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Hereditary spherocytosis (HS) is the most common cause of inherited red cell membranopathy, due to mutations in genes encoding for membrane or cytoskeletal proteins, including band 3, ankyrin, spectrin, band 4.1 or band 4.2. Membrane instability results in membrane surface area loss and generation of spherocytic red cells with elevated MCHC, decreased cellular deformability and reduced red cell survival, due to splenic sequestration. Clinical management of the hemolytic anemia due to HS depends on the age of the patient and the severity of anemia. Splenectomy is indicated in children with symptomatic anemia. A classic diagnostic test for HS is the incubated osmotic fragility: this test highlights the crucial role that ATP content plays in maintenance of normal RBC function including membrane stability: it is generally believed that the increased fragility of HS is the result of abnormal ATP depletion over the 24hr incubation. We explored the hypothesis that pyruvate kinase activator, mitapivat, by modulating ATP content could have potential beneficial effects for HS RBCs in band 4.2-/- mice, a well-established model of HS (Peters LL et al JCI 103: 1527, 1999). 4.2-/- mice exhibit moderate anemia which recapitulates most of the features of typical human HS without showing the profound anemization seen in other mouse models. Oral AG-348 administration to band 4.2-/- mice at dosages of 200 mg/kg/day over 6 months resulted in (i) improvement of anemia with reduced reticulocyte count (Hb 11.6±0.035 g/dL, n=17 vs 13.104±0.09 g/dL, n=9; P Disclosures Kung: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Kosinski:Agios Pharmaceuticals Inc: Current Employment, Current equity holder in publicly-traded company. Dang:Agios Pharmaceuticals Inc.: Current Employment, Current equity holder in publicly-traded company. Brugnara:Sysmex America Inc.: Consultancy; American Journal of Hematology: Other.

To the editor: KIR3DL2, a killer immunoglobulin-like receptor normally expressed by a subset of natural killer (NK) cells, is aberrantly expressed in Sezary syndrome (SS), an aggressive cutaneous T-cell lymphoma (CTCL). In SS patients, the detection of KIR3DL2 expression on circulating tumor cells

Diagnosis of gastrointestinal GVHD (GI-GVHD) is based on clinical symptoms and histologic findings. No biomarkers predicting responses to treatment are routinely available even though 30% to 50% of patients will not respond to corticosteroids. In this study, we aimed to evaluate fecal calprotectin, α-1-antitrypsin (α1-AT), and elastase at the time of first symptoms as diagnostic and prognostic tools for GI-GVHD in 72 consecutive patients, of whom 51 developed GI-GVHD. The prognostic value of markers was evaluated by their association with complete response (CR) and steroid-resistant (SR) GVHD. Calprotectin and α1-AT concentrations increased with GI-GVHD initial stages but patients with initial stage 1 GI-GVHD had similar marker levels to patients without GI-GVHD, so sensitivity to diagnose GI-GVHD was weak. In contrast, calprotectin and α1-AT were predictors for SR-GVHD and CR. Multiple regression modeling identified calprotectin and α1-AT concentration as independently predicting SR-GVHD together with initial stage > 2 GI-GVHD. Our results showed that fecal calprotectin and α1-AT levels at the time of diagnosis are predictive for responses to treatment but are not diagnostic markers for initial stage 1 to 3 GI-GVHD.

In rodent graft-versus-host disease (GVHD) models, anti–IL-21 neutralizing mAb treatment ameliorates lethality and is associated with decreases in Th1 cytokine production and gastrointestinal tract injury. GVHD prevention was dependent on the in vivo generation of donor-inducible regulatory T cells (Tregs). To determine whether the IL-21 pathway might be targeted for GVHD prevention, skin and colon samples obtained from patients with no GVHD or grade 2 to 4 GVHD were analyzed for IL-21 protein expression. By immunohistochemistry staining, IL-21 protein-producing cells were present in all gastrointestinal tract samples and 54% of skin samples obtained from GVHD patients but not GVHD-free controls. In a human xenogeneic GVHD model, human IL-21–secreting cells were present in the colon of GVHD recipients and were associated with elevated serum IL-21 levels. A neutralizing anti–human IL-21 mAb given prophylactically significantly reduced GVHD-associated weight loss and mortality, resulting in a concomitant increase in Tregs and a decrease in T cells secreting IFN-γ or granzyme B. Based on these findings, anti–IL-21 mAb could be considered for GVHD prevention in the clinic.

Sickle cell disease (SCD) is a worldwide distributed hereditary red cell disorder characterized by chronic hemolytic anemia in association with acute and chronic life-threatening complications mainly related to acute vaso-occlusive events (VOCs) due to amplified inflammatory response and defective pro-resolving events. Imatinib is an oral Tyrosine (Tyr)-kinase inhibitor, developed for the treatment of chronic myeloid leukemia (CML). Few case reports on SCD patients with CML undergoing imatinib treatment highlight the beneficial impact of imatinib on severity and recurrence of acute VOCs in SCD. In red blood cells (RBCs), Imatinib has been shown to interfere with Tyr-phosphorylation state of the integral membrane protein band 3, affecting RBC microparticle formation. Here, we study the actions of Imatinib on a model of acute VOCs using humanized SCD mice (Hbatm1(HBA)TowHbbtm2(HBG1,HBB*)Tow). We treated SCD and control healthy mice (AA, Hbatm1(HBA)Tow Hbbtm3(HBG1,HBB)Tow) (n=6-7 animals in each group) with Imatinib 50 mg/Kg/d for 2 weeks before hypoxia/Reoxygenation (H/R) stress used to mimic acute VOCs. Under normoxia, we found that in SCD mice Imatinib significantly reduced (i) Tyr-phosphorylation state of sickle red cell membrane proteins; (ii) the amount of phosphatidyl-serine (PS)+ sickle RBCs; and (iii) the release of erythroid microparticles, which was associated with accumulation of hemichromes and a more efficient erythrophagocytosis compared to vehicle treated animals. In SCD mice exposed to H/R stress, imatinib significantly decreased the H/R-induced (i) increase in peripheral neutrophil count; (ii) lung inflammatory cell infiltrate; (iii) kidney inflammatory cell infiltrate. In the lung of H/R SCD mice, Imatinib inhibited the H/R induced NF-kB activation and prevented the up-regulation of (i) pro-inflammatory cytokines such as ET-1; (ii) vascular endothelial activation markers (VCAM-1, ICAM-1); (iii) pro-fibrotic markers such as PDGF-B and (iv) the activation of UPR system. In the kidney of H/R SCD mice, Imatinib significantly reduced H/R induced expression of ET-1, VCAM-1 and E-selectin, which were again associated with inhibition of NF-kB. In addition, Imatinib significantly upregulated the expression of the microRNAs miR200a/b, which has been described to reduce renal fibrosis. Finally, in isolated aorta from Imatinib treated SCD exposed to H/R stress, a significant reduction in vascular activation markers was observed compared to vehicle treated animals. Collectively, our data indicate that Imatinib acts on multiple targets, modulating signal transduction and reducing inflammatory vasculopathy and extracellular matrix remodeling process related to VOC in SCD. Thus, Imatinib might represent a new therapeutic tool in clinical management of SCD patients. Disclosures El Nemer: Imara: Research Funding.

