerodermatous GVHD and suggests a therapeutic strategy for intervention.

Description: Abstract 196 Fanconi anemia (FA), the most common inherited bone marrow failure syndrome, is characterized by progressive loss of hematopoietic stem cells, aplastic anemia, genomic instability and cancer predisposition. Induced pluripotent stem (iPS) cells are a promising source of cells for disease-specific investigations and genetic correction. It has been reported that human FA dermal fibroblasts are resistant to direct reprogramming without prior genetic correction (Raya et al., Nature, 2009), but the mechanism remains unclear. In this study we aimed to define the role of the FA pathway during the transition from fibroblasts to iPS cells in murine cells. We transduced Fanca-/- and wild type (wt) tail-tip fibroblasts with four defined factors (Oct3/4, Klf4, Sox2, c-Myc) and enumerated the number of iPS colonies that were derived from 1×105 cells. We noted a 〉10-fold decrease in the reprogramming efficiency of Fanca-/- cells compared to wt controls [median (range): wt 132 (0 to 1296) colonies, efficiency 0.328%, n=17; Fanca-/- 4 (0 to 80) colonies, efficiency 0.019%, n=10, p

Description: Suppressor of cytokine signaling-3 (SOCS3) is the main intracellular regulator of signaling by granulocyte colony-stimulating factor, an immune-modulatory cytokine used to mobilize stem cells for transplantation. We have therefore studied the contribution of SOCS3 to the spectrum of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (SCT). Grafts from SOCS3−/Δvav donor mice in which SOCS3 deficiency is restricted to the hematopoietic compartment had an augmented capacity to induce acute GVHD. With the use of SOCS3−/ΔLysM and SOCS3−/Δlck donors in which SOCS3 deficiency was restricted to the myeloid or T-cell lineage, respectively, we confirmed SOCS3 deficiency promoted acute GVHD mortality and histopathology within the gastrointestinal tract by effects solely within the donor T cell. SOCS3−/Δlck donor T cells underwent enhanced alloantigen-dependent proliferation and generation of interleukin-10 (IL-10), IL-17, and interferon-γ (IFNγ) after SCT. The enhanced capacity of the SOCS3−/Δlck donor T cell to induce acute GVHD was dependent on IFNγ but independent of IL-10 or IL-17. Surprisingly, SOCS3−/Δlck donor T cells also induced severe, transforming growth factor β– and IFNγ-dependent, sclerodermatous GVHD. Thus, the delivery of small molecule SOCS3 mimetics may prove to be useful for the inhibition of both acute and chronic GVHD.

Description: Tumor necrosis factor (TNF) is a key cytokine in the effector phase of graft-versus-host disease (GVHD) after bone marrow transplantation, and TNF inhibitors have shown efficacy in clinical and experimental GVHD. TNF signals through the TNF receptors (TNFR), which also bind soluble lymphotoxin (LTα3), a TNF family member with a previously unexamined role in GVHD pathogenesis. We have used preclinical models to investigate the role of LT in GVHD. We confirm that grafts deficient in LTα have an attenuated capacity to induce GVHD equal to that seen when grafts lack TNF. This is not associated with other defects in cytokine production or T-cell function, suggesting that LTα3 exerts its pathogenic activity directly via TNFR signaling. We confirm that donor-derived LTα is required for graft-versus-leukemia (GVL) effects, with equal impairment in leukemic clearance seen in recipients of LTα- and TNF-deficient grafts. Further impairment in tumor clearance was seen using Tnf/Lta−/− donors, suggesting that these molecules play nonredundant roles in GVL. Importantly, donor TNF/LTα were only required for GVL where the recipient leukemia was susceptible to apoptosis via p55 TNFR signaling. These data suggest that antagonists neutralizing both TNF and LTα3 may be effective for treatment of GVHD, particularly if residual leukemia lacks the p55 TNFR.

