ALBERT

Description: Heme oxygenase-1 (HO-1) is an intracellular enzyme that degrades heme and inhibits immune responses and inflammation in vivo. In most cell types, HO-1 is inducible by inflammatory stimuli and oxidative stress. Here we demonstrate that human monocyte-derived immature dendritic cells (iDCs) and several but not all freshly isolated rat splenic DC subsets and rat bone marrow-derived iDCs, spontaneously express HO-1. HO-1 expression drastically decreases during human and rat DC maturation induced in vitro. In human tissues, iDCs also express HO-1, whereas mature DCs do not. Induction of HO-1 expression with cobalt protoporphyrin (CoPP) in human and rat DCs inhibits lipopolysaccharide (LPS)-induced phenotypic maturation and secretion of proinflammatory cytokines, resulting in the inhibition of alloreactive T-cell proliferation. CoPP-treated DCs, however, retain the ability to produce the anti-inflammatory cytokine interleukin 10 (IL-10). Reactive oxygen species induced by LPS in DCs were inhibited by induction of HO-1. In conclusion, we identify, for the first time, the capacity of HO-1 to block maturation of DCs and to inhibit proinflammatory and allogeneic immune responses while preserving IL-10 production. This novel immune function for HO-1 may be of interest for the inhibition of immune responses in autoimmune diseases, transplantation, and other conditions involving activation of the immune system. (Blood. 2005;106:1694-1702)

Description: To realize the therapeutic potential of human embryonic stem cells (hESCs), it is necessary to regulate their differentiation in a uniform and reproducible manner. We have developed a method in which known numbers of hESCs in serum-free medium were aggregated by centrifugation to foster the formation of embryoid bodies (EBs) of uniform size (spin EBs). These spin EBs differentiated efficiently and synchronously, as evidenced by the sequential expression of molecular markers representing stem cells, primitive streak, and mesoderm. In the presence of hematopoietic growth factors, reproducible differentiation was achieved with blood cells formed in more than 90% of EBs. Using chimeric EBs generated from mixtures of green fluorescence protein–positive (GFP+) and GFP– hESCs in a clonogenic assay, hematopoietic precursor frequency was estimated to be approximately 1:500 input cells. This method of EB formation provides a generally applicable means for modulating and objectively monitoring the directed differentiation of hESCs.

Description: When adopting basic principles learned in mice to clinical application in humans, it is often difficult to distinguish whether a “translation” fails because of an invalid target in the human disease or because the therapeutic agents are not optimal for the human target. It is, therefore, desirable to develop preclinical models to optimize therapies for human targets using in vivo settings. Although anti–mouse CTLA-4 antibodies are known to enhance immune responses in vivo, their effect on T-cell activation in vitro ranges from enhancement to inhibition. Here we use the hu-PBL-SCID mouse model of Epstein-Barr virus (EBV)–associated lymphoma development to screen a panel of anti–human CTLA-4 monoclonal antibodies (mAbs) for their effect on human lymphocytes in an in vivo “humanized” environment. We report significant heterogeneity of anti–human CTLA-4 mAbs in enhancing the expansion of human T cells in mice, and this heterogeneity cannot be attributed to immunoglobulin isotypes or affinity for CTLA-4. These data validate the development of additional screening tools, such as the one described, to further characterize functional activity of antihuman antibodies before proceeding with clinical translation to human studies.

Description: The metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motif) converts the hyperreactive unusually large (UL) forms of von Willebrand factor (VWF) that are newly released from endothelial cells into less active plasma forms by cleaving a peptide bond in the VWF A2 domain. Familial or acquired deficiency of this metalloprotease is associated with thrombotic thrombocytopenic purpura (TTP). ADAMTS13 belongs to the ADAMTS metalloprotease family, but, unlike other members, it also contains 2 C-terminal CUB domains (complement component Clr/Cls, Uegf, and bone morphogenic protein 1). Mutations in the CUB region have been found in congenital TTP, but deletion of the region did not impair enzyme activity in conventional in vitro assays. We investigated the functions of the CUB domain in ADAMTS13 activity under flow conditions. We found that recombinant CUB-1 and CUB-1+2 polypeptides and synthetic peptides derived from CUB-1 partially blocked the cleavage of ULVWF by ADAMTS13 on the surface of endothelial cells under flow. The polypeptide bound immobilized and soluble forms of ULVWF, and blocked the adhesion of ADAMTS13-coated beads to immobilized ULVWF under flow. These results suggest that the CUB-1 domain may serve as the docking site for ADAMTS13 to bind ULVWF under flow, a critical step to initiate ULVWF proteolysis.

Description: Severe ADAMTS13 deficiency in thrombotic thrombocytopenic purpura (TTP) is either constitutional and caused by ADAMTS13 mutations, or acquired and most often due to ADAMTS13 inhibitory autoantibodies. In strongly hemolytic serum of a pediatric patient, diagnosed with TTP postmortem, ADAMTS13 activity was less than 3%. Both parents had an ADAMTS13 activity of approximately 50%. Sequencing of the ADAMTS13 gene revealed an intronic 687-2A〉G substitution affecting exon 7, homozygous in the propositus and heterozygous in both parents, confirming constitutional ADAMTS13 deficiency. ADAMTS13 activity of normal plasma was inhibited by incubation with the propositus' serum, suggesting alloantibody formation to ADAMTS13. However, immunoglobulin purified from serum had no ADAMTS13 inhibitory effect, whereas the immunoglobulin-depleted hemolytic serum inhibited ADAMTS13 activity of normal plasma, suggesting an inhibitory effect of hemolysis products. Incubation of hemoglobin, recombinant and from lysed erythrocytes, with normal plasma revealed an ADAMTS13 inhibitory effect at hemoglobin concentrations of 2 g/L or higher.

Description: Background: A variety of ophthalmologic findings have been reported in patients with anemia. Aim: To determine the effect of beta-thalassemia minor on the optic nerve head topographic analysis. Methods: A total of 39 beta-thalassemia minor patients were divided into 2 groups. Patients with iron, folic acid, or vitamin B12 deficiency were ruled out. Group 1 comprised 20 patients with anemia, and group 2 comprised 19 patients without anemia. One eye of each patient was included into the study. All subjects underwent complete ocular examination. Optic nerve head topographic analysis was performed by using a confocal scanning laser ophthalmoscope type Heidelberg retina tomograph (HRT). The following stereometric parameters were evaluated: disc area, area and volume of cup, area and volume of neuroretinal rim, measure of cup shape, and mean retinal nerve fiber layer thickness. Results: The mean age of group 1 and 2 were 26.8±7.6 and 25.6±4.5 years, respectively (P=0.91). Their mean disc areas were 2.01±0.3 mm2 and 2.53±0.6 mm2, respectively (P =0.009). The differences between groups for area and volume of cup, area and volume of neuroretinal rim, cup shape measure, and mean retinal nerve fiber layer thickness were insignificant (p〉0.05). There was no significant difference between mean intraocular pressure of both groups (p=0.93). Conclusion: In beta-thalassemia minor, patients with anemia, optic disc area showed a statistically significant reduction compared to the patients without anemia. Further clinical trials on ocular blood flow and optic nerve oxygenation changes may highlight the role anemia in the optic nerve head of the beta-thalassemia minor patients.

Description: Although significant advances have been made over the last decade with respect to our understanding of stem cell biology, progress has been limited in the development of successful techniques for clinically significant ex vivo expansion of hematopoietic stem and progenitor cells. We here describe the effect of Notch ligand density on induction of Notch signaling and subsequent cell fate of human CD34+CD38– cord blood progenitors. Lower densities of Delta1ext-IgG enhanced the generation of CD34+ cells as well as CD14+ and CD7+ cells, consistent with early myeloid and lymphoid differentiation, respectively. However, culture with increased amounts of Delta1ext-IgG induced apoptosis of CD34+ precursors resulting in decreased cell numbers, without affecting generation of CD7+ cells. RNA interference studies revealed that the promotion of lymphoid differentiation was primarily mediated by Delta1 activation of Notch1. Furthermore, enhanced generation of NOD/SCID repopulating cells was seen following culture with lower but not higher densities of ligand. These studies indicate critical, quantitative aspects of Notch signaling in affecting hematopoietic precursor cell-fate outcomes and suggest that density of Notch ligands in different organ systems may be an important determinant in regulating cell-fate outcomes. Moreover, these findings contribute to the development of methodology for manipulation of hematopoietic precursors for therapeutic purposes.

Description: Background: Combined modality treatment consisting of chemotherapy (CT) followed by involved field radiotherapy (IF-RT) is the standard treatment for early unfavourable Hodgkin’s lymphoma (HL). Despite high complete remission (CR) rates, failures are common. We thus compared the baseline-dose BEACOPP regimen with ABVD and 20 with 30 Gy IF-RT in a prospectively randomized trial (HD11) in an attempt to improve outcome in this group of patients. Methods: Between May 1998 and January 2003, 1570 patients (pts) aged 16–75 with untreated intermediate stage HL (CS I, IIA with risk factors or IIB with elevated ESR and/or ≥3 nodal areas only) were randomized according to a factorial design between 4 cycles of ABVD followed by 30 Gy IF-RT (arm A - standard treatment), 4 ABVD + 20 Gy IF-RT (arm B), 4 baseline-dose BEACOPP + 30 Gy IF-RT (arm C) and 4 baseline-dose BEACOPP + 20 Gy IF-RT (arm D). Results: In the fifth preplaned interim analysis, 1293 pts were evaluable for the chemotherapy comparison and 1274 for the radiotherapy comparison. Patient characteristics were well balanced between the treatment arms. 95% of patients treated reached CR, 2% had pogressive disease, 8% relapsed and the total mortality rate was 4% with no significant differences between treatment arms for either endpoint. The most frequent haematological toxicities during chemotherapy were leucopenia observed in 32% of pts (ABVD: 25%, BEACOPP: 39%) and anemia in 4% of pts (ABVD

Description: The application of allogeneic stem cell transplantation (alloSCT) is limited by graft-versus-host disease (GVHD). GVHD can be divided into acute and chronic forms that likely have different requirements for initiation and pathogenesis mechanisms. In prior studies we demonstrated that residual host antigen-presenting cells (APCs) were required to initiate acute GVHD (aGVHD) mediated by CD8 T cells. In contrast, here we demonstrate that either donor or host APCs can initiate CD4-mediated GVHD in a model that has features of chronic GVHD (cGVHD). Both donor and host APCs must provide CD80/86-dependent costimulation to elicit maximal cGVHD, and there is no GVHD when both donor and host lack CD80/86. Finally, we were surprised to find that, although either donor or host APCs are sufficient to stimulate skin cGVHD, donor APCs play a dominant role in intestinal cGVHD. Both CD40 and CD80/86 are critical for donor APC function in intestinal cGVHD, but only CD80/86 is required for skin cGVHD. Thus, there are target-tissue–specific differences in APC requirements. These results identify differences in APC requirements between CD8-mediated aGVHD and CD4-mediated cGVHD. They further highlight donor APCs as additional targets for GVHD therapy.

Description: Eradication of minimal residual disease (MRD) during the first months of treatment for patients (pts) with ALL is associated with improved disease-free survival (DFS). We hypothesized that Alemtuzumab, a humanized monoclonal antibody directed against CD52, might be an effective, novel agent for eradication of MRD in ALL based on data demonstrating strong CD52 expression in other lymphoid malignancies and in several ALL cell lines, and from case reports of clinical activity in advanced ALL. In CALGB 10102, to define the percentage of CD52+ cases and to demonstrate feasibility, we tested dose escalation of Alemtuzumab in sequential cohorts to a target dose of 30 mg administered sc 3X/week for 4 weeks (12 doses) during post-remission therapy. Pts are eligible to receive Alemtuzumab if lymphoblast CD52 expression at diagnosis is ≥ 10% as determined in a CALGB reference laboratory. The 10102 therapy is composed of monthly treatment modules outlined below: Treatment module sequence is: A,B,C, D, A, B, C followed by maintenance therapy for a total of 2 years. Antimicrobial prophylaxis for cytomegalovirus (CMV) (e.g. acyclovir 800 mg qid) and pneumocystis carinii is mandated. Weekly quantitative monitoring for CMV viremia is performed. 150 pts with untreated ALL have enrolled: Median age is 48 yrs. 124 (83%) pts have precursor-B; 19(13%) have precursor T-ALL and 7 (4%) have biphenotypic or bilineal ALL. Of 139 evaluable pts, 95 (68%) had CD52 ≥ 10%. By immunophenotype, 72% of precursor B and 61% of precursor T pts were eligible to receive Alemtuzumab. Phase I dose escalation was recently completed. Dose limiting toxicity (DLT) for Phase I was defined as the inability to proceed with protocol treatment within 6 weeks of the last dose of Alemtuzumab. Non-heme toxicities have been mild and sc Alemtuzumab administration was well tolerated. Hematologic and infectious toxicities are summarized below: Myelosuppression was transient and use of G-CSF was permitted during Module D. 2 pts were treated for CMV viremia due to rising CMV titers in 2 sequential assays. 6 other pts had transient CMV elevations during or immediately following completion of Alemtuzumab that did not require treatment. 22/24 phase I pts received all 12 doses of Alemtuzumab. There were 2 DLTs reported: 1 pt in cohort 2 due to CMV viremia requiring gancyclovir following completion of Alemtuzumab; and 1 pt in cohort 3 due to ANC 〈 1500 six weeks after Alemtuzumab (pt was not given G-CSF). Based on these Phase I data, the targeted dose of 30 mg Alemtuzumab was recommended for further study in the ongoing Phase II study. In summary, we report for the first time that CD52 is expressed in the majority of ALL cases and demonstrate the feasibility of employing Alemtuzumab in front-line therapy. Ongoing accrual to the phase II study will evaluate the efficacy of Alemtuzumab in eradication of MRD in adult ALL. Module A Module B Module C Module D (Alemtuzumab) Maintenance Cytoxan Cytoxan Methotrexate (IV, PO, IT) 10 mg* cohort 1 Vincristine Daunorubicin Cytarabine Vincristine 20 mg* cohort 2 Dexamethasone Vincristine Vincrisitne 6-MP 30 mg* cohort 3 6-MP Dexamethasone L-asparaginase *Phase I dose escalation Methotrexate L-asparaginase IT-Methotrexate Alemtuzumab cohorts N Myelosuppression (grades 3 or 4) Lymphopenia CMV viremia 10 mg 6 2 1 2 20 mg 10 1 1 4 30 mg 8 1 0 2