In an experimental model we demonstrated that endothelial cells of all organs are targets of the alloimmune reaction. Here, in 68 digestive biopsies, we found endothelial lesions by immunohistochemistry and ultrastructure in patients with severe acute graft-versus-host disease (GVHD). In contrast, no such endothelial cell alterations were found either in patients without GVHD or in nongrafted controls. In the biopsies with severe GVHD lesions, ultrastructure showed rupture of the capillary basal membrane and extravased red blood cells. These pericapillary hemorrhages were highly correlated with GVHD severity. In a separate cohort of 39 patients who underwent an allogeneic transplantation after a nonmyeloablative conditioning, 8 patients had intestinal biopsies. Three of these latter patients had both severe pathologic lesions of GVHD and similar endothelial lesions, thus, strengthening the concept that endothelial lesions are linked to GVHD severity and not to the intensity of the conditioning regimen. (Blood. 2004;103:4681-4684)

Fas (CD95) is a death receptor involved in apoptosis induction on engagement by Fas ligand (CD95L). Although CD95L-mediated apoptosis has been proposed as a pathogenic mechanism in a wide range of diseases, including graft-versus-host disease, systemic CD95 engagement in mice by agonistic CD95-specific antibodies or by soluble multimeric CD95L (smCD95L), though lethal, has been reported to cause apoptosis only in a limited range of cell types, that is, hepatocytes, hepatic sinusoidal endothelial cells, and lymphocytes. Another member of the tumor necrosis factor (TNF)/CD95L family, TNF-α, induces disseminated vascular endothelial cell apoptosis, which precedes apoptosis of other cell types and lethal multiorgan failure. Here we show that systemic CD95 engagement in vivo by agonistic CD95-specific antibody or smCD95L causes rapid, extensive, and disseminated endothelial cell apoptosis throughout the body, by a mechanism that does not depend on TNF-α. Disseminated endothelial cell apoptosis was also the first detectable lesion in a murine model of acute tissue damage induced by systemic transfer of allogeneic lymphocytes and did not occur when allogeneic lymphocytes were from CD95L-defective mice. Both vascular and additional tissue lesions induced by agonistic CD95-specific antibody, smCD95L, or allogeneic lymphocytes were prevented by treatment with an inhibitor of caspase-8, the upstream caspase coupled to CD95 death signaling. Vascular lesions are likely to play an important role in the pathogenesis of allogeneic immune responses and of other diseases involving circulating CD95L-expressing cells or smCD95L, and the prevention of CD95-mediated death signaling in endothelial cells may have therapeutic implications in these diseases.

Digestive tract damage during graft-versus-host reaction (GVHR) causes high morbidity and mortality. Diagnosis is often late because biopsies are performed when clinical signs are severe and pathologic markers of early inflammatory lesions are lacking. Eosinophils are inflammatory cells, cytotoxic in vitro to digestive epithelium; they are found in biopsy specimens taken during acute flare-ups of inflammatory bowel disease. We performed systematic duodenal biopsies immediately after digestive symptoms occurred and found a digestive GVHR incidence of 73.1% (n = 93), higher than that found when digestive biopsies were performed immediately after severe clinical signs. Eosinophils were only present when there were histologic signs of GVHR; eosinophil presence correlated with GVHR severity. Electron microscopy with immunogold staining showed pathologic signs of in situ eosinophil activation, such as cytoplasmic granule alterations, and eosinophil peroxidase release in all patients. Interleukin-5 presence in activated eosinophils suggests eosinophil recruitment in digestive GVHR is an autocrine mechanism. Eosinophil density also correlated with GVHR severity, whether in acute or chronic clinical phases. Tissue eosinophils could thus be a marker of acute inflammatory flare-ups in GVHR. Systematic duodenal biopsy performed at the onset of digestive symptoms should allow early GVHR detection, and pathologic signs of GVHR, together with eosinophil density, might help modulate immunosuppressive therapy.

We previously reported a fusion between TEL and JAK2 in a t(9;12)(p24;p13) chromosomal translocation in childhood acute T-cell leukemia. This fusion gene encodes a TEL-JAK2 chimeric protein in which the 336 amino-terminal residues of TEL, including its specific self-association domain, are fused to the kinase domain of JAK2. TEL-JAK2 exhibits constitutive activation of its tyrosine kinase activity which, in turn, confers growth factor-independent proliferation to the interleukin-3-dependent Ba/F3 hematopoietic cell line. To elucidate the properties of TEL-JAK2 in primary cells and to create an animal model for TEL-JAK2-induced leukemia, we generated transgenic mice in which the TEL-JAK2 complementary DNA was placed under the transcriptional control of the EmuSRalpha enhancer/promoter. TEL-JAK2 founder mice and their transgenic progeny developed fatal leukemia at 4 to 22 weeks of age. Selective amplification of CD8-positive T cells was observed in blood, lymph nodes, thymus, spleen, and bone marrow. Expression of a tyrosine-phosphorylated TEL-JAK2 protein and activation of STAT1 and STAT5 (signal transducer and activator of transcription) were detected in leukemic tissues. TEL-JAK2 diseased mice also displayed invasion of nonhematopoietic organs, including liver, brain, lung, and kidney, by leukemic T cells. Leukemic organs of founder and transgenic progeny contained a monoclonal/oligoclonal T-cell population as analyzed by the rearrangement of the TCRbeta locus. Transplantation of TEL-JAK2 leukemic cells in nude mice confirmed their invasive nature. We conclude that the TEL-JAK2 fusion is an oncogene in vivo and that its expression in lymphoid cells results in the preferential expansion of CD8-positive T cells. (Blood. 2000;95:3891-3899)

T-cell prolymphocytic leukemia (T-PLL) is a rare form of mature T-cell leukemia associated with chromosomal rearrangements implicatingMTCP1 or TCL1 genes. These genes encode two homologous proteins, p13MTCP1 and p14TCL1, which share no similarity with other known protein. To determine the oncogenic role of MTCP1, mice transgenic for MTCP1under the control of CD2 regulatory regions (CD2-p13 mice) were generated. No abnormality was detected during the first year after birth. A late effect of the transgene was searched for in a cohort of 48 CD2-p13 mice aged 15 to 20 months, issued from 3 independent founders. Lymphoid hemopathies, occurring in the three transgenic lines, were characterized by lymphoid cells with an irregular nucleus, a unique and prominent nucleolus, condensed chromatin, a basophilic cytoplasm devoid of granules, and an immunophenotype of mature T cells. The molecular characterization of Tcrb rearrangements demonstrated the monoclonal origin of these populations. Histopathological analysis of the cohort demonstrated early splenic and hepatic infiltrations, whereas lymphocytosis and medullar infiltrations were found infrequently. The engraftment of these proliferations in H2-matched animals demonstrated their malignant nature. Cumulative incidence of the disease at 20 months was 100%, 50%, and 21% in F3, F4, and F7 lines, respectively, and null in the control group. The level of expression of the transgene, as estimated by Western blotting in the transgenic lines correlated with the tumoral incidence, with the highest expression of p13MTCP1 being found in F3 mice. CD2-p13 transgenic mice developed an hemopathy similar to human T-PLL. These data demonstrate that p13MTCP1 is an oncoprotein and that CD2-p13 transgenic mice represent the first animal model for mature T-PLL.

bcl-xL, a member of the Bcl-2 family, exerts an antiapoptotic effect on lymphocytes. To assess its clinical significance in patients with follicular lymphoma, realtime quantitative reverse transcription–polymerase chain reaction (RT-PCR) analysis of bcl-xL gene expression was investigated in whole lymph node sections and laser-microdissected lymphoma cells of 27 patients. Compared with 10 patients with reactive follicular hyperplasia, the bcl-xL gene was overexpressed in patients with follicular lymphoma at a higher level in microdissected lymphoma cells. The bcl-xL gene level correlated with the number of apoptotic lymphoma cells labeled by terminal deoxytransferase-catalyzed DNA nick-end labeling (TUNEL) assays (r = -0.7736). Clinically, a high bcl-xL level was significantly associated with multiple sites of extranodal involvement (P = .0020), elevated lactate dehydrogenase level (P = .0478), and an International Prognostic Index indicating high risk (P = .0235). Moreover, bcl-xL gene overexpression was linked to short overall survival times (P = .0129). The value of bcl-xL gene expression as a prognostic marker in follicular lymphoma should thus be considered.

Th17 cells have never been explored in human graft-versus-host disease (GVHD). We studied the correlation between the presence of Th17 cells with histologic and clinical parameters. We first analyzed a cohort of 40 patients with GVHD of the gastrointestinal tract. Tumor necrosis factor (TNF), TNF receptors, and Fas expression, and apoptotic cells, CD4+IL-17+ cells (Th17), and CD4+Foxp3+ cells (Treg) were quantified. A Th17/Treg ratio less than 1 correlated both with the clinical diagnosis (P < .001) and more than 2 pathologic grades (P < .001). A Th17/Treg ratio less than 1 also correlated with the intensity of apoptosis of epithelial cells (P = .03), Fas expression in the cellular infiltrate (P = .003), TNF, and TNF receptor expression (P < .001). We then assessed Th17/Treg ratio in 2 other independent cohorts; a second cohort of 30 patients and confirmed that Th17/Treg ratio less than 1 correlated with the pathologic grade of GI GVHD. Finally, 15 patients with skin GVHD and 11 patients with skin rash but without pathologic GVHD were studied. Results in this third cohort of patients with skin disease confirmed those found in patients with GI GVHD. These analyses in 96 patients suggest that Th17/Treg ratio could be a sensitive and specific pathologic in situ biomarker of GVHD.