Description: Abstract 4257 Iron in the plasma is bound to transferrin. The total iron binding capacity (TIBC) represents the maximum amount of iron that can be bound and is directly related to transferrin concentration. TIBC and serum transferrin concentration are increased in iron deficiency and decreased in iron overload. Recently, an association was reported between single nucleotide polymorphisms (SNPs) in the transferrin gene, TF, on chromosome 3q22.1, and serum transferrin levels (Benyamin et al. Am J Hum Genet. 2009;84:60-65). In the current study, we investigated whether the association between SNP rs3811647 in TF and transferrin levels (assessed by measurement of TIBC) is attributable to an effect on regulation of body iron status. The Personalized Medicine Research Project (PMRP) is the largest population-based biobank in the US containing genetic, phenotypic and environmental information on approximately 20,000 people. PMRP is part of the NHGRI-funded eMERGE (www.gwas.net) network. Previously, genotyping was performed on selected PMRP samples with the Illumina Human660W-Quad BeadChip platform. Eligible participants in the current study were 491 white men age ≥ 25 y and 747 white women ≥ 50 y with serum ferritin (SF) values collected between 1985 and 2010. Exclusion criteria included a diagnosis of celiac disease and previous phlebotomy treatment for hemochromatosis. Using TIBC as a marker of serum transferrin for eligible participants having multiple measurements, mean TIBC and median serum ferritin were considered in analyses. Subsets of participants included cases of iron deficiency with multiple measurements of SF ≤ 12 μg/L and iron-replete controls (all measurements of SF 〉 100 μg/L in men, all SF 〉 50 μg/L in women). Regression analysis was used to examine the association between outcomes (case-control status, natural log of serum ferritin, TIBC) and each of 54 SNPs, adjusted for gender. These SNPS included three in iron genes (rs3811647 in TF, rs1800562 in HFE, and rs2302591 in FLVCR2) and were selected for analysis on the basis of a GWAS of iron-related measures conducted in a separate study of iron deficient cases and iron-replete controls identified in the Hemochromatosis and Iron Overload Screening (HEIRS) Study. Statistical significance was defined as a SNP showing a p-value for association less than 0.001; the threshold is based on a nominal alpha of 0.05 with Bonferroni multiple test correction for the total number of SNPs analyzed. Genotypes were coded as 0, 1, or 2, indicating the number of copies of the less frequent of the two alleles in the genotype. Values for mean TIBC were analyzed for 1175 individuals (726 women, 449 men); median SF was analyzed for 1143 participants (693 women, 450 men). In the subset analyses, there were 258 cases with iron deficiency and 505 controls. The strongest statistical evidence for association with TIBC was found for SNP rs3811647 in the TF gene (observed p-value = 6.05 × 10-6, adjusted for gender). The minor allele frequency for SNP rs3811647 was 0.34. The regression slope parameter was 14.5, indicating that increasing copies of the minor allele were associated with increasing levels of TIBC. In contrast, there was no significant association with SF (observed p=0.22) or case vs. control status (odds ratio 1.26, observed p=0.21), adjusted for gender. For the C282Y mutation in the HFE gene, increasing copies of the minor allele were associated with decreasing levels of TIBC (observed p-value = 0.002, adjusted for gender). The fact that SNP rs3811647 in the TF gene was associated with TIBC levels but showed no significant association with either serum ferritin or the presence of iron deficiency does not support a role for the SNP in regulation of body iron status. Thus, the SNP may affect TIBC independently of iron status. Elevation of transferrin levels could help withhold iron from microorganisms, conferring protection from infection. Use of TIBC as an index of iron deficiency may be confounded by the existence in the population of the minor allele in the rs3811647 genotype, resulting in elevation of TIBC without a corresponding decrease in body storage iron. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 415 Background: The ability to cure patients (pts) with advanced Hodgkin's Lymphoma (HL) with combination chemotherapy (CC) (MOPP and variants, ABVD and variants) represented a major milestone in oncology research, and CC became a paradigm for other malignancies. Further, the rationale for combined modality therapy (CMT) (radiation (RT) and CC) in HL evolved based on the high frequency of relapse in initially involved sites. As response rates and survival improved, newer treatments such as the combined modality Stanford V regimen were developed to shorten the duration of chemotherapy, add RT to sites of disease and reduce toxicity while maintaining or improving the cure rate. Indeed, the Stanford V regimen was tested and validated in a Phase II co-operative group trial (E1492) (J Clin Oncol 2000; 18:972). In order to investigate this approach against “standard” therapy, we conducted a randomized Phase III Intergroup trial of ABVD vs. the Stanford V regimen for patients with locally extensive or advanced HL. Objectives: The trial was designed to detect a 33% reduction in the failure free survival (FFS) hazard rate with Stanford V compared with ABVD, which corresponds to a difference in five-year FFS of 64% vs. 74%. Method: Patients with locally extensive (defined as clinical Ann Arbor Stage I-IIA/B and bulky mediastinal disease (BMD) (mass 〉 1/3 maximum intrathoracic diameter on standing postero-anterior chest x-ray or 〉/−10 cm on computerized tomography) or advanced (Ann Arbor Stage III or IV) HL were randomized to receive either ABVD × 6–8 cycles (C) (51% had 6 C, 35% had 8 C, 14% had 5cm or for macroscopic splenic disease). The log-rank test was used to compare FFS for all eligible patients stratified on extent of disease (locally extensive vs. advanced), and number of International Prognostic Factor Project (IPFP) risk factors (0–2 vs. 3–7). An extended Cox model was also used to address non-proportional hazard between the two arms. Results: 854 pts enrolled from April, 1999 to June, 2006 and 812 were eligible for analysis. 404 pts were randomized to ABVD and 408 to Stanford V. Median age was 33 yrs in both arms (range 16–83). 53% were men and 47% women; 4% had Stage I, 31% had Stage II, 39% had Stage III and 25% had Stage IV disease by Ann Arbor criteria. 35% of pts on ABVD and 35% on Stanford V had BMD. Three % of pts had nodular lymphocyte predominant HL, 77% of pts had nodular sclerosis HL, 14% had mixed cell HL. Age, stage, pathology and risk factors (0–2 vs. 3–7) were similar in both arms. In total, 65% were IPFP score 0–2 and 33% were 3–6. Response rate. There was no difference in response rates (RR) between the two arms (ABVD=72% CR+ CCR, 7.7% PR, 7.9% SD; Stanford V= 69 % CR +CCR, 7% PR and 10 % SD. 8% were not evaluable for response on ABVD and 9% on Stanford V. Toxicity was similar in both groups. The most frequent Grade 3 + 4 toxicity was neutropenia, and was similar between the 2 groups (76% Grade 3 + 4 in ABVD and 70% Grade 3+ 4 in Stanford V). Grade 5 toxicity was 5 cm and macroscopic splenic disease). There was more Grade 3 lymphopenia (p〈 0.0001) and more Grade 3 + 4 sensory neuropathy (p〈 0.0001) on Stanford V. Thus ABVD (plus RT for BMD) remains the standard of care because Stanford V did not meet the objective of 33% improvement in FFS. For some patients, Stanford V, when given as described with RT, remains an acceptable alternative. Disclosures: Friedberg: Genentech: Honoraria. Blum:Seattle Genetics: Research Funding; Novartis: Research Funding; Celgene: Research Funding. Horning:Genentech: Employment.