Description: Introduction Several components of the haemostatic system are altered in sickle cell anaemia(SCA). The sum of these alterations is manifested as a state of hypercoagulability. Many therapeutic approaches including anticoagulants has been tried in managing the acute painful crisis of SCA along with the analgesics. Aim This trial aimed at assessing the efficacy and safety of low molecular weight heparin Tinzaparine (innohep®) as an adjuvant therapy in the management of acute painful vaso-occlusive crisis. Material and Methods This two arm pilot, randomised multi-centre controlled prospective trial has incorporated 253 patients admitted through emergency in acute painful crisis, with no other complications of SCA. Subjects were consecutively randomized to the study or control group. In the study group 127 patients (mean age 22,83 years) received tinzaparine 175 i.u, s.c once daily along with analgesics, while in the control group 126 patients (mean age 21,64 years) received placebo and the same analgesics. Subjects were followed for improvement using the numerical pain scale (NMS), assessing the number of days in which the patient experienced the severest degree of pain on the NMS, the hospitalization period, the duration of painful crisis, and the rate at which the pain intensity declined over time. The maximal treatment period was seven days. The complications encountered are treated and estimated in both groups. The results were analysed using “SSPS-V10” statistical tests Results The results displayed in the table.1 showed statistically significant reduction in the duration of morbidity in the study group. The drop in the pain intensity is sharper, and the complications experienced in the study group were two events of minor bleeding treated conservatively. Patient Control No. of days with severest pain score on the NMS (Days) 1,28 1,74 Duration of painful crisis(Days) 2,57 4,35 Total duration of hospitalization(Days) 7.08 12,06 Conclusion The results indicate a favourable outcome in the study group compared to the control group. The use of tinzaparine in the acute painful crisis of SCA as an adjuvant to the strong analgesics is recommended due to its proven efficacy and safety in shortening the period of the agonizing pain at presentation, the period of painful crisis and the hospital stay.

Description: Recently, inhibitory cytokine pathways for leukocyte chemoattraction and activation have been identified, but there is little insight into the operational mechanisms except for models that rely on simple receptor antagonism. We have previously identified the existence of a murine eosinophil inhibitory pathway mediated by the CXC chemokine ligand 9 (CXCL9, Mig [monokine induced by interferon-γ]) that impressively blocks eosinophil chemoattraction and function, but the mechanism has remained elusive. We now demonstrate that Mig's inhibitory action extends beyond receptor antagonism alone. Notably, in addition to inhibiting eotaxin-induced filamentous actin (F-actin) formation and chemoattraction, Mig potently blocks platelet activating factor (PAF)– and leukotriene B4 (LTB4)–induced responses. Remarkably, Mig-treated eosinophils display an abnormal F-actin assembly in the absence of agonist stimulation. Additionally, Mig pretreatment inhibits eotaxin-induced activation of the Rho–guanosine triphosphatase (GTPase) Rac, and Rac2-deficient eosinophils demonstrate an impaired transmigration and actin polymerization response to eotaxin stimulation. Furthermore, Mig was unable to inhibit eotaxin-induced responses in Rac2-deficient eosinophils. Finally, using CCR3 gene–targeted cells, Mig's inhibitory activity is demonstrated to be mediated by CC chemokine receptor 3 (CCR3). Thus, by altering agonist-induced signaling and abrogating cytoskeletal reorganization by a Rac2-dependent mechanism, Mig markedly inhibits eosinophil responses to diverse stimuli. These results establish evidence that distinct chemokines can use CCR3 to induce opposing signals in eosinophils.

Description: Recent studies by our laboratory have demonstrated unequivocally that factor V is endocytosed by megakaryocytes from plasma to form the platelet-derived factor V pool. In the current study, we have determined the time-dependent acquisition of factor V by platelets in a 67 year old factor V-deficient patient, previously shown to be completely devoid of plasma- and platelet-derived factor V. The patient now receives weekly tranfusions of two units of FFP to prevent gastrointestinal bleeding. Plasma and platelet lysate samples were prepared from fresh, whole blood drawn prior to (0 hrs), and following (2, 6, 24, 96, 168 hrs) patient transfusion. A factor V radioimmunoassay (RIA), which can detect factor V concentrations as low as 0.075 μg/mL, a standard factor V clotting-based activity assay, and/or western blotting analyses, which utilize a mixture of anti-human factor V light chain and heavy chain mAbs, were used to evaluate the appearance, and subsequent disappearance, of factor V in these two blood compartments. Prior to transfusion (t = 0 hr), the patient’s plasma-derived factor V could not be detected by any of the three factor V assays. The patient’s plasma-derived factor V level peaked immediately following transfusion (t = 2 hr) reaching a concentration of 1.3 +/− 0.08 μg/mL as measured by RIA, and declined until it reached an undetectable level at 96 hrs post transfusion. These data were confirmed by the results of both the clotting-based assays and western blotting analyses. As the patient’s platelet-derived factor V levels were below the sensitivity of both the RIA and clotting assay, they were analyzed by western blotting. Such analyses confirmed that subsequent to its endocytosis, the patient’s platelet-derived factor V is stored in a partially protelytically activated form similar to that of a control individual. Due to the partially proteolytically activated state of the platelet-derived cofactor, platelet lysates treated with thrombin to activate the factor V to factor Va were used for quantative western blotting. Interestingly, and perhaps consistent with the patient’s receipt of weekly FFP transfusions, factor V/Va could still be detected in platelets immediately prior to transfusion. Subsequent to transfusion and in marked contrast to changes in the plasma-derived cofactor pool, a significant increase in the patient’s platelet-derived factor V/Va level was not observed until 24 hrs post transfusion. These data are consistent with previous studies demonstrating that platelets do not endocytose factor V. Although the concentration of the platelet-derived factor V/Va pool decreased over the subsequent 6 days, antigen remained detectable even though the plasma-derived pool had been depleted 3 days earlier. These combined observations indicate that, subsequent to FFP administration, the patient’s megakaryocytes acquire and proteolytically process plasma-derived factor V normally. Furthermore, the consistent presence of factor V/Va within the patient’s platelets is in all likelihood preventing gastrointestinal bleeding in this individual, which supports the concept that platelet-derived factor V represents the hemostatically relevant factor V pool.

Description: The zebrafish system is an excellent vertebrate genetic model to study hemostasis and thrombosis because saturation mutagenesis screens can identify novel genes that play a role in this vital physiologic pathway. To study hemostatic mutations, it is important to understand the physiology of zebrafish hemostasis and thrombosis. Previously, we identified zebrafish thrombocytes and have shown that they participate in arterial thrombus formation. Here, we recognized 2 populations of thrombocytes distinguishable by DiI-C18 (DiI) staining. DiI+ thrombocytes have a high density of adhesive receptors and are functionally more active than DiI– thrombocytes. We classified DiI+ thrombocytes as young and DiI– thrombocytes as mature thrombocytes. We found young and mature thrombocytes each formed independent clusters and that young thrombocytes clustered first. We have also shown that young thrombocytes initiate arterial thrombus formation. We propose that due to the increased adhesive receptor density on young thrombocytes, they adhere first to the subendothelial matrix, get activated rapidly, release agonists, and recruit more young thrombocytes, which further release more agonists. This increase in agonists activates the less active mature thrombocytes, drawing them to the growing thrombus. Since arterial thrombus formation is a fundamental hemostatic event, this mechanism may be conserved in mammals and may open new avenues for prevention of arterial thrombosis.

Description: Hepatic transdifferentiation of bone marrow cells has been previously demonstrated by intravenous administration of donor cells, which may recirculate to the liver after undergoing proliferation and differentiation in the recipient's bone marrow. In the present study, to elucidate which cellular components of human bone marrow more potently differentiate into hepatocytes, we fractionated human bone marrow cells into mesenchymal stem cells (MSCs), CD34+ cells, and non-MSCs/CD34- cells and examined them by directly xenografting to allylalcohol (AA)-treated rat liver. Hepatocyte-like cells, as revealed by positive immunostaining for human-specific alpha-fetoprotein (AFP), albumin (Alb), cytokeratin 19 (CK19), cytokeratin 18 (CK18), and asialoglycoprotein receptor (AGPR), and by reverse transcription-polymerase chain reaction (RT-PCR) for expression of AFP and Alb mRNA, were observed only in recipient livers with MSC fractions. Cell fusion was not likely involved since both human and rat chromosomes were independently identified by fluorescence in situ hybridization (FISH). The differentiation appeared to follow the process of hepatic ontogeny, reprogramming of gene expression in the genome of MSCs, as evidenced by expression of the AFP gene at an early stage and the albumin gene at a later stage. In conclusion, we have demonstrated that MSCs are the most potent component in hepatic differentiation, as revealed by directly xenografting into rat livers. (Blood. 2005;106:756-763)

Description: The hepatic peptide hepcidin is the key regulator of iron metabolism in mammals. Recent evidence indicates that certain forms of hereditary hemochromatosis are caused by hepcidin deficiency. Juvenile hemochromatosis is associated with hepcidin or hemojuvelin mutations, and these patients have low or absent urinary hepcidin. Patients with C282Y HFE hemochromatosis also have inappropriately low hepcidin levels for the degree of iron loading. The relationship between the hemochromatosis due to transferrin receptor 2 (TFR2) mutations and hepcidin was unknown. We measured urinary hepcidin levels in 10 patients homozygous for TFR2 mutations, all with increased transferrin saturation. Urinary hepcidin was low or undetectable in 8 of 10 cases irrespective of the previous phlebotomy treatments. The only 2 cases with normal hepcidin values had concomitant inflammatory conditions. Our data indicate that TFR2 is a modulator of hepcidin production in response to iron. (Blood. 2005;105:1803-1806)

Description: Down-regulation of immune responses by regulatory T (Treg) cells is an important mechanism involved in the induction of tolerance to allo-antigens (Ags). Recently, a novel subset of Ag-specific T-cell receptor (TCR)αβ+ CD4-CD8- (double-negative [DN]) Treg cells has been found to be able to prevent the rejection of skin and heart allografts by specifically inhibiting the function of antigraft-specific CD8+ T cells. Here we demonstrate that peripheral DN Treg cells are present in humans, where they constitute about 1% of total CD3+ T cells, and consist of both naïve and Ag-experienced cells. Similar to murine DN Treg cells, human DN Treg cells are able to acquire peptide–HLA-A2 complexes from antigen-presenting cells by cell contact-dependent mechanisms. Furthermore, such acquired peptide-HLA complexes appear to be functionally active, in that CD8+ T cells specific for the HLA-A2–restricted self-peptide, Melan-A, became sensitive to apoptosis by neighboring DN T cells after acquisition of Melan-A–HLA-A2 complexes and revealed a reduced proliferative response. These results demonstrate for the first time that a sizable population of peripheral DN Treg cells, which are able to suppress Ag-specific T cells, exists in humans. DN Treg cells may serve to limit clonal expansion of allo-Ag–specific T cells after transplantation.

Description: We identified regulator of G-protein signaling-5 (RGS-5) as an angiogenic pericyte marker at sites of physiologic and pathologic angiogenesis. In a mouse model of pancreatic islet cell carcinogenesis, RGS-5 is specifically induced in the vasculature of premalignant lesions during the “angiogenic switch” and further elevated in tumor vessels. Similarly, RGS-5 is overexpressed in highly angiogenic astrocytomas but not in hypoxia-inducible factor-1α (HIF-1α)–deficient tumors, which grow along preexisting brain capillaries without inducing neovessels. Elevated levels of RGS-5 in pericytes are also observed during wound healing and ovulation indicating a strong correlation between RGS-5 expression and active vessel remodeling beyond tumor angiogenesis. Moreover, antitumor therapy, which reverses tumor vasculature to an almost normal morphology, results in down-regulation of RGS-5 transcription. Taken together, these data demonstrate for the first time a factor that is specific for “activated” pericytes. This further supports the notion that pericytes, like endothelial cells, undergo molecular changes during neovascularization that makes them a novel target for antiangiogenic therapy.

Description: The myelodysplastic syndromes (MDSs) are a group of clonal hematopoietic stem-cell disorders characterized by ineffective hematopoiesis and dysplasia. A wide spectrum of genetic aberrations has been associated with MDS, including chromosomal translocations involving the NUP98 gene. Using a NUP98-HOXD13 fusion gene, we have developed a mouse model that faithfully recapitulates all of the key features of MDS, including peripheral blood cytopenias, bone marrow dysplasia, and apoptosis, and transformation to acute leukemia. The MDS that develops in NUP98-HOXD13 transgenic mice is uniformly fatal. Within 14 months, all of the mice died of either leukemic transformation or severe anemia and leucopenia as a result of progressive MDS. The NUP98-HOXD13 fusion gene inhibits megakaryocytic differentiation and increases apoptosis in the bone marrow, suggesting a mechanism leading to ineffective hematopoiesis in the presence of a hypercellular bone marrow. These mice provide an accurate preclinical model that can be used for the evaluation of MDS therapy and biology.