DNA vaccination and all-trans retinoic acid (ATRA) result in a survival advantage in a mouse model of acute promyelocytic leukemia (APL). Depletion of CD4+ or CD8+ cells abolished this effect. CD4+ depletions of long-term survivors resulted in relapse and death within 3 months, thus demonstrating the need of both CD4+ and CD8+ subsets for the generation of DNA-driven antileukemic immune responses and underscoring a crucial role of CD4+ cells in the maintenance of durable remissions. Degranulation and cytotoxic carboxyfluorescein diacetate succinimidyl ester–based assays showed major histocompatibility complex–restricted APL-specific T cell–mediated immune responses. Sorted APL-specific CD8+CD107a+ T cells showed an increase of antileukemic activity. Effectors from ATRA + DNA–treated mice were shown to secrete interferon-γ when stimulated with either APL cells or peptides from the promyelocytic leukemia-RARα vaccine-derived sequences as detected by ELISpot assays. Our results demonstrate that DNA vaccination with ATRA confers the effective boosting of interferon-γ–producing and cytotoxic T cells in the leukemic mice.

The positive regulatory domain I (PRDM1) is a master regulator of terminal B-cell differentiation. However, PRDM1 is not B-cell specific. To determine its role in T-cell lymphoma, PRDM1 expression was investigated in 60 patients. PRDM1α and PRDM1β transcripts were detected in laser-microdissected T-lymphoma cells in 27 and 14 patients, respectively, mostly in cases with IRF4 expression. PRDM1β was associated with increased c-MYC expression. PRDM1β-positive patients displayed advanced Ann Arbor stage and high-risk International Prognostic Index and were linked to short survival times. In vitro, PRDM1β was related to resistance to chemotherapeutic agents and could be down-regulated by the proteasome inhibitor bortezomib. Kinetic studies showed that bortezomib down-regulation of PRDM1β preceded decreased IRF4 and c-MYC expression. An earlier retaining of cytoplasmic IκBα in bortezomib-treated cells was revealed, concomitant with blockade of NF-κB nuclear translocation. These results demonstrate the involvement of PRDM1β in T-cell lymphoma, with possible therapeutic interference by the proteasome inhibitor.

The positive regulatory domain I (PRDM1) is a master regulator in the differentiation of mature B lymphocytes to plasma cells. It has 2 isoforms, PRDM1alpha and PRDM1beta, and is regulated by the transcriptional regulator nuclear factor kappa (NF)-kappaB. PRDM1 protein expression was recently demonstrated in a subset of diffuse large B-cell lymphoma (DLBCL) with aggressive behavior, a type of lymphoma for which rituximab associated with chemotherapy (R-CHOP) is now widely indicated. Using laser microdissection combined with reverse transcription-polymerase chain reaction (RT-PCR) amplification, PRDM1 gene expression was assessed in 82 DLBCL patients. The results showed that both PRDM1alpha and PRDM1beta transcripts were expressed in microdissected lymphoma cells only in the non-germinal center B-cell-like (non-GCB) subtype of DLBCL. PRDM1beta gene expression was correlated with short survival time in the non-GCB patients treated with CHOP but not with R-CHOP. In vitro, B-lymphoma cells resistant to chemotherapy expressed PRDM1beta. Rituximab suppressed PRDM1beta expression, which was concomitant with NF-kappaB inactivation. The value of PRDM1beta expression as a prognostic marker in non-GCB DLBCL might thus be considered. This study confirms the efficiency of rituximab on DLBCL and allows a better understanding of one of its biologic actions.

Graft-versus-host disease (GvHD)–induced apoptosis of the skin targets both epidermal keratinocytes and dermal endothelial cells. We studied the donor-versus-recipient origin of GvHD of these target cells in skin of 18 sex-mismatched hematopoietic stem-cell transplant (HSCT) recipients. Combining XY fluorescence in situ hybridization (FISH) and double immunostaining, and further 3D tissue Z-stack analysis, we found keratinocytes and endothelial cells of donor origin, but only in patients with GvHD. Using terminal dUTP nick-end labeling (TUNEL) assay on sister sections, we found a correlation between the numbers of chimeric and apoptotic epidermal and endothelial cells. Moreover, donor-derived cells were more numerous and preferentially distributed in the areas of severe GvHD damage in biopsies performed early in the course of GvHD, whereas they were less numerous and found in the whole epidermis in late biopsies. Because donor-derived cells were found at the site and at the time of maximum tissue damage, they could contribute to epidermal and microvessel repair.

To assess whether Fanconi anemia (FA) patients might be at risk for acute graft-versus-host disease (AGvHD) despite using low-intensity conditionings, we retrospectively analyzed the incidence of AGvHD and its impact on outcome in 37 FA patients and 73 patients with acquired aplastic anemia (AAA) that received transplants at Saint Louis Hospital from HLA-genotypic identical siblings with similar conditionings (thoraco-abdominal irradiation plus cyclophosphamide 20 [FA] or 150 mg/kg [AAA]). Despite being younger, FA patients had an increased risk of grades II to IV AGvHD (relative risk [RR], 2.00; P = .021), especially in younger patients (RR, 7.93; P = .014). The risks of requiring systemic corticosteroids to treat AGvHD and experiencing cortico-resistant AGvHD were significantly increased in FA patients. Although non-FA and FA patients had similar 10-year outcomes, acute and chronic GvHD had a biphasic effect on FA patient outcome with an additional cluster of lethal events starting by 5 years after transplantation. This late survival fall, restricted to FA patients, was closely related to head and neck carcinomas (15-year incidence: 53%). FA patients represent a group at risk regarding AGvHD when using irradiation-based conditionings. The impact of AGvHD on survival may not be limited to the early posttransplantation period and may be a major risk factor for head and neck carcinomas and late mortality in FA patients.

The gastrointestinal (GI) tract is a major target in graft-versus-host disease (GvHD). In rodents both tumor necrosis factor (TNF) and Fas-dependent apoptosis have been shown to play a major role in GvHD lesions, but data in humans on TNF and Fas in situ expression are scarce. More recently, the role of non-T cells as GvHD effectors has also been suggested in experimental models. Here we report a detailed quantitative pathologic analysis in 95 patients who underwent gastroduodenal biopsy. This analysis included characterization and quantification of the cellular infiltrate, TNF, TNF receptors, and Fas in situ expression analyses and quantification of apoptotic cell numbers. TNF was expressed in all biopsies and it was highly specific for acute GvHD. In multivariate analysis, including pathologic factors only, increased early transplantrelated mortality (TRM) was associated with the presence of more than 20 neutrophils per field. Factors affecting early and late TRM were then assessed by multivariate analyses including both pathologic and clinical factors. Increased day-90 TRM was associated with the presence of more than 5 apoptotic bodies per field within the cellular infiltrate, and with stage II or higher acute liver GvHD. One-year TRM associated with the same 2 factors and with chronic GvHD.

Donor origin of epithelial intestinal cells has been studied in animals and humans after transplantation and has been used as evidence of hematopoietic stem cell (HSC) plasticity. However, in the human gastrointestinal tract, no study used X- or Y-chromosome detection by fluorescence in situ hybridization (FISH) coupled with immunologic stainings to characterize cell types on the same tissue section. Here, we combined these techniques on the same section of duodenal epithelium in 6 patients with acute graft-versus-host disease. Donor-derived lymphoid cells were detected in the epithelium and the lamina propria, as expected. However, using our stringent criteria, no donor-derived cells could be proven to be epithelial.