Description: Abstract 577 Trisomy 8 (+8), the most frequent numerical aberration in AML, occurs in approximately 10% of pts. In one-third of them, +8 is the sole chromosome (chr) anomaly; it is considered to confer an intermediate/adverse prognosis but further studies are required to define the clinical and biologic significance of this cytogenetic abnormality. We assessed the associations of sole +8 with prognostic gene mutations, outcome, and gene and miR expression profiles by comparing a relatively large cohort of sole +8 de novo AML pts (n=80; median, 63 years [y]; range, 18–84 y) with cytogenetically normal (CN) de novo AML pts (n=483; median, 60 y; range, 18–83 y). CN pts constitute the largest cytogenetic group in AML, with an overall intermediate prognosis modified by molecular markers. Markers analyzed in the present sole +8 cohort included mutations in NPM1, FLT3 (FLT3 internal tandem duplication [ITD], FLT3 tyrosine kinase domain), CEBPA, WT1, IDH1/2, N/KRAS and RUNX1. All pts were enrolled on frontline cytarabine/daunorubicin based CALGB protocols. No pt included in outcome analyses received allogeneic stem cell transplant in 1st complete remission (CR). Median follow-up was 7.1 y for pts alive. Compared with CN pts, sole +8 pts had lower platelet counts (P

Description: Graft-versus-host disease and graft rejection are major complications of allogeneic HLA-mismatched stem cell transplantation or organ transplantation that are caused by alloreactive T cells. Because a range of acute viral infections have been linked to initiating these complications, we hypothesized that the cross-reactive potential of virus-specific memory T cells to allogeneic (allo) HLA molecules may be able to mediate these complications. To analyze the allo-HLA reactivity, T cells specific for Epstein-Barr virus, cytomegalovirus, varicella zoster virus, and influenza virus were tested against a panel of HLA-typed target cells, and target cells transduced with single HLA molecules. Eighty percent of T-cell lines and 45% of virus-specific T-cell clones were shown to cross-react against allo-HLA molecules. The cross-reactivity of the CD8 and CD4 T-cell clones was directed primarily against HLA class I and II, respectively. However, a restricted number of CD8 T cells exhibited cross-reactivity to HLA class II. T-cell receptor (TCR) gene transfer confirmed that allo-HLA reactivity and virus specificity were mediated via the same TCR. These results demonstrate that a substantial proportion of virus-specific T cells exert allo-HLA reactivity, which may have important clinical implications in transplantation settings as well as adoptive transfer of third-party virus-specific T cells.