Description: Patients with aggressive T-cell non-Hodgkin’s lymphoma (NHL) treated with chemotherapy have a prognosis that is significantly inferior to that of B-cell NHL. Chemotherapy combined with CD20 monoclonal antibody therapy further improves the outcome for patients with B-cell NHL. The paradigm of combining monoclonal antibodies and chemotherapy has improved outcome for a variety of malignancies. Monoclonal antibodies directed against T cell antigens are available and it is important to evaluate their efficacy in combination with chemotherapy for the treatment of T-cell malignancies. We are conducting a single-center phase I dose escalation trial of Campath-1H with dose-adjusted EPOCH (DA-EPOCH) infusional chemotherapy to assess the maximum tolerated dose (MTD) and safety in patients (pts) with CD52-positive aggressive NHL. A single infusion of Campath-1H (30, 60, or 90 mg) is given over 12 hours before each cycle of chemotherapy; pts are premedicated with Prednisone 12 hrs before the Campath infusion is initiated. DA-EPOCH was initiated immediately following completion of the Campath infusion. Toxicity during the first cycle of treatment was used to determine Campath dose escalation. Pts were required to be chemotherapy naive and to express CD52 on the malignant T-cells. 17 pts were evaluated for eligibility and 14 pts treated; 3 were ineligible due to absence of expression of CD52 as assessed by flow cytometry on the malignant T-cells (2 with NK-T cell nasal lymphoma and 1 with CD30 positive peripheral T-cell lymphoma). 14 pts were entered (6 Peripheral T-cell lymphoma (NOS), 4 Adult T-cell leukemia/lymphoma, 2 Angioimmunoblastic T-cell lymphoma, 2 hepatosplenic T-cell lymphoma); 8 pts were treated at dose level 1 (30 mg), 3 at dose level 2 (60 mg), and 3 at dose level 3 (90 mg). The median age of treated pts was 35 years (range 17–77), median IPI 3 (range 0–5). A total of 56 cycles of treatment were administered. 2 of the 14 pts treated experienced grade 3 hypersensitivity reactions; most pts experienced grade 1–2 allergic and infusional reactions manifested as fever, chills, and urticaria. Although not originally incorporated into the definition of dose-limiting toxicity, bone marrow suppression with reversible bone marrow aplasia prevented the administration of further treatment in 2 pts at the 60 mg dose level (cycle 3 and 5) and 2 pts at the 90 mg dose level (cycle 4 and 5) of Campath. 3 of these 4 pts were CMV antigen positive and were treated with oral or intravenous gangciclovir. Grade 4 neutropenia was observed in all pts (12 during cycle 1) and grade 4 thrombocytopenia in 4 pts (3 in cycle 1). All but one pt developed grade 4 lymphopenia. Documented infections were observed in 11 pts and included bacterial, fungal and viral pathogens. 5 pts were CMV antigen positive during treatment; 2 pts developed hemorrhagic cystitis associated with BK virus infection that resolved despite continued treatment. Half of the pts entered have died due to progressive lymphoma, 5 are in complete remission (17, 10, 9, 7, 3 months) and two are too early to evaluate. Accrual at the 30 mg dose level of Campath is ongoing. Bone marrow suppression that prevented completion of therapy has not been observed in any patient treated at the 30 mg dose of Campath and appears safe for combination with DA-EPOCH for phase II evaluation.

Description: Shp2 tyrosine phosphatase plays a critical role in hematopoiesis, and dominant active mutations have been detected in the human gene PTPN11, encoding Shp2, in child leukemia patients. We report here that although no such mutations were detected in 44 adult leukemia patients screened, Shp2 expression levels were significantly elevated in primary leukemia cells and leukemia cell lines, as compared with normal hematopoietic progenitor cells. The Shp2 protein amounts correlated well with the hyperproliferative capacity but were inversely associated with the differentiation degree of leukemia cells. Suppression of Shp2 expression induced apoptosis and inhibition of leukemic cell clonogenic growth. Notably, the majority of Shp2 was preferentially localized to the plasma membrane and was constitutively phosphorylated on tyrosine in leukemia cells, and also in normal hematopoietic cells following mitogenic stimulation. Based on these results, we propose that aberrantly increased expression of Shp2 may contribute, collaboratively with other factors, to leukemogenesis.

Description: Chelation therapy is the conventional treatment for transfusional iron overload, which leads to complications in the heart, liver and endocrine glands. A 1-year, open-label, multicenter, Phase III study compared the investigational, once-daily, oral iron chelator deferasirox (DSX) with deferoxamine (DFO) in adult and pediatric β-thalassemia patients aged ≥ 2 years. Patients with liver iron concentration (LIC) of 2–3, 〉3–7, 〉7–14 and 〉14 mg Fe/g dw were randomized to receive daily DSX 5, 10, 20 and 30 mg/kg or DFO 20–30, 25–35, 35–50 and ≥ 50 mg/kg, respectively. In total, 586 patients received treatment; 299 (51%) were aged

Description: In most inherited red blood cell (RBC) disorders with high gene frequencies in malaria-endemic regions, the distribution of RBC hydration states is much wider than normal. The relationship between the hydration state of circulating RBCs and protection against severe falciparum malaria remains unexplored. The present investigation was prompted by a casual observation suggesting that falciparum merozoites were unable to invade isotonically dehydrated normal RBCs. We designed an experimental model to induce uniform and stable isotonic volume changes in RBC populations from healthy donors by increasing or decreasing their KCl contents through a reversible K+ permeabilization pulse. Swollen and mildly dehydrated RBCs were able to sustain Plasmodium falciparum cultures with similar efficiency to untreated RBCs. However, parasite invasion and growth were progressively reduced in dehydrated RBCs. In a parallel study, P falciparum invasion was investigated in density-fractionated RBCs from healthy subjects and from individuals with inherited RBC abnormalities affecting primarily hemoglobin (Hb) or the RBC membrane (thalassemias, hereditary ovalocytosis, xerocytosis, Hb CC, and Hb CS). Invasion was invariably reduced in the dense cell fractions in all conditions. These results suggest that the presence of dense RBCs is a protective factor, additional to any other protection mechanism prevailing in each of the different pathologies. (Blood. 2005; 105:4853-4860)

Description: FLT3 (fms-like tyrosine kinase 3) is constitutively activated in about 30% of patients with acute myeloid leukemia (AML) and represents a disease-specific molecular marker. Although FLT3-LM (length mutation) and TKD (tyrosine kinase domain) mutations have been considered to be mutually exclusive, 1% to 2% of patients carry both mutations. However, the functional and clinical significance of this observation is unclear. We demonstrate that FLT3-ITD-TKD dual mutants induce drug resistance toward PTK inhibitors and cytotoxic agents in in vitro model systems. As molecular mechanisms of resistance, we found that FLT3-ITD-TKD mutants cause hyperactivation of STAT5 (signal transducer and activator of transcription-5), leading to upregulation of Bcl-x(L) and RAD51 and arrest in the G2M phase of the cell cycle. Overexpression of Bcl-x(L) was identified as the critical mediator of drug resistance and recapitulates the PTK inhibitor and daunorubicin-resistant phenotype in FLT3-ITD cells. The combination of rapamycin, a selective mTOR inhibitor, and FLT3 PTK inhibitors restored the drug sensitivity in FLT3 dual mutant–expressing cells. Our data provide the molecular basis for understanding clinical FLT3 PTK inhibitor resistance and point to therapeutical strategies to overcome drug resistance in patients with AML.

Description: Objective: In this pilot study we evaluated the clinical activity, toxicity and mobilizing capacity of a new short-course (bi-weekly), dose intensive, cytoreductive/mobilizing salvage regimen (R-GIFOX) combining the cross-synergistic agents Gemcitabine (G), Ifosfamide (Ifo), Oxaliplatin (Ox) and Rituximab (R), in patients with relapsed and refractory CD20+ NHL. Based on the predicted clinical activity, tolerability and synergy among drugs, the R-GIFOX regimen may offer an effective and less toxic alternative to Cisplatin/ARA-C-based salvage regimens, also for patients aged or unfit for high-dose procedures. Patients and methods: Patients were scheduled to receive three courses of therapy followed by mobilization and ASCT or three more courses if ineligible for ASCT. Therapy was delivered on a compassionate basis after written informed consent. R-GIFOX consisted of R (375 mg/m2, d 1), G (1000 mg/m2, d 2), Ox (130 mg/m2, d 3) and Ifo (5 g/m2, d 3), as a 24-hour single infusion in patients aged ≤ 65 years, or fractionated over 3 days (dd 3–5) in older patients. Treatment was given every two weeks with G-CSF support (5 mcg/kg/day, dd 6–11; 10 mcg/kg/day at the 3rd course for stem cells mobilization). Responses were evaluated after three courses by the integrated FDG-PET/IWC criteria, and reassessed at the end of the entire program. Results: Fourteen patients (median age 63 years, range 37–78 years) with relapsed (n = 9) or primary progressive (n = 5) aggressive [diffuse large cell (n=7), mantle cell (n=4), follicular G3b (n=3)], advanced (stage IV = 71%), poor risk (IPI 3–5 = 50%; median number of previous therapy=2, r 1–4) NHL, were accrued. Forty-nine total courses were delivered (median 4, range 1–6); thirteen patients completed at least 3 courses of therapy and were evaluable for response. Actual dose intensity of the first 3 courses was 81%, 83.5%, and 86.5% for G, Ifo and Ox, respectively. CTCAE v3.0 G3/G4 thrombocytopenia was present in 26 % of courses, G3/G4 neutropenia in 22%; febrile neutropenia and infections in 8% and 6% of cycles, respectively. The ORR assessed after three courses of R-GIFOX was 77%, with 7 complete responses (54%; CR=5; CRu=2) and 3 partial; CRu converted to CR at BM biopsy after 6 courses. According to age, the ORR was 67% (4 CR, 2 PR) and 80% (3 CR, 1 PR) for patients aged ≤ 65 years and those older, respectively. According to disease status, the ORR was 40% (1 CR, 1 PR) and 89% (6 CR, 2 PR) for primary refractory and relapsed patients, respectively. Among CRs no patient has relapsed at a median time of 5 months (range, 2+ − 10+). Effective CD34+ cell mobilization was obtained in 4 out of 6 eligible patients and 2 already received ASCT. Failure free survival was 79.6%. In mantle cell lymphoma 2 CRs and 1 PR were obtained, including 2 molecular remissions (BM, PB). Conclusions: Based on the results of this pilot study, R-GIFOX was feasible, tolerable, effective and able to mobilize peripheral stem cells in patients with reccurrent aggressive NHL. It enabled the achievement of effective dose-intensities and high response rates also in the older patients. Finally, the R-GIFOX regimen showed clinical activity also in ’difficult’ histotypes such as mantle cell lymphomas.

Description: Context: A substantial clinical need exists for an alternate to vitamin-K-antagonists for treating deep-vein thrombosis in many patients. Long-term low-molecular-weight heparin, body-weight adjusted, avoids anticoagulant monitoring and may be associated with less harm due to bleeding. Objective: Considerable evidence suggests that reporting of harms-related data* from randomized clinical trials needs improvement. In accordance with CONSORT* we present a harm analysis regarding hemorrhagic complications. Design: Randomized clinical trial using objective outcome measures. Setting: 30 centres across Canada. Participants: Acute symptomatic proximal-vein thrombosis patients. Intervention: Therapeutic tinzaparin subcutaneously once-daily, or usual-care intravenous unfractionated heparin/vitamin-K-antagonists for 3 months. Main Outcome Measures: Benefit was assessed by assessing the frequency of symptomatic objectively documented recurrent venous thromboembolism and harm by objectively documented hemorrhagic complications. Results: A broad-spectrum of patients was randomized. Of 737 patients, 18 of 369 receiving tinzaparin (4.9 percent) suffered recurrent venous thromboembolism at three months compared with 21 of 368 (5.7 percent) receiving usual-care; (absolute difference, −0.9 percent, 95 percent confidence interval −4.1 to 2.4). Hemorrhagic complications occurred less frequently in the low-molecular-weight heparin group; 48 of 369 patients, (13.0 percent) versus usual-care 73 of 368, (19.8 percent), (absolute difference −6.8 percent; p=0.011; risk ratio = 0.66). The prevalence of major bleeding according to the duration of study treatment favored low-molecular-weight heparin therapy. Major bleeding persisted throughout study treatment for patients in the usual-care group receiving warfarin but ceased early by day 23 in the low-molecular-weight heparin group (p=0.034). Twenty-five patients (6.8 percent) in the low-molecular-weight heparin group and 24 patients (6.5 percent) receiving usual-care died (absolute difference 0.3 percent, 95 percent confidence interval −3.4 to 3.9). Conclusion: Our findings are clinically relevant and important, offering the clinician an alternate therapy to vitamin-K-antagonist therapy which does not require anticoagulant monitoring in a wide range of patients with proximal venous thrombosis. Self-management with once-daily subcutaneous tinzaparin was at least as effective as usual-care and vitamin-K-antagonist therapy and offers a safety advantage with less harm due to bleeding.