We would first like to underline that our study did not focus on the assessment of PRDM1β expression as a prognostic marker in diffuse large B-cell lymphomas (DLBCL), but on its significance on the therapeutic response. Tam et al, using quantitative real time–polymerase chain reaction (RT-PCR)

Abstract 678 Background and aims: BCL-2 activation plays a role in the progression of MDS to AML and BCL 2 inhibition may represent a therapeutic target in such patients. Using our double transgenic mouse model MRP8[NRASD12/BCL2], in which the transgenes induce MDS progressing to AML with dysplasia (Omidvar Cancer Res, 2007), we assessed the effect of ABT-737, a small molecule and mimetic inhibitor binding the BH3 domain of the BCL-2 family of proteins, on survival and leukemia initiating cells (LIC) in this mouse model. Methods: In this MRP8[NRASD12/hBCL2] double transgenic mouse model 2-week old mice have MDS with a mean of 6% bone marrow (BM) blasts (compared with 3% in the normal wild type littermates), while adult mice have AML, with a mean of 60% marrow blasts. In the present study, double transgenic mice were treated just after weaning and genotyping at 3 weeks of age with 75 mg/kg dose of ABT-737 (MDA & Abbott) 3 times weekly for 30 days. A cohort of mice (60 untreated and 35 treated) was followed for survival. Mice were sacrificed and BM harvested after treatment and Giemsa stained for BM analysis by microscopy (n=6 in each group), for LICs characterized as part of the lineage negative (Lin-)/Sca1+/cKit+ (LSK) population by flow cytometry (in 8 treated and 12 untreated mice), and for progenitor assays (n=4 in each group). Hematoxylin and eosin stained liver sections were examined and apoptosis assessed by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assays of liver sections (n=4 per group). RNA was extracted from Sca+ enriched spleen cells from untreated and treated mice (n=3 from each group) and assayed for gene expression profiling using exon specific arrays (Affymetrix). Results: Survival from birth of 35 treated mice was significantly longer than for 60 untreated mice (p Conclusions: ABT-737 extends lifespan in NRASD12/BCL2 transgenic mice, a preclinical model of high risk MDS/AML. ABT-737 targets the leukemia initiating cell, and regulates, amongst several, cell cycle (proliferation), differentiation and apoptosis pathways. These data suggest that clinical trials in high risk MDS and AML patients are warranted. Disclosures: Grange: Genosplice Technology: Employment.

Abstract 3191 Resveratrol is a polyphenolic stilbene with anti-oxidant, anti-inflammatory and anti-tumoral bioactivities. High concentrations of resveratrol (50 μM) have been reported to induce HbF synthesis in an in vitro model of normal and beta-thalassemic erythropoiesis (Fibach E. Int J Mol Med 2012; Rodrigue CM. BJH 2001) and to improve erythropoiesis in a mouse model for Fanconi Anemia (Zhang Q. Blood 2010). Beta thalassemia (b-thal) is characterized by ineffective erythropoiesis and increased cellular oxidative stress. We studied the effects of resveratrol (5 μM) on erythropoiesis in vitro from peripheral CD34+ cells of healthy and b-thal subjects. Erythroid maturation was evaluated at 7, 9, 11 and 14 days of culture by cytofluorimetric analysis using the CD71-GPA-CD36 strategy that allows to separate CFU-E, Pro-E, Int-E and Late-Erythroblasts (Merryweather-Clarke AT. Blood 2011). Resveratrol reduced cell growth in both cell types, with a reduction of CFU-E, increased Int-E at day 7 and 9, and increased Int-E and Late-E at 11 and 14 days. The early maturation of erythroid progenitors was confirmed by morphological analysis of the cells. We sorted CFU-E cells (at 7 days) from resveratrol treated and untreated cells and analyzed the cell cycle, cyclinD1 and p21 expression. In both cell types resveratrol induced increased frequency of S-G2/M cells compared to untreated cells with increased p21 levels, suggesting decreased cycling of CFU-E with increased maturation of erythroblasts. No changes of gamma chain mRNA levels were present in cells treated with resveratrol (5 μM). Since FOXO3 is a key regulator of erythroid redox required for normal erythroid maturation (Marinkovic D. JCI 2007), FOXO3 expression and activity was assessed in sorted CFU (7day) and Int-E (11 day) with and without resveratrol. FOXO3a mRNA levels were increased in resveratrol treated cells in both sorted cell populations. We used nuclear localization as a surrogate assay for FOXO3a activity and found resveratrol increased the overall expression of FOXO3 protein in the nucleus without impacting significantly the nuclear/cytoplasmic ratio. Interestingly, resveratrol did not appear to modify FOXO1 expression or subcellular localization. These results suggest that resveratrol enhances specifically expression of FOXO3 in human erythroblasts. Dietary resveratrol supplementation (2.4 mg/Kg) was studied in wild-type and Hbb3th+/− mice (2 months of age) for 6 months. In resveratrol Hbb3th+/− treated mice increased Hb levels (8.3±0.6 vs 10.3±0.5 g/dL, n=12; P Disclosures: Cappellini: Novartis Pharmaceuticals: Honoraria, Research Funding.