Description: Heparin therapy for any indication, including venous thromboembolism (VTE), can be complicated by heparin-induced thrombocytopenia (HIT). The purpose of this retrospective study was to characterize the clinical experience of patients in whom HIT complicated heparin therapy for VTE and who were switched to argatroban therapy. From the previously reported prospective, multicenter, historical-controlled Argatroban-911 and Argatroban-915 studies of argatroban therapy in HIT, we identified all patients who developed HIT while on heparin therapy for pulmonary embolism and/or deep venous thrombosis and in whom heparin was discontinued and argatroban therapy initiated. The primary study end point was a composite of death, amputation, or new thrombosis within 37 days of argatroban initiation; we also evaluated a 37-day composite end point of thrombosis-associated events, including death due to thrombosis, amputation secondary to HIT, or new thrombosis. A total of 145 patients with VTE and HIT were included in our analysis. During heparin therapy before HIT was diagnosed, platelet counts decreased from daily mean values greater than 175x109/L to a mean±SD nadir of 78±67x109/L over the course of 5 days, and new thrombosis developed in 75 (52%) patients. After heparin was discontinued, patients received argatroban (mean dose 2.1±1.2 mcg/kg/min) for 6.8±4.3 days achieving mean activated partial thromboplastin times during therapy of 63±12 s. By day 6 of argatroban therapy, the mean platelet count had risen to 〉150x109/L. The primary end point occurred in 41 (28.3%) patients, and the thrombotic composite in 23 (15.9%) patients (Table 1). Seventeen (11.7%) patients, including 12 who had also experienced thrombosis while on heparin, developed new thrombosis after argatroban initiation, typically on the day argatroban was discontinued or later (n=10). Death due to thrombosis occurred in only 1 (0.7%) patient. Seven (4.8%) patients experienced major bleeding. We conclude that in heparin-treated patients with VTE, HIT-associated thrombosis often occurs before HIT is recognized, emphasizing the importance of platelet count monitoring and a high degree of suspicion for HIT in this setting. For VTE patients with HIT, argatroban provides effective anticoagulation, with outcomes comparable with those reported for argatroban-treated patients in whom HIT developed following heparin therapy for any indication. New thrombosis occurring after switching to argatroban therapy more typically occurs in patients with existing HIT-associated thrombosis and at/after argatroban discontinuation. Clinical Outcomes n(%) Outcome n=145 Primary (all cause)* Thrombosis-related†,¥ *All-cause death, all-cause amputation, or new thrombosis within 37 days. †Death due to thrombosis, amputation secondary to HIT, or new thrombosis within 37 days. ¥More than 1 outcome may have occurred in a single patient. Composite end point 41 (28.3) 23 (15.9) Individual components Death 19 (13.1) 1 (0.7) Amputation 8 (5.5) 6 (4.1) New thrombosis 17 (11.7) 17 (11.7)

Description: Background The prophylactic use of systemic antifungal agents reduces morbidity and mortality after allogeneic hemopoietic stem cell transplantation (HSCT). However, the efficacy of this approach in pts receiving intensive chemotherapy or autologous HSCT is yet unproven. This trial was designed to evaluate the efficacy of L-AmB prophylaxis in high-risk neutropenic pts. Methods: 231 pts with hematological malignancies and expected neutropenia (N) of more than 10 days (d) following intensive chemotherapy or autologous HSCT were enrolled, 219 pts became neutropenic and were randomized to receive either 50 mg L-AmB i.v. every second d (arm A) or no systemic antifungal prophylaxis (arm B). Treatment with L-AmB started 1–3 d prior onset of N and was continued until neutrophil recovery, breakthrough IFI, intolerable toxicity or death. The level of significance was 0.05 (two-sided) for all statistical analyses. Calculations were performed using commercially (SPSSWIN 12.0) software, the calculations for GEE were performed using the software MAREG. Results Pt. characteristics: Eligible pts 219; arm A: 110; arm B: 109. Reasons for exclusion were: Absence of N (8), infection prior N (3) and pts decision (1). Baseline characteristics were balanced for age (mean 53.8 years), underlying disease (119 AML, 27 ALL, 64 NHL, 9 other), duration of N (mean 14.8 D) and treatment modality (primary 149, secondary 42, transplant 28). Primary endpoint: The incidence of proven and probable IFI was 5 of 110 pts (4.6%) in arm A and 22 of 109 pts (20.2%) in arm B (p = 0.001, RR = 2.9, CI 1.3 – 6.5). Key secondary endpoints: Pneumonia of unknown origin occurred in 6 pts (5.5%) vs. 28 pts (25.7%) (p 〈 0.001), the incidence of possible, probable and proven IFI was 11 pts (10.2%) vs. 42 pts (39.6%) (p 〈 0.001), systemic antifungals were used in 24 pts (22%) vs. 64 pts (59%) (p 〈 0.001), and death occurred in 4 pts (3.7%) vs. 9 pts (8.2%) (p = 0.16) in arm A vs. arm B. Toxicity: No grade 3 or 4 toxicity was noted. Laboratory abnormalities, including creatinine and liver function tests, were not different between the treatment groups. Conclusion: Intermittent application of low dose L-AmB is save. The significant lower incidence of IFI in pts treated with L-AmB prophylaxis supports its use in prolonged N.

Description: Background: A subset of children with cITP is at increased risk for severe bleeding and functional limitations. We evaluated the HRQL of children with cITP as part of a multicenter, open label Phase I/II clinical trial of four weekly doses of Rituximab, evaluated for response at 12 weeks. Responders had a ≥ 50,000/mm3 platelet count over weeks 9–12 without other supportive therapy. Methods: All participating parents were invited to complete the Child Health Ratings Inventories (CHRIs) at baseline and 12 wks. The CHRIs, a generic HRQL questionnaire, contains 20 items, forming four domains in the areas of physical, role, and emotional functioning, and school. Each item has a five-point Likert response scale. Domain scores range from 0–100, with higher scores connoting better functioning. Of the 36 study participants, 24 parents opted to participate in the HRQL evaluation; 22 of 24 (91%) completed both the baseline and 12-week assessments. The baseline clinical profile and 12-week Rituximab response rates of HRQL participants and non participants (n=12) were compared. Potential clinical covariates (e.g., duration of illness, worst bleeding score, platelet count, number of prior therapies) were examined in a univariate analysis (UVA) with baseline HRQL domain scores. Regression analyses (MVA) adjusting for age and gender were used to assess the effects of treatment response and clinical covariates on the mean change in scores between baseline and week 12. Results: While HRQL participants had more previous treatments and lower platelet counts than nonparticipants, their clinical profile was otherwise similar; Rituximab response rates did not differ. Mean HRQL domain scores (standard deviation) at baseline were as follows: physical-52.1 (36.2); role-78.6 (23.7); emotional-63.4 (18.3); school-64.1 (25.8). Lower platelet count and greater number of prior therapies were significantly associated with worse baseline HRQL scores on UVA. By week 12, 36% (8/22) responded to Rituximab. Clinically meaningful improvements in HRQL scores (≥ 0.5 standard deviation improvement) were observed in responders; no change was detected in non-responders. In MVA, responder status contributed the predominate effect on the HRQL change scores, with the greatest improvement noted in emotional functioning (p=.002). No other clinical variables emerged in this limited sample size. Conclusions: The pattern of parent-reported HRQL scores for these children with severe cITP mirrored scores from other severe, pediatric health conditions. Significant improvements in HRQL were observed among Rituximab responders over the 12-week period while non-responders had no change. Further overall characterization of HRQL in the cITP pediatric population is needed.

Description: Objective: To observe the effect of allogeneic peripheral blood stem cell transplantation (allo-PBSCT) on hematopoietic reconstitution of chronic myelogenous leukemia (CML) and reversion of myelofibrosis (MF). Methods: A 39 years old male patient diagnosed with CML complicated with MF and hepatitis B carrier condition (HBsAg+)underwent allo-PBSCT. The donor was the patient’s sibling, his 48 years old sister. Six loci on HLA-A, B, and DRB1 were completely matched with that of the recipient and HbsAb(+). Four days after the administration of G-CSF (250ug/day), PBSCs were collected from the donor for 3 consecutive days. A total volume of 168 ml was harvested. A median nucleated cell (MNC) count of 8.54 ×108/kg with 5.1 ×106 /kg of CD34+ cells were actually administered to the recipient. The conditioning regimen was busulfan/cyclophosphamide (BU/CY). The recipient was started on intravenous infusion (IV) of cyclosporine A (CSA) on Day -1 and a plasma concentration of 150–250 ng/L was maintained. IV methotrexate (MTX) 10mg/m2 was given on Days +1, +3, +6, and +11. Oral mycophenolate mofetil (MMF)was given from Days +1 to +28 for prophylaxis of acute graft-versus-host disease (GVHD). If the concentration of hemoglobin (Hb) decreased to less than 70 g/L and/or platelet count (BPC) less than 10×109 /L, the recipient was transfused with 60Co 25cGy irradiated and leuko-depleted red blood cells (RBCs) and/or single-donor platelets. Started from Day +3, the recipient was given G-CSF until white blood cells (WBC) increased to at least 3.0× 109 /L. EPO and Interleukin-11 were also given to stimulate the proliferation of the progenitors of erythrocytes and megakaryocytes. The recipient also received anisodamine with a 24 hour-maintenance dose to improve the microenvironment of hematopoiesis in bone marrow. Results: DNA-STR on Day +30 indicated a complete chimerism in the recipient. Bone marrow biopsy on Day +50 showed a dramatic decrease of myelofibrosis in the ground substance and active hematopoiesis. Reticulinfibers reduced to 1+. No significant change on splenomegaly was observed but the hepatitis B antigen had turned negative and HBsAb had appeared. Conclusion: 1. In order to prevent aggravation of MF, Allo-PBSCT needs to be considered as early as possible once the diagnosis of CML complicated with MF is made. 2. Transfusion with a large quantity of donor hematopoietic stem cells is beneficial for engraftment. 3. Strict Immunoinhibition of the recipient will decrease the incidence and degree of GVHD and improve engraftment. 4. The addition of agents which improve the microenvironment of hematopoiesis will enhance engraftment. 5. Recovery of the platelet count is promising post-transplantation whereas significant changes on splenomegaly was not observed. 6. Allo-PBSCT may be a promising therapy for hepatitis B.

Description: Introduction. An ideal biopsy needle consistently recovers representative specimens of adequate length with minimal dependence on patient characteristics. We have shown that bone marrow biopsy needle gauge influences the length and quantity of recovered specimens. A needle that performs independently of patient characteristics should yield specimens demonstrating a normal distribution of specimen lengths. We evaluated the influence of age and needle gauge on the specimen length distribution curves of patients undergoing bone marrow biopsies using SNARECOIL needles. Methods. 88 bone marrow core specimens were recovered from 72 patients using 11G SNARECOIL bone marrow biopsy needles. The mean age of the patients was 61.4 and the m/f ratio was 45/27. 53 specimens were recovered from 39 patients (mean age = 47.1, m/f = 27/12) ≤ 64 and 35 specimens were recovered from 33 patients (mean age = 78.6, m/f = 18/15) ≥ 65. 106 patients underwent 127 bone marrow biopsy procedures using 8G SNARECOIL needles. The m/f ratio was 56/50 and the mean age of the patients was 63.1 years. 72 specimens were recovered from 56 patients (mean age = 51.4, m/f = 30/26) ≤ 64 years old and 55 specimens were recovered from 49 patients (mean age = 76.1, m/f = 26/23) ≥ 65. 40 additional specimens were prospectively recovered from 39 patients ≥ 65 (mean age = 74.3, m/f = 19/20) with 8G SNARECOIL needles. Results. The mean specimen lengths of the 11G and 8G specimens were statistically the same (mean±SEM, 1.97±0.07 cm vs. 1.99±0.05 cm, respectively, p = 0.8). However the 11G specimen length frequency distribution curve deviated markedly from a normal distribution (skewness(skw) = 0.52) while the 8G specimen distribution was nearly normal (skw = 0.04). While the frequency distribution curves of older patients (≥ 65) biopsied with 8G needles showed minimal deviation from normality (skw = 0.12,) the distribution curve of older patients (≥65) biopsied with 11G needles deviated markedly from a normal curve (skw = 0.64). 40 specimens prospectively obtained from patients ≥ 65 demonstrated a mean specimen length of 1.77±0.09 cm with a near normal specimen length frequency distribution curve (skw = 0.07). Conclusions. 1. Although commonly reported, mean specimen lengths do not completely characterize the performance of biopsy needles. 2.0 Specimen length frequency distribution curves provide added characterization of needle performance in defined patient cohorts. 3.0 In the older patient population, near-normal 8G specimen frequency distributions and skewed 11G distributions suggest that 8G specimens may provide more representative sampling.

Description: The 26S proteasome plays a vital role in degrading regulatory proteins such as p53, p21, p27, NF-kB, I-kB, and bcl-2, that govern cell cycle, transcription factors activation, apoptosis and cell trafficking. Thus, the mechanisms by which bortezomib elicits its antitumor activity may vary among tumor types, and the extent to which each affected pathway is critical to the inhibition of tumor growth could also differ. The aim of this study was to determine the efficacy and toxicity of bortezomib in previously pretreated patients with peripheral T-cell lymphoma unspecified (PTCLU) and cutaneous T-cell lymphomas (CTCL). Each patient had to meet the following inclusion criteria to be enrolled in the study: histologically confirmed PTCLU or CTCL (according to REAL/WHO classification); any stage, any IPI, any bone marrow status; second or more relapse or refractory disease; age ≥ 18; ECOG performance status ≤ 2; Hb ≥ 10 g/dL, ANC ≥ 1.5x109/L and platelets ≥ 100 x 109/L; normal hepatic, renal and cardiac functions; and voluntary written informed consent. Bortezomib was given at 1.3 mg/m2 IV push on days 1, 4, 8 and 11 every 21 days. Restaging was done every 2 cycles. Patients were treated for up to a total of 6 cycles unless removed from study for failure to respond or toxicity. The response criteria were those recommended by NCI sponsored Working Group. A total of 30 patients will be enrolled; so far 10 patients entered and preliminary results of this trial will be presented.

Description: Background Diagnosis and prognosis of patients with established MDS/CMML is currently based on WHO criteria and the IPSS prognostic Index. High variation of survival in subgroups warrants the search for additional criteria. A comparison of CFU-C cultures with WHO criteria and IPSS score has not yet been done in a large patient group. Patients and methods We analyzed in a single center retrospective cohort study 93 untreated consecutive patients (55 male/ 38 female; median age: 66 years; range: 13 – 88 years) admitted between July 1992 and June 2002 and diagnosed as MDS/CMML (RA/RARS(4), MDS-U (2), RCMD (26), RAEB I/II (44) and CMML I/II (17). All patients had an unequivocal diagnosis of MDS or CMML according to WHO criteria. Simultaneous examinations of blood, bone marrow (cytology and biopsy), BM-cytogenetics and BM-and PB cultures for CFU-GM and BFU-E were done. Culture results were scored blindly and classified either as “Low risk CFU-C“ including normal growth (N=2), no colony growth (N=6) or reduced growth of normal colonies (N=19) or as „High risk CFU-C’s“ including excess normal growth in PB termed “MPS pattern” (N=5), discrete leukemic cluster growth (N=22), abundant leukemic cluster growth (N=14) or the „CMML pattern“ defined as giant“ pseudonormal“ CFU-GM and strongly reduced BFU-E (N=23). Culture score was compared with the WHO diagnosis and with the IPSS score in all 93 patients and survival was assesed in 82 patients treated with conventional therapy (12 allografted patients were excluded) after minimal observation time of 3 years in July 2005. Results Comparison of WHO diagnosis with culture score Low risk CFU-C score High risk CFU-C score RA/RARS/RCMD/MDS-U 15 16 RAEBI/II 12 30 CMML-I/II 0 17 The typical CMML pattern was observed in 13 of 17 patients with CMML (Specificity 94%). Comparison of IPSS Score with culture score low risk CFU-C score low risk CFU-C score High risk CFU-C score High risk CFU-C score IPSS Score alive/dead mean survival(d) alive/dead mean survival(d) Low and Int-1 4/4 2022+/−543 8/39 965+/−143 Int-2 and High 1/1 1024+/−701 1/26 565+/−92 Mean survival time for patients with a low/intermediate-1 IPSS score was less than half if they had a high risk CFU-score (p

Description: Chronic lymphocytic leukemia (CLL) is a disease in which two-thirds of patients will require therapy, usually with the alkylating agent, chlorambucil (CLB) or the nucleoside analog, fludarabine (FLU). TRAIL is a death receptor ligand that has shown selective cytotoxicity towards a variety of malignancies. CLL cells, however, are relatively resistant to this agent. We have previously demonstrated that CLB- and Flu-induced apoptosis is partially mediated through activation of the TRAIL apoptotic pathway and this is related to the up-regulation of the TRAIL death receptors, DR4 and DR5 (Oncogene, 22:8356–8369, 2003). Combining CLB or Flu with TRAIL produced a synergistic apoptotic response in CLL cells. In contrast, the upregulation of DR5 and the synergistic apoptotic response was not observed in normal lymphocytes. We have subsequently demonstrated that the up-regulation of DR5 is mediated by transcription factors, nuclear factor κB (NFκB) and p53, and histone acetylation. Using chromatin immunoprecipitation assays, we have found that the p65 subunit of NFκB and p53 are bound to the DR5 gene in CLL cells following either CLB or Flu treatment. In addition, histone 3 is acetylated following CLB and Flu treatment corresponding to histone acetylase p300 binding to the DR5 gene. Histone deacetylase 1 (HDAC1) also binds to the DR5 gene following CLB or Flu treatment, but generally at later time points. Overall histone acetylation was also found to be increased in CLL cells, as compared to normal lymphocytes. Treatment with HDAC inhibitors, which are being evaluated in clinical trials, resulted in increased binding of p53 and NFκB, but not HDAC1, to the DR5 gene and increased DR5 mRNA levels in cells. Furthermore, enhanced apoptosis was also observed in CLL cells treated with combinations of SAHA (another HDAC inhibitor) and TRAIL. These findings suggest that histone acetylation is important in regulating DR5 expression and that HDAC inhibitors increase DR5 expression mediated by p53 and NFκB. This could provide a mechanism to sensitize CLL cells to TRAIL-induced apoptosis.

Description: Backgrounds: The International Prognostic Index (IPI) is the most commonly used survival parameter for patients with Non-Hodgkin’s lymphoma (NHL). To investigate an another factor to predict survival, we studied the role of tumor necrosis factor receptor with molecular weight of 75 kd (p75-R-TNF) and extracellular protein kinase A (ECPKA). TNF has a central role in inflammatory processes, and its receptor is constitutively found in the circulation and is elevated in a variety diseases. The cAMP-dependent protein kinase (PKA) is critically involved in the regulation of metabolism, cell growth and differentiation, and gene expression. PKA is a predominantly intracellular enzyme, but it has been shown that cancer cells of various cell types excrete PKA into the conditioned medium. This extracellular form i.e., ECPKA is known to be upregulated in the serum of cancer patients as compared with normal serum. The aim of this prospective study was to evaluate p75-R-TNF and ECPKA as feasible prognostic factors for patients with NHL. Methods: From October 2003 to May 2005, chemotherapy-naive patients with NHL who were planned to receive CEOP-B or R-CHOP chemotherapy at Guro Hospital, Korea University were enrolled. Blood sampling for p75-R-TNF and ECPKA was done before chemotherapy initiation and was stored at −70°C until the assay. The level of p75-R-TNF was measured using ELISA kit. ECPKA was measured by RIA method. A total of 20 serum samples from normal people were used as control. Results: A total of 45 patients were enrolled. The male to female ratio was 22:23, and the median age was 58 years old (range: 29–87). Indolent histologic type and aggressive type were 4 and 41 patients, respectively. Twenty-two patients were limited stage (stage I, II) and 23 patients were advanced stage (stage III, IV). The level (mean±SD) of p75-R-TNF was 1066.6±1174.9 pg/ml for patient group and that of control group was 678.2±312.4 pg/ml. ECPKA activity of patient group was 87.6±21.1 mU/ml as compared with 40.6±21.5 mU/ml in control group. During the median follow-up period of 8.5 months, 12 patients died and 33 patients were alive. As a result of univariate analysis, serum albumin (p=0.005), hemoglobin (p=0.054), the IPI score [0–2 vs 3–5] (p=0.006), occurrence of febrile neutropenia (p=0.01), and level of p75-R-TNF (p=0.003) were significantly associated with survival. By logistic regression testing, p75-R-TNF level was identified as an independent predictive factor for survival (p=0.037). Median survival of patients with elevated p75-R-TNF (cutoff: 678.2 pg/ml) was 10.5 months. For patients with p75-R-TNF 〈 678.2 pg/ml, median value was not reached yet. Conclusions: This study indicates that in addition to the IPI, high baseline levels of p75-R-TNF can predict the survival of patients with NHL. The levels of ECPKA were elevated in patient group than those of normal controls, but its level could not predict the prognosis.

Description: In a previous intramural study using tumor registry data, we found no difference in survival for all 222 patients diagnosed with low-grade (LG) or intermediate-grade (IG) non-Hodgkin’s lymphoma (NHL) during 1980–1989 compared to all 404 such patients who were diagnosed during 1990–1999 (ASH 2002). However, the anti CD20 monoclonal antibody rituximab, which was introduced in 1998, has been associated with improved survival in randomized trials of patients with intermediate grade B cell lymphomas. In this study we carried out two analyses. The first compared relative survival for 368 Hoag lymphoma patients diagnosed during 1995–2000 (median age 65 years) to national data from the Surveillance Epidemiologic and End Result (SEER) program. The 5-year relative survival rate for Hoag patients compared to SEER was 72% vs. 59% for 1995–2000, in sharp contrast to the relative 5-year survival rates observed during 1989–1994 which were only 46% for Hoag lymphoma patients vs. 51% for SEER. The second comparison was for observed survival for all 362 Hoag patients with LG or intermediate-grade IG B cell NHL, who were diagnosed during 1998–2003 (median age 65, 105 deaths), compared to 352 Hoag patients with LG or IG B-cell NHL, who were diagnosed during 1990–1997 (median age 63, 175 deaths). The respective 5-year survival rates were 58% vs. 51% (p=.032, 2-tailed log-rank test). We conclude that survival has improved for Hoag patients with lymphoma both by intramural historical comparisons and extramural contemporary comparisons. Improvement took place during the time that rituximab was adopted as standard therapy at Hoag, and suggests that the survival benefits that have been observed in randomized trials are also taking place in the general population of B cell lymphoma patients.

Description: The goal of this study is to compare the efficacy and safety of an original DAF regimen: daunorubicine (DNR) 60 mg/m2/d iv, days 1–3; cytarabine (AraC) 200 mg/m2/d ci, d 1–7, and fludarabine 25 mg/m2 2h inf. iv d 1–5 versus previously studied DAC regimen* (DNR, AraC, Cladribine), and versus standard DA in de novo acute myeloid leukemia (AML) patients below 60. Primary objective is complete remission (CR) rate after single course of induction and overall survival, secondary objectives - overall CR rate, toxicity, leukemia-free survival rate, assessment of lymphocyte subpopulations levels and survival in patients submitted to bone marrow allotransplantation (alloBMT) immediately after CR assessment. Patients achieving CR and did not submit to alloBMT received two courses of subsequent intensive consolidation: 1) HAM (HD AraC, mitoxantrone) 2) HD AraC. In case of partial remission (PR) after the first induction course the same regimen was repeated, Patients with no remission (NR) or PR/NR after 2 induction courses were withdrawn with the study. We are planning to enrol to the study 600 patients in 3 years. Between 09.2004 and 07.2005, 147 adult AML patients, aged 48 (19–60)y, sex: male 57, female 90, treated in 16 co-operating Polish Adult leukemia Group (PALG) centres were randomised to either DAF (n=44), DAC (n=49) or DA (n=54) arm. PML/RAR alfa positive - FAB M3 cases were excluded. Both study groups were well balanced in respect of age, sex, FAB subtype, and WBC. The final CR rate and CR rate after the first induction course equalled: for DAF 65% and 60%, for DAC 70% and 60%, and for DA 55% and 47%, respectively (p=NS). The median times to ANC recovery 〉 0.5 G/L, and PLT 〉50 G/L in each arms were similar (22–26 d.) (p=NS). All patients developed WHO grade IV thrombocytopenia and agranulocytosis. The frequency and severity of infections, mucositis, vomiting, diarrhea, alopecia, polyneuropathy as well as of cardiac, liver or kidney failure were comparable in each treatment arms. Early death was noted in 6% (n=2) in DAF, in 9% (n=3) in DAC, and in 13% (n=5) in DA group, because of bacterial sepsis in every cases. In conclusion, this original study proves that the addition of fludarabine to the standard DNR+AraC regimen (DAF) comparing to DAC and DA regimen is a potent antileukemic treatment without increased toxicity.

Description: Granulocytic Sarcomas are extramedullary tumors composed of immature myeloid cells, these tumors can be seen in myeloid malignancies such as chronic myelogenous leukemia, myelodysplastic syndrome and acute myelogenous leukemia. They can be seen at the time of diagnosis of the myeloid malignancy, prior to diagnosis, or after treatment as a first sign of relapse. We report here the case of a young woman who presented with a 6 month history of a slowly enlarging nasal mass. Biopsies of the lesion had been inconclusive until a CBC revealed abnormal blasts in the peripheral blood. Subsequently bone marrow biopsy revealed more than 59% erythroid precursors with a preponderance of immature erythroblasts and 33% myeloblast consistent with AML (M6). Reanalysis of the nasal biopsy demonstrated positive staining for CD68, Myeloperoxidase, Lysozyme, and CD117, but negative for CD34, suggestive of granulocytic sarcoma. The patient received induction chemotherapy with idarubicin and cytarabine and achieved bone marrow remission along with complete resolution of the nasal mass. To our knowledge this is the first report of a granulocytic sarcoma in association with AML M6.

Description: MG-0103 is a novel non-hydroxamic acid inhibitor of human histone deacetylases (HDACs), with selectivity for the cancer-associated isoforms of class I HDACs. Deacetylation of histones by HDACs is postulated to inactivate tumour suppressor genes leading to neoplastic transformation, and therefore inhibition of this enzyme may result in antineoplastic activity. To study the safety and activity of MG-0103, we have developed a phase I open-label dose escalation study of MG-0103 administered orally, three-times weekly in patients with leukemia or MDS, with the primary endpoints being the determination of the maximum tolerated dose (MTD) and the pharmacokinetic (PK) and pharmacodynamic (PD) characteristics of MG-0103. Eligibility criteria included appropriate performance status and renal and hepatic functions. Patients with relapsed/refractory leukemia or MDS and older patients with untreated AML/MDS were eligible. Eight patients have been enrolled at two dose levels (20 &40 mg/m2) so far. Their characteristics are: median age 70 years (range 33–79); all patients so far treated have AML; median number of prior therapies is 1 (range 0–3). All patients had complex cytogenetics including one elderly patient with t(8;21) that had not achieved CR with high dose ara-C-based therapy. MG-0103 has been well tolerated with no dose-limiting toxicities. No drug-related adverse events (AEs) 〉= Grade (Gr) 3 have been observed. The only Gr 2 drug-related AE has been heartburn (1 patient). All other drug-related AEs were grade 1. No cardiac abnormalities have been observed so far. Two of 3 patients at the first dose level have been treated for 3 or more cycles. PK evaluations are shown in the table below. Analysis of peripheral blood cell HDAC activity indicates that HDAC inhibition occurs in a dose-dependent manner. Enrolment is currently proceeding at a dose of 80 mg/m2. In summary, MG-0103 is a well-tolerated HDAC inhibitor in patients with AML at doses and exposures that result in target inhibition in peripheral blood. Dose 20 mg/m2 40 mg/m2 Pharmacokinectic characteristics AUC (0-∞) ng.hr/ml 260 +/− 10.8 528 +/−275 T 1/2 h 8.09 +/− 2.98 6.24 +/− 0.373

Description: Patients during cancer treatment and cancer survivors frequently utilize complementary and alternative medicine (CAM) therapies. While the beliefs and knowledge regarding CAM of many cancer-specific patient groups have been well studied such as breast cancer patients and prostate cancer patients, no specific evaluation of lymphoma survivors and their beliefs and knowledge about CAM has been undertaken. Because CAM can yield both risks such as toxicity and displacement of efficacious therapy as well as potential benefits such as improvement in quality of life and mood, we surveyed lymphoma survivors in a pilot study to ascertain their current beliefs, knowledge, and utilization of CAM. Using the Mayo Tumor Registry, we identified eligible patients who were 16 years or older at diagnosis, U.S. residents, first diagnosed with Hodgkin or non-Hodgkin lymphoma from 1984–1998, diagnosed and/or initially treated at Mayo Clinic Rochester, and survived for 5 to 20 years (N=2,485). In October of 2004, we mailed a 23-page survey to 95 randomly selected patients; 7 were found to be ineligible (deceased or too ill). Of the 88 remaining patients, we were able to find a correct address for 82, and 57 completed a survey for a 70% participation rate. Complete data were available on 54 patients at the time of this analysis. The mean age at completion of the questionnaire was 60.8 years (26.1–86.7). The mean time since diagnosis was 12.0 years (6.3–19.9), and 52% survived more than 11 years. The histologies included 22 (39%) Hodgkin lymphoma, 21 (38%) diffuse large B-cell, 3 (5%) follicular, 1 (1%) high grade, 5 (9%) peripheral T-cell lymphoma, and 4 (7%) other. A majority of patients expressed no knowledge about the use of CAM cancer care, while only 4% of patients responded that CAM could both cure cancer and that it was perfectly safe. Ten to twenty percent of patients felt that CAM could assist other therapeutic interventions, relieve symptoms, assist the body to heal or increase quality of life. Fifteen percent of patients reported that CAM utilization increased the feeling of control, and 24% reported that CAM could have side effects. With respect to CAM utilization, overall 32% of patients had ever used CAM, but no patients reported that CAM usage was directed specifically towards their lymphoma. The most commonly used CAM modalities were chiropractic (39%), massage (21%), relaxation therapy (7%), meditation (5%) and acupuncture (5%). Overall usage of dietary supplements was relatively low, with green tea, garlic, flax seed, and echinacea being the only dietary supplements used by more than 10% of respondents. Five percent had used St. John’s Wort and 7% had used shark cartilage. In conclusion, lymphoma long-term survivors appear to use CAM at a rate similar to the general population, which does not follow the typical pattern seen in other cancer survivorship populations. The use of St. John’s Wort has potential risks if not identified prospectively. At the same time, lack of access to potentially beneficial modalities was also identified, and these observations suggest the opportunity for further study of targeted educational interventions regarding the use of CAM in this population.