Abstract 3835 Background: Activation of the RAS pathway plays an important role in the pathogenesis of myeloid malignancies. RAS mutations occur in some 10% of MDS and acute myelogenous leukemia (AML) cases. We previously showed in mouse models of NRASD12/BCL-2 MDS/AML that RAS and BCL-2 co-operate in vivo. These genes co-localize at the plasma membrane in the MDS model (MRP8NRASD12/MMTVLTRTetoBCL-2) with increased apoptosis and in mitochondria in the AML post-MDS model (MRP8[NRASD12/BCL-2] with reduced apoptosis (Omidvar Cancer Res 2007). Here, we screened MDS patients for the cellular co-localizations of RAS:BCL-2 and the consequent apoptosis status. Methods: 39 MDS and AML post-MDS patients were studied, including (WHO classification): 3 RCMD-RS, 3 RCMD, 1 RARS, 8 RAEB I, 9 RAEB II, 4 CMML I, 1 CMML II, 10 AML post-MDS and 2 normal individuals. 17 patients had cytogenetic abnormalities. Bone marrow (BM) mononuclear cells (MNC) were assayed for RAS:BCL-2 localization by confocal and immunofluorescence microscopy. IL-3 dependent mouse hematopoietic FDCP-1 cells were infected with NRASD12 or BCL-2 alone or in combination. Co-localization was measured using anti-NRAS, anti-BCL-2, anti-mitochondrial (Tom 20) and anti-ezrin or anti-wheat germ agglutinin (WGA) plasma membrane antibodies. Apoptosis in patient samples was detected using the Mebstain apoptosis kit and visualized using FITC-dUTP on cytospun cells; apoptosis in the mice was detected by single photon emission computed tomography (SPECT) using technicium labeled annexin-V (99mTc-AnnexinV). NRAS mutations were analyzed by exon direct sequencing or by WAVE, a denaturing high-performance liquid chromatography (DHPLC) based assay. Lineage negative Lin-/Sca-1+/c-Kit+ (LSK) spleen cells were purified by automacs followed by flow sorting. Western blots were probed with anti-caspase 3 and 9 specific antibodies. Results: RAS and BCL-2 were co-expressed in the BM (MNC) of all 39 cases. The intensity (low or high) of RAS:BCL-2 co-localization measured by confocal microscopy significantly correlated with % BM blasts (p=0.0283) and WHO classification (p15% marrow blasts. The intensity of the complex was independent of karyotype and NRAS mutation (found in 3 of 30 (10%) patient samples). The 2 normal samples had low intensities of RAS:BCL-2 co-localizations and wild type NRAS. Further analysis of RAS and BCL-2 co-localizations was carried out in 13 of these patients. In 6 of the 7 patients with BM blasts Conclusion: These findings illustrate the prognostic impact of the intensity of the RAS:BCL-2 complex in MDS/AML post-MDS patients, and further suggest that localization of RAS:BCL-2 at the plasma membrane correlates with less advanced disease while its presence at mitochondria correlates with more advanced MDS. Therapeutic targeting of the RAS:BCL-2 complex could potentially prevent transformation in MDS patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 2339 Introduction: Graft versus host disease (GVHD) is a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HSCT). Gastrointestinal (GI) GVHD occurs in 10 to 40% of patients. Its diagnosis is based upon histological findings of per-endoscopic mucosal biopsies. The aim of our study was to compare endoscopic appearance of the mucosa and histological results and assess the outcomes of transplanted patients who underwent duodenal biopsies according histological and endoscopic findings. Patients and methods: We performed a retrospective analysis including allografted patients who underwent duodenal biopsies because of digestive GVHD suspicion. For each patient we recorded clinical data, endoscopic signs, and then we reviewed histological findings. Diagnosis of GVHD was histologically defined as the presence of single-cell necrosis. The severity of disease was graded on a four-point scale from 0 (absent) to 4 (very severe), according the number of single-cell necrosis, the degree of mucosal infiltration, the presence of neutrophils and eosinophils, epithelial rupture, and vascular involvement. Endoscopic findings were described and graded as normal mucosa (0), presence of erythema or oedema with congestive mucosa (1), ulceration (2), and extensive mucosal involvement (3). Agreement between endoscopy and histology was measured by to kappa statistics. Outcomes were assessed by Kaplan-Meyer survival curves. Results: Between 1995 and 2008, 270 duodenal biopsies in 228 patients have been performed in patients. We focused only on the first one. Indication for HSCT was acute leukaemia or myelodysplastic syndrome in 133 patients (58.3%), NHL/HD in 35 (15.4%), CML or myeloproliferative disease in 35 (15.4%), other diagnosis in 25 (10.9%). Donor was identical sibling in 48% of cases, unrelated donor in 52% (HLA matched 21% and mismatched 31%). Stem cell source was bone marrow in 46%, peripheral blood in 42% and cord blood in 12%. Finally most of patients (72%) received a myeloablative conditioning whereas the others had reduced intensity conditioning. The median time from transplantation to biopsy was 28 days. More than two thirds of patients (77%) presented an acute GVHD occurring before day 100. Upper digestive endoscopy was performed at the onset of digestive symptoms; nausea or vomiting in 25% of cases, diarrhea in 75%. Pathological GVHD was diagnosed in 77% of patients, graded minimal (1) in 29%, mild (2) in 25%, severe (3) in 10% and very severe (4) in 12%. Concerning endoscopic findings, normal macroscopic aspect was noted in 80% of cases (n=176), erythema or oedema was reported for 12% of patients, mucosal ulceration in 6%, and extensive mucosal involvement in 2%. There was no agreement between histological diagnosis and endoscopic findings with a kappa coefficient of -0.08, [95%CI: -0.17;-0.01], even for severe histological stages. Then 80% of patients with normal endoscopy had a diagnosis of GVHD, which was graded as histologically severe or very severe in 39%. The median follow up was 47 months (31-68), the 4 years overall survival was 47% [IC95% 41%; 54%]. Severe (n=24) and very severe (n=27) histological GVHD was significantly associated with mortality, with respectively HR 3.23 [95%CI: 1.82; 5.72] p Conclusion: Since digestive GVHD is a pan-intestinal process, systematic duodenal biopsies in allografted patients suffering from high or low digestive symptoms permit a diagnosis of GI GVHD in more than three quarters of cases. The early realization of endoscopic examination leads most often to a normal macroscopic aspect of small bowel mucosa and there is no agreement between histological and endoscopic findings. Mucosal injury could be more likely linked to a viral involvement. Nevertheless if endoscopic findings do not lead to GVHD diagnosis, presence of ulceration and extensive involvement is predictive of mortality, as well as histological severity of GVHD. Disclosures: No relevant conflicts of interest to declare.

Abstract 2714 Poster Board II-690 Adult T cell leukemia (ATL) is one of the rare human cancers initiated by a transforming retrovirus, HTLV-I. After many years of controversy, it is now accepted that the viral transactivator protein Tax plays a critical role in initiating the leukemic process, because Tax transgenics develop a disease with striking ATL features. Long-term prognosis of ATL patients remains extremely poor, but we recently reported that the combination of As2O3, interferon-a (IFN), and zidovudine yielded unprecedented response rates. Indeed, in 10 chronic ATL patients, a 100% response rate was observed, including 7 complete remissions (CR), 2 CR but with more than 5% circulating atypical lymphocytes, and one partial response. We demonstrate that the As2O3/IFN combination, previously shown to degrade Tax, cures Tax-driven murine ATLs. Unexpectedly, this combination immediately abrogates leukemia transplantation into secondary recipients, while the primary tumor continues to grow and only exhausts much later. Leukemia initiating cell (LIC) clearance is reversed by proteasome inhibition, demonstrating that LICs are addicted to continuous Tax expression. Most anticancer treatments fail to target LIC. These results establish that As2O3/IFN/zidovudine acts through Tax targeting and predict a favorable long-term outcome for responsive patients. Thus, oncogene degradation can selectively target LICs, explaining this very recent success of a similar regimen in patients. Disclosures: No relevant conflicts of interest to declare.

Background; The recent discovery of CD4+ characterized by secretion of IL-17 (Th17) and the regulatory Foxp3 CD4 cell (Treg) has had a major impact on our understanding of immune processes. Th17 and Treg have been implicated in the pathogenesis of human autoimmune disease, including inflammatory bowel disease (IBD). Data from murine model of autoimmune disease suggest that there is a reciprocal relationship between Th17-induced pathology and Treg regulatory role which prevent tissue inflammation and mediate self-tolerance. Today few murine data have been published on the role of Th17 in GvHD and, to the best of our knowledge, none in human GvHD. Aim; We took the advantage of stored biopsies from one of our previous study on human GI GvHD (Blood2004; 103: 50–57), to look for the expression of Il-17 and of Foxp3 in CD4+ cells in the same biopsies we previously studied. Methods; In addition to characterization and quantification of the cellular infiltrate, TNF, TNF receptors, and Fas in situ expression analyses, quantification of apoptotic cell numbers by TUNEL, double immunohistochemistry staining and quantification of CD4+/IL17+ cells (Th17) and of CD4+/Foxp3+ cells (Treg) was analyzed. The sensitivity and specificity of the Th17/ Treg ratio to the clinico-pathological diagnosis of GvHD and the correlation of this ratio to previously described parameters (Blood2004; 103: 50–57) were then analyzed. Results; 40 patients (pts) with suspected GvHD of the GI tract (nausea/vomiting or diarrhea) were enrolled and had biopsies before any steroid treatment. All underwent transplantation after a myeloablative conditioning regimen. 18 pts had severe pathological grade GvHD. The density of CD4+ expressing IL-17 or Foxp3 was not high needing high magnification for quantitative estimates; the numbers of Th17 cells per field range from 0 to 8 per field with a median of 1(IQR 0.25-4), and that of Treg was 4 (range 0–11, IQR 1–6), with a mean Th17/ Treg ratio of ½. The number of Th17 cells in patients with pathologically proven GvHD ranged between 0 and 2 in 27pts, even in 15/18 pts with pathological grade 2. On the contrary, among 34 patients with pathological GvHD median of 5 Treg cells per field was found. A Th17/ Treg ratio< 1 correlated both with the clinical diagnosis (sensitivity 64%, specificity 94%, p Conclusions; Contrary to IBD models and few clinical data in pts with rheumatoid arthritis and Crohn’s disease our data do not support increased tissue damage associated with Th17 presence in human GvHD of the GI tract. However our data perfectly fit with a recent study in a murine model of GvHD demonstrating that absence of donor Th17 exacerbated acute GvHD (Blood Prepublished Jul 2, 2008; doi:10.1182/blood-2007-12-126987).

Ischemic/reperfusion (I/R) injury plays a critical role in the pathogenesis of several clinical manifestations of sickle cell disease (SCD) and it results from complex interactions between cells and plasma factors. Hepatic dysfunction has been described in SCD and the pathophysiology of liver I/R damage in SCD is only partially known. The liver is highly vascular organ with high metabolic rate, susceptible to stress-induced by hypoxia. Here, we studied the effects of acute and chronic hypoxia on liver function and pathology in transgenic sickle mice (SAD mice). We evaluated the following parameters at baseline, at 4, 48 and 168 hours (hrs) of exposure to hypoxia (8% oxygen) followed by 2 hrs reoxygenation (21% oxygen): complete blood counts with retic, red cell density, serum AST and ALT levels, liver pathology and expression by RT-PCR of the following genes: TNF-a, IL-1b as inflammatory response markers; TgfB1 (involved in control cell growth and extracellular matrix formation), mmp9 (marker of hepatic matrix remodeling), NfKb (transcription effector involved in liver cell death and regeneration), Icam1 (endothelial damage and vascular remodeling marker), in addition to heme oxygenase-1 (Homx1), eNOS (Nos3) and iNOS (Nos2). SAD mice at baseline exhibited liver infiltrates of inflammatory cells, no thrombi and a mild hepatic injury (pathological score: 1.4±0.02; n=5); up-regulation of TgfB1, Icam1, Nos3 and down-regulation of Nfkb compared to normoxic WT mice. One SAD mice died at 48 hrs hypoxia and 2 SAD mice died at 96 hrs hypoxia. In SAD mice I/R induced: increased red cell density at 48 and 168 hrs of hypoxia; increased peripheral neutrophil count at 48 and 168 hrs; increased retic count at 168 hrs; increased serum levels of AST/ALT; increased liver cell inflammatory infiltrate at 48 hrs (n=5) and 168 hrs (n=6); increased pathological score at 48 hrs (2.3±0.02 n=5) and at 168 hrs (3.2±0.05 n=6; P In SAD mice I/R induced changes in the expression of the following genes: 4hrs: up-regulation of Nfkb, Ilb1, Nos2 and down-regulation of Tgfb1, Icam1, homx1 and Nos3; 48 hrs: up-regulation of Icam1, Nos2, Tnfa and down-regulation of TgfB1, Nos3, homx1, Nfkb, Ilb1; 168 hrs: up-regulation of TgfB1, Icam1, Nos 3, homx1, Nfkb, Ilb1, Nos2 and down-regulation of Nos2. In WT mice I/R induced increased neutrophils and retic count at 168 hrs; increased serum levels of AST/ALT at 48 and 168 hrs; increased liver cell inflammatory infiltrate at 168 hrs (n=7) and pathological score at 168 hrs and modulation of the following genes at 4hrs: up-regulation of Nfkb, Icam1, Ilb1, Nos2, mmp9 and down-regulation of TgfB1 and homx1; at 48 hrs: up-regulation of TgfB1, Icam1, homx1, Nos2, Tnfa, mmp9 and down-regulation of Ilb1; at 168 hrs: up-regulation of Icam1, Nfkb, Nos 3, Nos2, Tnfa, mmp9 and down-regulation of Il1b, homox1, TnfB1. Then, we investigated the effects of a PDE-4 inhibitor (Rolipram) on hepatic I/R injury in sickle cell SAD mice, since cAMP cell content has been recently described to be crucial in I/R liver damage and PDE-4 is a key regulator of cAMP metabolism. In SAD mice, Rolipram reduced the hypoxia induced peripheral neutrophilia and serum AST/ALT increase, prevented the I/R liver injury as supported by reducing cell inflammatory infiltrate, pathological score (1.8±0.02, n= 6, P

OBJECTIVES: Activating RAS mutations and over-expression of BCL-2 are prognostic features of myelodysplastic syndromes/acute myeloid leukemia (MDS/AML) transformation. Using NRASD12 and BCL-2, we created two distinct transplantable in vivo models of MDS and AML. Expression of hBCL-2 in a primitive compartment by MMTV-LTR results in a disease resembling human MDS with bone marrow blasts of 15% with increased apoptosis assayed by TUNEL on liver sections, whilst the myeloid MRP8 promoter induces a disease with characteristics of human AML with marrow blasts of up to 90% with liver apoptosis patterns similar to wild type. In MDS mice RAS and BCL-2 do not co-localize in the mitochondria, but localize to the plasma membrane, where active pro-apoptotic RAS is normally located, whereas in the AML disease RAS and BCL-2 co-localize in the mitochondria, where BCL-2 is normally found; consistent with its anti-apoptotic properties. We next examine the mouse hematopoietic FDCP-1 in vitro model with stable exogenous expression of NRASD12, hBCL-2 or NRASD12 and hBCL-2. Furthermore, we report the in vivo effects of the BH-3 mimetic inhibitor ABT-737. RESULTS: FDCP-1 lines demonstrate that whilst hBCL-2 alone prevents staurosporine-induced mitochondrial-dependent (caspase-9-mediated intrinsic) apoptosis, both NRAS-D12 and NRASD12/hBCL-2 cell lines were pro-apoptotic. No significant difference was observed in cell-death receptor (caspase-8-mediated extrinsic) apoptosis between the cell lines following pharmacological induction with Fas-ligand or TRAIL. Annexin-V/Propidium Iodide flow cytometry and caspase activity assays on hematopoietic primary cells from the mouse models recapitulated the findings; namely the MDS-model demonstrates increased caspase-9-mediated apoptosis (within the Lin−/Sca-1+/KIT+ (LSK) sub-population), whilst the AML-model illustrate anti-apoptotic activity. This is concordant with the signaling profile where phosphorylated AKT is reduced in the RAS-mediated MDS mice and active-AKT is increased in the BCL-2 mediated AML disease. Expanded leukemic stem cell LSK populations had increased hBCL-2 expression in the RAS-GTP complex in both MDS/AML diseases. Thus we suggest that this complex can be a specific target for therapy. When hBCL-2 is switched off with doxycycline in the conditional MDS mice, only partial reversal of the phenotype was observed as RAS recruits endogenous mouse BCL-2 to remain active; thus demonstrating the role of the complex in the disease. In order to target both human and mouse BCL-2 the efficacy of in vivo treatment with the specific BH-3 mimetic inhibitor ABT-737 was determined. We show that the treatment significantly reduced the progression of the disease, increased the peripheral blood platelet counts, decreased the bone marrow blast and cleared the tissue invasion in the MDS mice or reduced tissue infiltration of the AML. In vivo imaging by single-photon emission computed tomography (SPECT) using 99mTc-labelled Annexin-V shows that ABT-737 induces apoptosis of the blast cells that infiltrate the liver and the spleen of the treated mice, which was confirmed by TUNEL on liver sections. Additionally, ABT-737 can reduce the myeloid colony growth and the LSK expansion in these mice; whilst abrogating BCL-2:RAS-GTP complex formation illustrated via biochemical and confocal assays. CONCLUSION: This represents the first in vivo progression model of MDS/AML dependent on the formation of a BCL-2:RAS-GTP complex rescued by ABT-737 via intrinsic apoptosis and thus support the case for BH-3 mimetic therapy.

OBJECTIVES: DNA vaccines can be effective in the acquisition of humoral and cellmediated immune responses. Our studies on an acute promyelocytic leukemia (APL) mouse model show that DNA vaccination combined with all-trans retinoic acid (ATRA) results in a survival advantage with a significant increase in the Th1 cytokine IFNg. ATRA alone can act as an adjuvant to induce immune responses as measured by an increase in anti-RARa antibody production, which correlated with improved survival in mice. Similar increases in antibody production have been observed in our patients after maintenance therapy. The aim of this study is to use immunomonitoring and functional assays to evaluate the presence of activated T-cells and to demonstrate APL-specific killing and to determine if the protective effect of DNA vaccination is CD4+ and CD8+ mediated. METHODS: Using an APL transplant model in FVB/N mice, CD107a, expressed on the surface of lytic granules of activated T-cells, was measured by flow cytometry. A flow based CFSE assay was used to measure APL specific cell killing by cytotoxic T-cells (CTLs). As FVB/N mice have H2q haplotyes, blocking anti-H2q antibodies were used to determine if the cytotoxic activity was MHC restricted. Immunophenotyping by measuring CD4+ and CD8+ absolute counts were conducted. Mice injected with APL cells were depleted of CD4+ or CD8+ cells with anti-CD4 or anti-CD8+ antibody treatment initiated the day after DNA vaccination (early) and continued every 5 days and assayed for efficacy of the DNA+ATRA combined therapy or CD4+ or CD8+ cells were depleted in long term survivors (>100 days, late). RESULTS: Th1 cytokines TNFa and IFNg were increased indicative of DNA effects and specific activated CD3/CD8 T cells were detected and observed to release cytotoxic granules in the presence of APL cells in long term survivors. A dose dependent decrease in CFSE positive cells was observed assaying effectors from spleens of ATRA alone, ATRA+DNA treated mice and CD107a+ sorted cells from the latter using APL cells as targets. This effect was MHC restricted as anti-H2q antibodies reduced the specific cytotoxic activity. CD4+ absolute numbers measured on day 38 significantly correlated with survival (p=0.005). Although not significant a similar trend was observed for CD8+ counts. The CD4+ or CD8+ depleted mice treated with DNA + ATRA died earlier compared with the undepleted animals. When DNA + ATRA treated long term survivors were depleted of CD4+ or CD8+ cells, the CD4+ depleted mice relapsed and died in 3 months whereas the CD8+ depleted mice survived for a further 3 months when the experiment was terminated. These data are consistent with an increase in anti-RARa antibody production previously measured in other protocols. Interestingly the late depletions show that CD4+ cells are required for the maintenance of the remissions and show that memory T-cells are required. CONCLUSION: Therefore we have been able to detect protective cellular and humoral responses in mice with the combined treatment of DNA+ATRA, which correlates with outcome.