Description: Bcl-2 is the prominent member of a family of proteins responsible for dysregulation of apoptosis, prevention of cancer-cell death, and resistance to chemotherapy and radiotherapy. Overexpression of Bcl-2 protein was shown to result in resistance to a variety of apoptosis-inducing signals in acute leukemia. Arsenic trioxide (As203)induces clinical remission in acute promyelocytic leukemia with minimal toxicity and apoptosis in APL-derived NB4 cells at low (0.5 to 2 μmol/L) concentration. Short interfering RNA (siRNA) has been evaluated as an attractive and effective tool for suppressing a target protein by specifically digesting its mRNA. In this study, we studied the effect of transfection with siRNA targeting Bcl-2 in NB4 cells in terms of proliferation and induction of apoptosis. Further,we investigated whether the Bcl-2 siRNA may enhance the sensitivity of NB4 cells to arsenic trioxide. Synthetic siRNA was transferred against Bcl-2 into NB4 cells using Oligofectamine transfection protocol in the presence or absence of arsenic trioxide. RT-PCR and Western blotting analysis of Bcl-2 expression in NB4 cells was performed after transfection. The inhibition of cell growth was assessed by a modified MTT assay, counting cell number. Apoptosis was determined by morphological observation and flow cytometric analysis. When synthetic Bcl-2 siRNA was delivered into NB4 cells, no Bcl-2 transcripts were detected at 24h, and Bcl-2 protein levels were reduced by approximately 72% for 48h after siRNA transfer. Bcl-2 siRNA treatment for 72h led to 36% apoptosis of cells,as compared to control siRNA treatment. Control siRNA had no significant effect on growth and apoptosis of cells. Simultaneous administration of As203 and Bcl-2 siRNA was able to reduce the mRNA and protein expression of Bcl-2 significantly compared to Bcl-2 siRNA alone demonstrating a synergistic effect of combined As203 and siRNA treatment (P

Description: Acquisition of drug resistance in tumor cells in children with T-cell acute lymphoblastic leukemia (T-ALL) during chemotherapy results in relapse and poor outcome. T-ALL cell lines that have acquired resistance to chemotherapeutics are therefore critical tools for the study of acquired resistance, yet there is a paucity of cell lines available for study. In this study, we hypothesize that drug resistant T-ALL cells can be produced by prolonged exposure to chemotherapeutics and that microarray analysis can be employed to identify the gene products responsible for acquired drug resistance. By incrementally increasing the drug concentration in growth media, we have produced T-ALL cell lines (Jurkat and Sup T1) that grow well in the presence of therapeutic concentrations of L-asparaginase (ASNase) and daunorubicin (DNR). The genetic profiles of the drug-resistant cell lines were compared to their parental progenitors using the Affymetrix HG-U133Plus2 GeneChip platform, capable of hybridizing ~54,000 genes and ESTs/chip. Signal intensity was normalized using the robust multi-array average (RMA) technique in GeneSpring 7.2. The Sup T1 and Jurkat ASNase-resistant cell lines increased their IC50s 26-fold (0.044 IU/mL to 1.14 IU/mL) and 320-fold (0.003 IU/mL to 0.96 IU/mL), respectively. The IC50 of the Jurkat DNR resistant cell line increased 77-fold (30 nM to 2300 nM), and 4.0-fold, (0.46 nM to 1.85 nM), respectively. Notably, DNR resistant Jurkat cells were also resistant to therapeutic concentrations of vincristine and prednisolone, but not ASNase. In contrast, the ASNase resistant cell lines remained sensitive to DNR, vincristine, and prednisolone. Microarray data comparing DNR-resistant and parental cell lines showed 288 genes upregulated 〉1.5-fold in the resistant line. Two sets of genes were the most upregulated in the drug resistant cells in comparison to parental cells. ABCB1 (ABC transporter P-glycoprotein) was upregulated ~940-fold and genes coding for 6 different killer-cell immunoglobulin-like receptors (KIRs) were upregulated 〉6-fold. In the case of the ASNase-resistant cell lines, 96 genes were found to be upregulated 〉1.5-fold in both Jurkat and Sup T1 lines. The most highly upregulated gene in both cell lines was argininosuccinate synthase (ASS), 32-fold upregulated in Jurkat and 6.5-fold in Sup T1. All expression results were confirmed by qRT-PCR. These genes have previously been implicated in the acquisition of drug resistance: ASS is critical for responding to asparagine depletion caused by ASNase. ABCB1 acts as a molecular pump capable of lowering intracellular concentrations of substrate chemotherapeutics such as DNR, vincristine, and prednisolone, consistent with our observation of multi-drug resistance in that cell line. To our knowledge, this is the first description of DNR and ASNase resistant Jurkat and Sup T1 T-ALL cell lines. In addition, our results suggest that microarray technology is a valid method for elucidating the genetic nature of drug resistance in T-ALL cell lines, making it a productive approach to identify mechanisms of chemotherapy resistance. Finally, these cell lines will serve as useful tools for studying mechanisms of chemotherapeutic resistance in T-ALL.

Description: Hsp90 inhibitor has shown promising anti-tumor activity through the destabilization and eventual degradation of Hsp90 client proteins critical for cell survival. In this study, we examined the in vitro effects of Hsp90 inhibitor on the phenotype and function of human T lymphocytes and NK cells. We observed no significant effects of Hsp90 inhibitor treatment on cell survivals. However, Hsp90 inhibitor treatment for 24 hours led to irreversible down-regulation of expression of critical T-cell surface antigens including CD3, CD4, CD8, CD28, CD154 (CD40L) and TCRab. Among the antigens evaluated, expression of CD4 antigen was most significantly downregulated (untrt vs. trt = 326 vs. 88 in Mean Fluorescence Intensity) following Hsp90 inhibitor treatment. Decreased CD3+ T lymphocytes proliferation (untrt vs. trt = 222839 cpm vs. 111102 cpm, 3[H]-thymidine incorporation) and reduced IFN-g secretion (untrt vs. trt = 77 vs. 48 pg/ml) was observed upon stimulation with allogeneic dendritic cells following 24 hrs treatments of T cells with Hsp90 inhibitor. Furthermore, CD3+ T-cell proliferation in response to mitogen stimulation, as measured by flow cytometry using CFSE was decreased following Hsp90 inhibitor treatment (untrt vs. trt = 41% vs. 3%, CFSE). Specifically, the CD4+CD28+ (untrt vs. trt = 32% vs. 1%) and CD8+CD28+ (untrt vs. trt = 27% vs. 17%) activated T-cell subpopulations displayed a significant decrease in proliferation in response to mitogen. Similarly, NK cells displayed decreased activation receptor expression including CD2, CD11a, CD94, NKp30, NKp44, NKp46, and KARp50.3 and reduced cytotoxic activity against multiple myeloma cells (untrt vs. trt = 49% vs. 11% against MM1S cells, 65% vs. 8% against ARP cells) following Hsp90 inhibitor treatment. These studies demonstrate that Hsp90 inhibitor treatment significantly affects phenotype and function of human T-lymphocytes as well as NK cells, and suggest the need to monitor immune functions in patients being treated with Hsp90 inhibitor in our future studies.

Description: The anemia of chronic disease-which encompasses inflammation, infection, tissue injury, and conditions associated with the release of proinflammatory cytokines (such as cancer)- is one of the most common forms of anemia seen clinically. Symptomatic anemia requires treatment. The two major forms of treatment are transfusions and erythropoietin. Arsenic trioxide (As2O3) used to treat human diseases for centuries in traditional Chinese medicine. Our recent studies suggest that low dose of As2O3 induces erythroid differentiation of K562 human leukemic cells and high dose of As2O3 induce apoptosis. In this study, we have investigated in vitro effect of As2O3 on the erythroid differentiation and it could inhibit TNF-α induced suppression of erythroid differentiation of human cord blood CD34+ cells. Expression of glycophorin A was 35.94 ± 7.94% after 7 days culture of human cord blood CD34+ cells and was decreased to 17.63 ± 7.33% when culture of human cord blood CD34+ cells with 100ng/mL of TNF-α. Expression of glycophorin A was increased in dose dependent manner after 7 days treatment with As2O3 and As2O3 increased percentage of glycophorin A in culture with TNF-α compared to TNF-α alone. The results of colony assay of CFU-MIX and BFU-E after culture with various conditions revealed similar patterns with expression of glycophorin A. These results suggest that As2O3 induces erythroid differentiation of human cord blood CD34+ cells and can reverse TNF-α induced suppression of erythroid differentiation of human cord blood CD34+ cells. The BFU-E colony assay of the human cord blood CD34+ cells after culture with TNF-α or/and Arsenic trioxide. The BFU-E colony assay of the human cord blood CD34+ cells after culture with TNF-α or/and Arsenic trioxide.

Description: The size of the Natural Killer (NK) cell pool is maintained through production and subsequently export from the bone marrow, peripheral survival and proliferation, and ultimately cell death. While there exists considerable knowledge about developmental stages of lymphoid, myeloid and erythroid cells, comparably little is known about NK cell intermediates and the genes required for their development. Most of the models of NK cell differentiation have been based on in vitro culture systems where NK cells could be generated from multipotent HSC precursors. However, this approach suffers from the problems inherent to in vitro cell manipulations. We have utilized conditional gene targeting in adult mice to examine the role of the bcl-x gene in the development of NK cells in bone marrow and after their export to the spleen. Bcl-x is an important member of the anti-apoptotic member of the Bcl-2 gene family, and a critical role for this gene in the survival of hematopoietic cells was demonstrated in bcl-x-deficient mice causing embryonic death due to massive apoptosis of immature hematopoietic cells and of neurons. Conditional deletion in erythroid cells lead to hemolytic anemia and extensive splenomegaly. Furthermore, bcl-x is critical for the maturation of pre-B cells to the pro B cell stage, while it is not essential for the development of effector and memory T cells. We have conditionally deleted the gene in the stem cells of adult mice by cross breeding them with the Mxi-cre deleter strain, which allows for induced expression of cre recombinase by injection with pIpC. As early as 9 days after the first injection of pIpC, the number of NK cells in the bone marrow of mice started to decline as demonstrated by multi-color FACS analysis staining for IL2 Receptor beta (CD122) and NKG2D, among other markers. Cultures of bone marrow and spleen cells in the presence of cytokines to generate lymphokine activated killer cells failed, while no such effect was observed in cultures from Mxi-cre mice that were subjected to pIpC injections and carried along as controls. Analysis of animals after 3 weeks of pIpC administration revealed absence of NK cell precursors in the bone marrow as demonstrated by the lack of CD122+/Lin- negative cells. This phenomenon was accompanied by a reduction in the number of mature NK cells in the spleen. To date, six stages of NK cell maturation are described with the acquisition of IL2 receptor beta expression marking commitment to the NK-cell lineage. IL2 Receptor beta as well as NKG2D are expressed throughout NK cell development and at all stages. In order to characterize the specific stage at which expression of bcl-x is essential for NK cell maturation, we employed multi-color FACS analysis staining for CD122, NKG2D, CD49b, and the integrins CD43 and Mac-1 after depletion of lineage positive cells, followed by sorting for defined populations. Real Time PCR on sorted cells demonstrated that Bcl-x mRNA is highly expressed throughout all stages of NK cell development. Taken together, the gradual reduction in the number of NK cell precursors eventually leading to complete loss of this lineage in the bone marrow and peripheral sites suggests that bcl-x is indispensable for the development of NK cells presumably from the earliest time point of commitment to this lineage.

Description: Hepcidin, a small peptide produced by the liver, is the principal iron-regulatory hormone. It acts by binding to the iron exporter ferroportin, inducing its internalization and degradation, thereby blocking cellular iron efflux. The bioactive 25 amino acid peptide has a hairpin structure stabilized by 4 disulfide bonds. We synthesized a series of hepcidin derivatives and determined their bioactivity in a cell line expressing ferroportin-GFP fusion protein, by measuring the degradation of ferroportin-GFP and the accumulation of ferritin after peptide treatment. Bioactivity was also assayed in vivo, by measuring hypoferremia in mice injected with hepcidin derivatives. Serial deletion of N-terminal amino acids caused progressive decrease in activity which was completely lost when five N-terminal aa were deleted. Synthetic 3-aa and 6-aa N-terminal peptides alone, however, did not internalize ferroportin, and did not interfere with ferroportin internalization by native hepcidin. Deletion of two C-terminal amino acids did not affect peptide activity. Removal of individual disulfide bonds by pairwise substitution of cysteines with alanines also did not impact peptide activity in vitro. However, these peptides were significantly less active in vivo, likely due to their decreased stability in circulation. Peptides with a substitution G71D or K83R, previously described in human subjects, were fully active in vitro and in vivo. Zebrafish hepcidin, which is only 60% similar to human hepcidin, but with conservative substitutions at the N-terminus, was as active as its human counterpart. Apart from the essential nature of the N-terminus, hepcidin structure appears permissive for mutations. Further studies of hepcidin structure in relation to its function are essential for the design of hepcidin antagonists and agonists which could be used for treatment of iron disorders.