Long-term survivors after allogeneic hematopoietic stem cell transplantation (HSCT) develop solid tumors. Chronic graft-versus-host disease (GvHD) increases the risk of squamous cell carcinoma (SCC). Five of 24 patients with HSCT and SCC, had sex-mismatched HSCT. In 3 of them, tumor sex karyotype studied by combined XY FISH and double immunostaining on the same SCC section, with 3D Z-stack analysis, was compatible with a cancer originating from donor hematopoietic cells. After control by short tandem repeats analysis (PCR-STR) of laser micro-dissected tumor and normal epidermal cells from the recipient and donor or PCR-STR and FISH analysis of a tumoral cell line from one SCC, a recipient origin was found in one case but in the two other cases the donor genotype of the tumor was confirmed. In a third step, PCR-STR of laser micro-dissected tumor in 2 cases after sex-matched transplant proved SCC to be of the donor origin. We thus obtain conclusive evidence that 4 epithelial tumors among 8 studied arose from the engrafted donor cells. Tissue stem cells were proposed to exhibit an unexpected level of plasticity, although issues on cell fusions have lead to some controversies. Only transplantation experiments using genetically distinct recipients and donors can unequivocally demonstrate these changes in cell fate. We have analyzed a unique set of squamous cell carcinomas arising in long terms survivors of allogeneic stem cell transplantation. In these patients, chronic graft-versus host disease greatly favors development of solid tumors, possibly as a consequence of mucosal inflammation and immunosuppression. Thus, donor-derived hematopoietic stem cells recruited to sites of chronic inflammation yielded epithelial tumors.

Although the incidence of HIV-associated Non-Hodgkin lymphoma (NHL) has decreased since the use of combination antiretroviral therapy (cART) and survival has improved, NHL still represents one of the most frequent and severe complications in HIV-infected patients. Out of the 361 patients included in a cohort of HIV-NHL from 1996 to 2006, 114 patients had undetectable (? 500 copies/ml) plasma HIV-RNA. These patients were included in a study to analyze the characteristics of NHL over time according to the duration of HIV infection control. All available histological samples were reviewed by a single pathologist. Tumor was defined as virus-associated (VposNHL) if EBV (ISH, IHC or PCR) or HHV-8 (IHC or PCR) were detected in the tumor cells, and not virus-associated (VnegNHL) if nor EBV nor HHV8 were detected. Comparisons were performed using non parametric tests. All patients were treated with cART, with a median duration of 24 months (IQR, 2–119). The median duration of HIV replication control was 9.3 months (IQR, 0.13–90.5). Most cases (63%) had occurred within 18 months of HIV control versus only 37% after M18. When comparing characteristics of NHL in patients with less or more than 18 months of HIV control, the patients were older (52 years versus 41 years, P18 months versus ?18 months. None of the NHL features (histology, phenotype, staging, ECOG, LDH, B symptoms) were different between the 2 groups. Virus-association could be assessed for EBV and HHV-8 in 96 cases (84%): 52 patients presented with a virus-associated tumor (31 EBV+, 13 HHV-8+, 8 both) and 43 tumors were not virus-associated. There was no difference in this repartition according to the duration of HIV control (P=.62). VposNHL VnegNHL VundeterNHL HIV-RNA 18 months 16 13 4 Median CD4 cell count (×106/L) 280 290 317 Virus-associated tumors were neither associated with CD4 cell count (P=.86), nor with CD8 cell count (P=.78). In conclusion, NHL still occur in patients with HIV controled infection (30% of HIV-NHL), but within the first 18 months after plasma HIV-RNA becomes undetectable in most patients. After 18 months, the incidence dramaticaly decreased. However, characteristics of NHL and specifically the association with EBV and/or HHV-8 were not different according to the duration of control of HIV infection.

Calcineurin (PP2B) is an ubiquitously expressed serine/threonine protein phosphatase dependent on calcium calmodulin. Calcineurin plays a pivotal role in many important biological processes, including the development and function of the immune system. Sustained signalling through calcium/calcineurin results in the activation of transcription factors of the NFAT family (Nuclear Factor of Activated T cells). Mouse genetic studies have demonstrated a strong epistatic relationship between calcineurin and NFAT activation and function in many developmental processes. In lymphoid cells, the calcineurin/NFAT signalling pathway is essential to specific aspects of T cell development and plays a central role in the activation of the immune response and in its homeostatic control. Despite their central role in T cell development, immune function and homeostasis, deregulation of calcineurin/NFAT pathway have not been shown so far to have a pro-oncogenic role in lymphomagenesis. Using our TEL-JAK2 transgenic mouse model of T-ALL, we investigated the activation status and importance of the calcineurin/NFAT signalling pathway in leukemogenesis. We found that the calcineurin/NFAT pathway is activated in leukemic cells of TEL-JAK2 transgenic mice. Western blot and EMSA experiments showed that all NFAT members expressed in the lymphoid lineage (NFAT1, 2 and 4) are found in an active hypophosphorylated form, they are located in the nucleus, and exhibit an increase in their DNA binding activity. In order to investigate whether constitutive calcineurin/NFAT activity was specific to TEL-JAK2 leukemia or whether it is a more general property of leukemic cells, we analyzed NFAT activation in leukemic cells of transgenic mice aberrantly expressing oncoproteins involved in lymphoma in human. This showed that calcineurin/NFATpathway is not only aberantly active in leukemic cells of TEL-JAK2 transgenic animals, but also in other mouse models of human leukemia. We also found that the activation of the calcineurin/NFAT pathway is only observed when cells are rapidly analyzed following their explantation and dissociation from diseased animals. Indeed, if cells are incubated in tissue culture for a short period of time (60–120min.), essentially stochiometric rephosphorylation, meaning inactivation, of NFAT is observed. This indicates that activation of the calcineurin/NFAT pathway depends upon signals generated in the in vivo tumor cell environment and is not -or not solely- under the control of the initiating oncogene. Using these mouse models we conducted pre-clinical studies that indicated that calcineurin/NFAT pathway is an attractive novel target of therapeutic interest in lymphoid malignancies.

The anti-CD20 monoclonal antibody rituximab has shown promising results in the clinical treatment of patients with B-cell non-Hodgkin’s lymphoma (B-NHL). However, its therapeutic effect could still be improved. This study examined the anti-tumor activity of rituximab combined with histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) in CD20-positivie B-NHL cell lines, as well as in primary B-NHL cells and a murine B-NHL model. The combination treatment sensitized B-NHL cells to apoptosis in a synergistic manner, concomitant with Bcl-2/Bcl-XL downregulation, mitochondrial instability, and caspase activation. Particularly in Daudi cells relatively insensitive to rituximab, these events were associated with nuclear factor-kB (NF-kB) inactivation. SAHA presented functional complementation with rituximab, through decreasing IKKa/b and IkBa phosphorylation, thus preventing NF-kB nuclear translocation. In addition, SAHA induced IkBa cleavage to a stable inhibitory form and caused NF-kB degradation in response to caspase-3 activation. As an independent approach, co-administration of rituximab and SAHA resulted in a clear decline in levels of ERK cascade members, including extracellular-signal-regulated kinase (erk) itself, upstream Raf-1, mitogen-activated protein kinase/ERK Kinase Kinase-1 (mekk1), and mekk2, as well as their downstream transcription factor target c-myc. By western blot, c-Myc protein was subsequently downregulated when treated with rituximab or SAHA, and the degradation was most significant in combination group. More importantly, rituximab-SAHA combination significantly promoted primary B-NHL cells apoptosis and improved the survival time of a SCID mouse lymphoma model established with intravenous injection of Daudi cells. Terminal deoxytransferase-catalyzed DNA nick-end labeling and ultrastructural study revealed increased apoptotic lymphoma cells on mice spleen sections of combination group. Taken together, these findings emphasized the value of targeting apoptosis signaling pathway in lymphoma therapy. Rituximab in conjunction with histone deacetylase inhibitor may represent a novel strategy in treating patients with B-NHL.