Description: Background: Numerous studies have shown that lymphoma B-cells are resistant to CTL-mediated death, however the underlying mechanism for this resistance is not clear. In previous work, we have identified a subset of CD4+CD25+ T-cells overrepresented in the tumor sites of B-cell NHL that display a phenotype compatible with regulatory T cells (Treg cells). These cells express high levels of Foxp3 and CTLA-4, and are capable of suppressing the proliferation and cytokine production of autologous infiltrating CD4+CD25- T cells in B-cell NHL. Goal: To explore whether Treg cells exert a suppressive effect on the induction and function of autologous tumor-infiltrating CD8+ T-cells in B-cell NHL. Results: Using fresh specimens obtained from patients with B-cell lymphoma, we found that intratumoral Treg cells had a significantly suppressive effect on autologous tumor-infiltrating CD8+ T-cells in B-cell NHL. When activated CD8+ T-cells were cocultured with infiltrating CD4+CD25- T-cells, they displayed less proliferation than CD8+ T-cells cultured alone. However, we found that with a 1:4 ratio of stimulating cells (Treg cells) to responding cells (CD8+ T-cells), intratumoral Treg cells completely inhibited the proliferation of autologous tumor-infiltrating CD8+ T-cells activated with PHA. Furthermore, we have observed that 20% of infiltrating CD8+ T-cells in fresh isolated samples from B-cell NHL coexpress perforin and granzyme B. When intratumoral Treg cells were cocultured with CD8+ T-cells, the Treg cells inhibited the activation-induced production of perforin and granzyme B of autologous tumor-infiltrating CD8+ T-cells in a dose-dependent fashion. We also found that cytokine treatment (IL-2 and IL-12) or PHA activation induced the capability of tumor-infiltrating CD8+ T-cells to kill lymphoma B-cells, which was accompanied by upregulation of expression of CD107a on the surface of cytotoxic CD8+ T-cells. Intratumoral Treg cells significantly inhibit cytotoxicity of activated infiltrating CD8+ T-cells to lymphoma B-cells. Finally, we found that there was an inverse correlation of cell frequencies between intratumoral Treg cells and tumor-infiltrating CD8+ T cells in patients with B-cell NHL, suggesting that Treg cells may inhibit the migration of cytotoxic T-cells to the sites of B-cell lymphoma. Conclusion: Intratumoral Treg cells dramatically suppress the proliferation and activation-induced production of granules in infiltrating CD8+ T-cells in B-cell NHL. These Treg cells also inhibit the ability of activated tumor-infiltrating CD8+ T-cells to kill lymphoma B-cells in vitro, and may prevent the migration of CD8+ cells to the sites of lymphoma. Taken together, these data indicate an important role for intratumoral Treg cells in CTL-mediated anti-tumor immunity in B-cell NHL.

Description: Notch activation has been suggested to promote T cell development at the expense of B cell commitment at the level of a common lymphoid progenitor prior to B cell commitment. Here, we explored the possibility that Notch activation might be able to switch the fate of already committed B cell progenitors towards T cell development upon Notch activation. To address this we overexpressed constitutively activated Notch-3 (N3IC) in B cell progenitors purified from transgenic mice in which human CD25 is expressed under control of the λ5 promoter. Strikingly, whereas untransduced and control transduced B220+λ5+CD3− B cell progenitors gave rise exclusively to B cells, CD4+ and CD8+ T cells but no B cells were derived from N3IC-transduced cells when transplanted into sublethally irradiated NOD-SCID mice. Gene expression profiling demonstrated that untransduced B220+ λ5+CD3− B cell progenitors expressed λ5 and CD19 but not the T cell specific genes GATA-3, lck and pTα, whereas CD3+ T cells derived from N3IC-transduced B220+λ5+CD3−cells failed to express λ5 and CD19, but were positive for GATA-3, lck and pTα expression as well as a and b T cell rearrangement. Furthermore, DJ rearrangements were detected at very low levels in CD3+ cells isolated from normal non-transduced BM, but were more abundant in the N3IC-transduced CD3+ BM cells. Noteworthy, N3IC-transduced B220+λ5+CD3−CD19+ proB cell progenitors failed to generate B as well as T cells, whereas N3IC-transduced B220+λ5+CD3−CD19− pre-proB cells produced exclusively T cells, even when evaluated at low cell numbers. In conclusion Notch activation can switch committed B cell progenitors from a B cell to a T cell fate, but this plasticity is lost at the Pro-B cell stage, upon upregulation of CD19 expression.

Description: Background: Erythropoiesis-stimulating proteins (ESPs) are commonly used to treat anemia in patients with cancer receiving chemotherapy. While it is well established that these agents increase hemoglobin levels and reduce the incidence of red blood cell (RBC) transfusions, the effects of ESPs on overall survival are inconclusive, with reports of both positive and negative impacts on survival. A recent Cochrane meta-analysis of double-blind randomized placebo-controlled trials (Bohlius et al, 2005) determined that the hazard ratio for overall survival associated with recombinant human erythropoietin (rHuEPO) in 19 trials with 2865 patients was 0.81 (95% CI: 0.67 to 0.99) for adjusted data and 0.84 (95% CI: 0.69, 1.02) for unadjusted data, suggesting no negative impact on survival. The objective of the present investigation was to conduct an analysis with identical methodology to that reported by Bohlius et al, but including trials of darbepoetin alfa, which were not part of the Cochrane meta-analysis. Trial inclusion was limited to those that addressed patient populations with chemotherapy-induced anemia (studies by Henke et al and Leyland-Jones et al, which reported decreased survival in patients treated with rHuEPO, were not included in this analysis as they were not conducted in anemic patients). Methods: Data from 4 randomized, placebo-controlled clinical trials of darbepoetin alfa in chemotherapy-induced anemia were analyzed using meta-analysis methodology as performed in the Cochrane analysis. The primary outcomes included hematological response (hemoglobin increase of ≥ 2 g/dL); change in hemoglobin from baseline; RBC transfusions; and overall survival. Survival was also analyzed by combining the 4 darbepoetin alfa trials with the rHuEPO trials used in the Cochrane analysis. Results: In the meta-analysis of 4 trials (N=759), darbepoetin alfa significantly reduced the risk of receiving a RBC transfusion (RR 0.69; 95% CI 0.59–0.81; p

Description: Thrombosis is a major complication of central venous access devices, and incidence depends on material, diameter, tip position, and tip surface. Catheters are usually cut to the appropriate length for accurate positioning. However, cutting is not recommended as rough surfaces can serve as a nidus for thrombi. This study was done to assess the roughness of the catheter tips as provided by various manufacturers, versus the roughness once cut and handled. Four types of catheters (Groschong, Hickman, Port-a-Cath, and Per Q Cath) were cut by scissors, iris scissors, or scalpel, and handled with debakey forceps, a needle driver, and adsons with or without teeth, to determine the damage created on the catheter. The manufactured tip was compared as a control. Scanning electron microscopy (SEM) was used to do imaging for all samples and roughness and section analysis was quantified using atomic force microscopy (AFM) for the cutting methods. SEM showed that scalpel-cut and manufactured ends appeared smoother relative to those cut with scissors or iris scissors (Fig 1). This complemented the roughness and section analysis by AFM (Fig 2). Catheters handled by debakey equipment and adsons with teeth showed the most roughness, visible as deep holes or a grainy surface when observed by high magnification SEM. Decreasing smoothness of catheters is in the following order: uncut surface, followed by surfaces cut by scalpel, scissors or iris scissors. Handling should be minimized and use of adsons with teeth, needle drivers and debakey forceps avoided, which can leave permanent damage. The least damage appeared to be adsons without teeth. Thus, the cutting of catheters is not recommended since rough surfaces can serve as a thrombotic nidus. Figure Figure

Description: Hyperuricemia is a commonly seen in patients with hematologic malignancies and high tumor burden, and is a major feature of tumor lysis syndrome (TLS). Hyperuricemia is conventionally treated with hydration, urine alkalinization, and allopurinol. Allopurinol inhibits the conversion of hypoxanthine to xanthine, and xanthine to uric acid (UA) by inhibiting xanthine oxidase. It has no direct effect on existing UA in the serum. Rasburicase lowers UA rapidly to very low levels at the approved dose of 0.15–0.2 mg/kg daily for 5 days by converting UA to allantoin which is excreted rapidly. Despite this dramatic effect on UA, rasburicase has not been shown to have any beneficial impact on survival in patients with hematologic malignancies. We administered 0.2 mg/kg ideal body weight rasburicase to an obese hyperuricemic patient (actual 120 kg, ideal 60 kg). Serum UA declined from 11 mg/dL to 1.4 mg/dL in 3 h and 0.4 mg/dL in 11 h. The serum UA level remained low for several days and a second dose was not needed. After seeing this dramatic and sustained response, variable low rasburicase doses were used in patients with malignant diseases with close monitoring of biochemical parameters (Trifilio et al, ASH 2004). Here, we present our experince with the use of a single 3 mg dose of rasburicase in 42 adult patients receiving chemotherapy or undergoing hematopoietic stem cell transplantation for hematologic malignancies. In addition to rasburicase, allopurinol and other supportive therapy including hydration were also administered. Serum UA levels (baseline 6.4–16.4 mg/dL, median 9.7) had declined by 12–95% (median 43%) 24 hours after rasburicase administration. Figure Figure The baseline serum creatinine was 1.0–8.6 mg/dL (median 2.1), and that 24 hours after rasburicase administration was 0.7–8.0 mg/dL (median 2). The median change in creatinine was a decline of 10%. Except for 3 myeloma patients who were already on chronic hemodialysis, renal impairment did not progress to hemodialysis in any patient - and substantial renal function improvement (at least 50% decline in serum creatinine) was seen in all patients with a baseline serum creatinine of ≥ 3 mg/dL. Based on the Red Book rasburicase cost of $387 for a 1.5 mg vial, the amount of money saved ranged from ~$10,000 to ~$30,000 per patient. Our data suggest that rasburicase is effective at a fraction of the recommended dose. A formal randomized comparison of low- and conventional-dose rasburicase is warranted to see if the recommended higher dose offers any clinical benefit.

Description: The pathophysiology of Diamond Blackfan Anemia (DBA) is not confined to erythropoiesis. Associated physical anomalies and growth retardation are common, and there is an increased risk of hemopoietic and non-hemopoietic cancers. Heterozygous mutations affecting RPS19, encoding ribosomal protein S19, are present in 26% of cases of DBA. The indistinguishable clinical phenotype of DBA patients with and without RPS19 mutations predicts that all DBA genes share a common function, or interdependence in a common pathway. If the known ribosomal role of RPS19 underlies the pathogenesis of DBA, as opposed to an as yet unknown erythroid specific extraribosomal role, then every cell type should be affected, by analogy with the Bst mouse, another mammalian example of a naturally occurring ribosomal protein haploinsufficiency. In addition, cells from DBA patients with and without RPS19 mutations should be similarly affected. EBV lymphoblastoid cell lines (LCLs) generated from peripheral blood lymphocytes from DBA patients were studied as an easily accessible source of non-erythroid DBA cells to test these predictions. As RPS19 haploinsufficiency may not affect ribosomal abundance in the steady state, cells were subjected to serum deprivation and refeeding to induce translational stress. Under these conditions, 6 DBA LCLs, including 2 with RPS19 mutations, showed impaired proliferation in comparison with control LCLs. A similar phenotype could be induced in control LCLs by low dose rapamycin or cycloheximide. On FACS analysis, two subpopulations of EBV LCLs can be distinguished. Staining with Pyronin Y or FITC showed the larger cells to have a tight RNA and total protein content, with no difference between normal and DBA LCLs, and unaffected by treatment with rapamycin or cycloheximide. The other population of smaller cells had a lower, more variable RNA and protein content. DBA LCLs or rapamycin treated control LCLs had a relatively lower proportion of the larger, actively proliferating cells. These results would be consistent with exit from cell cycle of DBA LCLs in response to translational stress. To determine whether the actively proliferating subpopulation of cells showed altered cell cycle kinetics, the cumulative entry into S phase of cells on refeeding was determined by the addition of BrdU at the time of serum refeeding. FACS analysis showed BrdU positive cells to be contained exclusively within the population of larger LCLs in control and DBA. The BrdU labeling index was reduced in DBA LCLs, and exacerbated by rapamycin. A reduction was also observed in rapamycin treated control LCLs. We have thus demonstrated an impaired growth phenotype and alteration in cell cycle kinetics in non-erythroid cells derived from patients with DBA with and without RPS19 mutations, supporting the hypothesis that ribosomal pathophysiology is the underlying common denominator in DBA. As there was no difference in the 28S:18S ratio in total cellular RNA extracted from control or 5 DBA LCLs, pulse-chase labeling with 3H-uridine was used to study nascent rRNA as an indicator of ribosomal biogenesis. Interestingly, in comparison with a control LCL, there was an apparent relative reduction in 18S nascent rRNA in one DBA LCL and in 28S rRNA in another, both without detectable RPS19 mutations, confirmed in repeat independent experiments. Further investigations are in progress to determine whether these findings reflect an epiphenomenon in the context of an immortalised cell line, or truly reflect abnormal ribosomal biogenesis in DBA.