Pulmonary hypertension (PH) is one of the major causes of morbidity and mortality for young-adult patients with sickle cell disease (SCD). Little is known about the role of vaso-active agents, pro-inflammatory and pro-fibrotic mediators in the genesis of PH in SCD. We developed a model for studying PH in transgenic sickle cell SAD mice. Wild-type (WT) and SAD mice aged between 4 and 6 months were divided in groups of 7 animals each. One group from each strain served as control under normoxia, while a second group from each strain was exposed to 7 days hypoxia (8% oxygen). The following parameters were evaluated in normoxic and hypoxic groups: lung histopathology, complete blood count, reticulocytes, bronchoalveolar lavage (BAL) total leokocyte and neutrophil count, BAL levels of IL-6, IL-10, IL-1β and TNF-α. The expression of nine genes was examined by quantitative RT-PCR. These included endothelin-1 (ET-1), endothelin-1 receptor (ET-1R), cycloxygenase-2 (COX2), NF-kBp65, transforming-growth-factor-1β (TGF-1β), metalloproteinase-2 and 9 (MMP-2, MMP-9), IL-10 and IL-1β. Two of the seven SAD mice exposed to hypoxia died at day fourth. The remaining five mice were sacrified at day seven and showed: a) increased pulmonary arterial wall thickness and α-actin staining, suggesting a vascular remodeling; b) lung injury compatible with PH in SCD; c) increased BAL total leukocyte and neutrophil count; d) increased BAL IL1β, IL10, IL6 and TNF-α; e) increased peripheral neutrophil and reticulocyte counts, hematocrit and hemoglobin levels. In lungs of hypoxic SAD mice, ET-1, ET-1R, TGF-1β, MMP-2, MMP-9, IL-1β and IL-10 gene expression were induced, with no significant changes in NF-Kbp65 gene expression. These data were consistent with a model of PH in SCD. We then assessed the effect of the PDE-4 inhibitor ROLIPRAM on the PH in seven hypoxic SAD mice. ROLIPRAM was administrated at the dosage of 30 mg/Kg/day by gavage. No deaths were observed in this group. ROLIPRAM a) protected SAD mice from hypoxia induced lung injury; b) decreased BAL total leukocyte and neutrophil count; c) decreased BAL IL-1β, IL-6 and IL-10 levels; d) reduced peripheral neutrophil count; d) normalized and/or modulated the expression of the hypoxia-induced genes. These data suggest that the PDE-4 inhibitor ROLIPRAM has anti-inflammatory properties and could have beneficial effects on the hypoxia-induced airway-remodeling and possibly PH in sickle cell disease.

Occurrence of an aggressive lymphoma in patients with chronic lymphocytic leukemia (CLL), clinically referred to as Richter’s syndrome, occasionally manifests as a lymphoproliferation resembling Hodgkin’s lymphoma (HL) and often containing the Epstein-Barr virus (EBV). Only a limited number of HL variants have been subject to informative analysis regarding EBV status and their clonal relation-ship to the CLL, with evidence of an identical clonal origin in some cases and of clonally unrelated neoplasms in most cases. We reported a clinical, pathological and molecular analysis of three new cases of Richter’s syndrome with HL features. The three patients (two males, one female), aged of 67, 70 and 74, have 4, 8 and 15-year history of CLL, respectively. At HL, clinical stages (CS) were IIIAa, IIBb and IIIBb, at HL diagnosis. All the three patients responded to HL specific chemotherapy but two of them died from infectious and cardiac complications and the last one died from HL relapse. Histologically the lesions were composite comprising areas of CLL, areas of CLL with Hodgkin Reed-Sternberg (HRS) cells and areas of HL. The HRS cells were CD45RC−/CD20−/CD79a−/CD30+/CD15+/J-chain-(all cases), Oct-2+ (case 2) and Pax-5+ (cases1 and 3). In all cases, HRS cells were positive for EBV with a latence 2 profile. Using single cell laser microdissection and PEP-PCR amplification of the immunoglobulin heavy chain gene (IgH) FR3A region of each cell type, we demonstrated a same clonal origin for the CLL cells and the HRS cells in all cases. These data were confirmed in two cases by Genescan reamplification with fluorescent FR3A primer and sequenced with BigDye Terminator chemistry. Clinicopathological presentation was similar to classical HL but persistent EBV infection is probably related to an underlined immunosuppression due to CLL and chemotherapy. Seven other cases have been reported in the literature using similar single cell techniques, four of them, EBV +, were clonally unrelated while the three other one, EBV−, were of same clonal origin. At the opposite and although our three cases are EBV+, we show that the HRS cells and CLL cells belong to the same clonal population. Our data do not support the hypothesis that EBV+ HL in CLL patients occurs as unrelated secondary neoplasm in immunocompromised patients.

Clinical manifestations of sickle cell disease are related to vaso-occlusive (VOC) events, which are responsible for acute and chronic organ damages. We have recently shown the beneficial effects of inhaled nitric oxide (NO) in both transgenic sickle cell (SAD) mice exposed to hypoxia/reoxygenation (H/R) and in sickle cell children with acute painful crisis (Blood102: 1097, 2003; JAMA289: 1136,2003). Polynitroxyl-albumine (PNA), nitrosylated at position 34, may represent a new therapeutic tool in acute sickle cell VOCs. In SAD mice, PNA was intraperitoneally (ip) administrated and a dose-response curve was determined. Methemoglobin (MetHb) levels and blood pressure were measured under normoxia. PNA, 300 mg/Kg twice a day (equal to 4.5 mM/Kg twice a d.), induced a significant but transitory decrease in blood pressure 60 min after ip injection, while MetHb levels did not change. The effects of PNA and human serum albumine (hsAlb) on ischemia/reperfusion (H/R) lung injury related to sickle cell disease were then studied. SAD mice were divided into 4 groups of 6 animals each: one served as normoxia untreated control. The other groups were exposed to 46 hrs hypoxia (8% O2) followed by 2 hrs normoxia: one served as untreated hypoxic group, one was treated with hsAlb (300 mg/Kg twice a day) and one with PNA (300 mg/Kg twice a day). We evaluated lung histopathology, complete blood counts, bronchoalveolar (BAL) fluid neutrophil counts, cytokine levels, which are activated by H/R stimulus and are modulated by inhaled NO (Blood102:1087, 2003). In addition to histological lung damage, H/R induced an increase in blood neutrophils, a market increase in BAL total leukocyte and neutrophil counts, an increase in BAL IL-6 and IL-1β, with no significant changes in BAL IL-10 and TNF-α levels. PNA prevented the H/R lung injury, determined a reduction in H/R mediated increase in blood neutrophils, prevented the increase in BAL total leukocyte and neutrophil counts and in BAL IL-6 and IL-1β levels, while no change was observed in BAL IL-10, TNF-α and MetHb levels. hsAlb ameliorates the H/R lung injury but it did not affect either the H/R induced blood neutrophils, and BAL cells and cytokines. These results show that the beneficial effects of PNA are similar to those observed with inhaled NO and that PNA could be therapeutically efficacious in sickle cell disease.

Basara et al[1][1] recently reported interesting data on the predictive value of eosinophilia in the evolution to acute graft-versus-host disease (GVHD) in a systematic prospective study of bone marrow smears and biopsies (n = 237). This is in accordance with our previous findings in patients