Description: Elevated levels of the molecular adaptor protein p21cip1/waf1 (p21) and of the IL-3 receptor α chain are correlated with chemoresistance and poor prognosis in acute myeloid leukemia (AML). p21 is a core regulator of many biological functions including cell cycle control, apoptosis and differentiation. Our laboratory has demo−nstrated that p21 undergoes dynamic changes in expression levels and subcellular compartmentalization during cytokine-induced granulocytic differentiation, suggesting that p21 may play an important role in myeloid development. Based on our observation that p21 protein levels decrease during granulocytic differentiation of CD34+ human progenitor cells, we hypothesized that p21 antagonizes granulopoiesis. The proliferative cytokine IL-3 is required to maintain the undifferentiated state in murine 32Dcl3 cells and has been shown to slow the kinetics of differentiation of normal human myeloid progenitors (Hevehan DL, 2000). Given that 32Dcl3 myeloblasts express high basal levels of p21, we also hypothesized that IL-3 inhibition of 32Dcl3 differentiation is mediated in part by p21. Our findings demonstrate that siRNA knockdown of murine p21 is correlated with premature expression of the primary granule proteins myeloperoxidase and proteinase-3 that are normally not abundant in cells maintained as myeloblasts by IL-3. Rescue of p21 knockdown myeloblasts with human p21 suppressed aberrant expression of granule proteins. The upregulation of myeloperoxidase and proteinase-3 occurred at a posttranscriptional level. These findings indicated that p21 prevented premature expression of primary granule proteins and may contribute to maintenance of the myeloblast phenotype. p21 knockdown was also found to accelerate morphologic granulocytic differentiation in 32Dcl3 cells stimulated with G-CSF, indicating that p21 antagonized the entire differentiation process rather than only suppressing primary granule proteins. We then determined how IL-3 maintains p21 expression in myeloblast cells. We demonstrated that IL-3 stabilized p21 mRNA in myeloblasts leading to high levels of p21 protein. This effect mapped to the 3′ untranslated region (UTR) of the p21 transcript. IL-3 also rescued the decrease in p21 mRNA stability noted during G-CSF-induced differentiation. This has been shown to coincide with differentiation blockade. p21 transcript stabilization by IL-3 was independent of PI3-kinase and ERK pathway signaling. In vitro binding assays provided evidence that distinct sets of RNA:protein interactions occur within the proximal 303 nucleotides of the p21 3′ UTR and are regulated by IL-3 and G-CSF signaling. Association of a ~60–65 kDa protein with p21 riboprobes correlated with IL-3 mediated p21 mRNA stabilization, whereas binding by a ~40–42 kDa protein was associated with destabilization of p21 transcripts in 32Dcl3 cells undergoing G-CSF-induced differentiation. These findings provide the first evidence for IL-3-mediated stabilization of mRNA transcripts in myeloid progenitor cells. The finding that p21 antagonized granulopoiesis is also novel. Because high levels of the IL-3 receptor and high p21 expression have separately been linked to poor outcomes in AML, IL-3 mediated p21 mRNA stabilization may contribute to differentiation blockade during AML pathogenesis.

Description: Murine models of transplantation established that nonmyeloablative conditioning using repeated low doses of irradiation targeted to lymphoid tissues (TLI) and depletive anti-T cell antibodies protects against GVHD by skewing residual host T cell subsets to favor regulatory natural killer (NK) T cells that suppress GVHD by polarizing donor T cells toward secretion of non-inflammatory cytokines such as IL-4. We recently translated the murine protocol to a clinical study using non-myeloablative TLI and ATG host conditioning with HLA matched related and unrelated donors, and showed a marked reduction in the incidence of acute GVHD while retaining graft anti-tumor activity (Lowsky et al., in Press NEJM). Engrafted donor CD4+ T cells showed a marked increase in IL-4 production as compared to CD4+ T cells from controls. We now adapted the TLI and ATG nonmyeloablative host conditioning regimen to a clinical study of allogeneic HCT using haploidentical matched (3/6 HLA matched) related donors to determine if it will result in donor hematopoietic cell engraftment and also protect against acute GVHD. Blood derived hematopoietic progenitor cells were collected by apheresis from donors mobilized with G-CSF and the product was T cell depleted using CD34+ selection. CD3+ T cells were added back to the donor inoculum according to a dose escalation schedule. The initial T cell dose was 1 x105 CD3+ cell/kg with designated increments based on clinical outcomes of up to a maximum of 1 x107 CD3+ cells/kg. The desired CD34+ cell dose was 〉5 x 106 CD34+ cells/kg for all patients. Seven patients were transplanted; the median age was 53 years (range 27 to 61 years). Five patients had acute myelogenous leukemia, two with disease in remission and three not in remission at the start of TLI and ATG, one with myelodysplastic syndrome, and one with progressive peripheral T cell lymphoma. The median follow-up for all patients is 265 days with three of seven patients alive and free of disease at the last observation period. Sustained donor hematopoietic cell engraftment was achieved in three of three patients only after the T cell dose was increased to 1 x107 CD3+ cells/kg. No patient developed acute GVHD. None of the three patients receiving the highest dose of T cells had any invasive fungal or viral infections. Monitoring of sorted host T cell subsets before TLI and ATG, and immediately after but before the infusion of donor cells, revealed in five of five patients a highly significant skewing of residual host T cells favoring invariant NK T (CD3+ CD161hi Va24 +Vb11 +) cells. The mean absolute number of host CD3+, and CD4+ and CD8+ T cells decreased by 99, 163 and 121 fold, respectively, immediately after conditioning compared to the absolute numbers before the start of TLI and ATG, whereas the mean absolute number of invariant NK T cells decreased by only 11%. In conclusion, we have determined the conditions for successful hematopoietic cell engraftment using a non-myeloablative regimen of TLI and ATG that appears associated with a reduced aGVHD risk yet retained graft anti-tumor activity. As in the pre-clinical model, we show direct evidence that the low incidence of aGVHD is associated with a significant alteration in residual host T cell subsets markedly favoring invariant NK T cells.

Description: Megakaryopoiesis is a highly specialized cellular process which sustains platelet production. At the end of megakaryopoiesis, megakaryocyte (MKs) fragments into platelets via long and thin cytoplasmic extensions called proplatelets. Proplatelet formation (PPF) is associated essentially with cytoskeleton changes, including actin dynamics. The Rho/Rock pathway is a well characterized regulator of the actin reorganization. In the present study, we have tried to understand the precise role of the Rho/Rock pathway in PPF from human CD34+ derived MKs. Our results show that Rho is expressed in MKs and that its expression and activity remain stable during megakaryopoiesis. Overexpression of a RhoA dominant negatif (RhoA N19) in MKs leads to an increase in PPF. Conversely overexpression of a RhoA spontaneous active (RhoA V14) in MKs leads to a decrease in PPF. These results indicate that Rho activation could inhibit PPF in vitro. It is known that Rho/ROCK promotes actin cytoskeleton dynamics by regulating myosin light chain 2 (MLC2) phosphorylation. To demonstrate that Rho/Rock inhibits PPF through MLC2 phosphorylation, we added MLC kinase inhibitor (P18), Rho inhibitor (TatC3) and ROCK inhibitor (Y27362) in MKs culture just before PPF. Western blot analysis shows that MLC2 phosphorylation was inhibited by these 3 compounds, in contrast, PPF was significantly increased. Moreover, the platelet produced have an identical size and ultrastructure as control platelets and could be normally activated. These results suggest that Rho/ROCK could inhibit PPF through MLC phosphorylation during megakaryopoiesis.

Description: We have recently shown that granulocyte–colony-stimulating factor (G-CSF)– and interferon-γ (IFN-γ)–activated human neutrophils accumulate and release remarkable amounts of soluble B-lymphocyte stimulator (BLyS) in vitro. In this study, we provide evidence that neutrophils migrating into skin window exudates (SWEs) developed in healthy volunteers and in patients with rheumatoid arthritis (RA), synthesized, and released BLyS in response to locally produced G-CSF. Accordingly, the concentrations of soluble BLyS in SWEs were significantly more elevated than in serum. Because the levels of SWE BLyS, but not SWE G-CSF, were higher in patients with RA than in healthy subjects, we examined the effect of CXCL8/IL-8, C5a, and other proinflammatory mediators that dramatically accumulate in RA SWEs and in inflamed synovial fluids. We show that CXCL1/GROα, CXCL8/IL-8, C5a, immune complexes, tumor necrosis factor-α (TNF-α), leukotriene B4, N-formyl-methionyl-leucyl-phenylalanine (fMLP), and lipopolysaccharide (LPS), which by themselves do not induce BLyS de novo synthesis, act as potent secretagogues for BLyS, which is mainly stored in Golgi-related compartments within G-CSF–treated neutrophils, as determined by immunogold electron microscopy. This action is pivotal in greatly amplifying neutrophil-dependent BLyS release in SWEs of patients with RA compared with healthy subjects. Collectively, our data uncover a novel mechanism that might dramatically exacerbate the release of BLyS by neutrophils during pathologic inflammatory responses.

Description: We elucidate the cellular and molecular kinetics of the stepwise differentiation of human embryonic stem cells (hESCs) to primitive and definitive erythromyelopoiesis from human embryoid bodies (hEBs) in serum-free clonogenic assays. Hematopoiesis initiates from CD45 hEB cells with emergence of semiadherent mesodermal-hematoendothelial (MHE) colonies that can generate endothelium and form organized, yolk sac–like structures that secondarily generate multipotent primitive hematopoietic stem progenitor cells (HSPCs), erythroblasts, and CD13+CD45+ macrophages. A first wave of hematopoiesis follows MHE colony emergence and is predominated by primitive erythropoiesis characterized by a brilliant red hemoglobinization, CD71/CD325a (glycophorin A) expression, and exclusively embryonic/fetal hemoglobin expression. A second wave of definitive-type erythroid burst-forming units (BFU-e's), erythroid colony-forming units (CFU-e's), granulocyte-macrophage colony-forming cells (GM-CFCs), and multilineage CFCs follows next from hEB progenitors. These stages of hematopoiesis proceed spontaneously from hEB-derived cells without requirement for supplemental growth factors during hEB differentiation. Gene expression analysis of differentiating hEBs revealed that initiation of hematopoiesis correlated with increased levels of SCL/TAL1, GATA1, GATA2, CD34, CD31, and the homeobox gene-regulating factor CDX4 These data indicate that hematopoietic differentiation of hESCs models the earliest events of embryonic and definitive hematopoiesis in a manner resembling human yolk sac development, thus providing a valuable tool for dissecting the earliest events in human HSPC genesis.

Description: Rho guanosine triphosphatases (GT-Pases) are recognized as critical mediators of signaling pathways regulating actin assembly, migration, proliferation, and survival in hematopoietic cells. Here, we have studied a recently identified hematopoietic-specific Rho GTPase, RhoH. Unlike most members of the Rho GTPase family, RhoH is GTPase deficient and does not cycle between GTP- and guanosine diphosphate (GDP)–bound forms, suggesting that regulation of RhoH expression may be critical in its activity. We found that RhoH is expressed in murine hematopoietic progenitor cells (HPCs) and fully differentiated myeloid and lymphoid lineages. In cytokine-stimulated HPCs, knockdown of RhoH expression via RNA interference stimulates proliferation, survival, and stromal cell-derived factor-1α (SDF-1α)–induced migration in vitro. Conversely, RhoH overexpression in these cells via retrovirus-mediated gene transfer is associated with impaired activation of Rac GTPases, reduced proliferation, increased apoptosis, and defective actin polymerization and chemotaxis. In vivo, HPCs with RhoH overexpression demonstrate defective hematopoietic reconstitution capability compared with control vector-transduced cells. Our results suggest that RhoH serves as a negative regulator of both growth and actin-based function of HPCs possibly via suppression of Rac-mediated signaling.

Description: BACKGROUND: Angiogenesis and activation of coagulation system in cancer patients are common and are thought to be unfavorable clinical parameters. Vascular endothelial growth factor (VEGF) and fibroblast growth factor (bFGF) are well-known angiogenic cytokines. The elevations of plasma fibrinogen and D-dimer level indicate coagulation and fibrinolysis activation. There may be links between angiogenic cytokines and coagulation - fibrinolysis factors in cancer. Possible specific interactions include releasing angiogenic factors, such as VEGF by activated platelets and binding of VEGF and bFGF to fibrin and fibrinogen resulting in an increase in endothelial cell proliferation. AIM: The purpose of our study was: (a) to analyze relations of VEGF, bFGF serum levels and fibrinogen, D-dimer plasma levels with stage of disease according to Ann Arbor Staging System (AASS); (b) to evaluate correlation between serum levels of angiogenic cytokines and plasma levels of coagulation-fibrinolysis factors in non Hodgkin’s lymphoma patients. MATERIAL AND METHODS: 52 non Hodgkin’s lymphoma patients (31 men, 21 women; median age 52,1 ± 14,7 years) in II, III or IV stage of disease according to AASS were assessed. In stage II were 15, in stage III- 10 and in stage IV- 27 persons. Serum VEGF, bFGF and plasma D-dimer levels were measured by enzyme-linked immunosorbent assay (ELISA). Plasma levels of fibrinogen were determined using Behring Coagulation System (BCS) equipment. RESULTS: Plasma level of D-dimer was elevated in majority of patients, mean plasma D-dimer levels [ng/ml] were in stage II: 1654,3 ± 1301,5, in stage III: 1816,6 ± 1370,7, in stage IV: 2747,1 ± 1410,8. There was significantly higher D-dimer level in IV stage of disease in comparison to stage II and III. p

Description: The incidence of acute myeloid leukemia (AML) increases with age. Because of poor performance status, co-morbidity and treatment-related side effects, a conventional dose chemotherapy containing anthracyclins may be toxic to the majority of elderly patients. In contrast, the administration of suboptimal dose of myelosuppressive chemotherapy could lead to an unsuccessful clinical outcome including lower complete remission (CR) rate. To evaluate the effect of attenuated dose of idarubicin, compared to the standard dose, on the clinical outcome and chemotherapy-related complications, we analyze the consecutive 32 elderly de novo AML patients (range, 60 – 74 years) with normal karyotype. Eleven patients received one cycle of conventional-dose remission induction chemotherapy (idarubicin, 12 mg/m2/day on days 1–3 and cytarabine 100mg/m2/day on days 1–7) (Group 1) and 21 patients received attenuated-dose idarubicn (8 mg/m2/day on days 1–3) and cytarabine (100mg/m2/day on days 1–7) (Group 2). Six cases (54.5%) in Group 1 and 12 cases (57.1%) in Group 2 had CR. The difference of CR between the two groups was not significant (P = 0.59). The intervals from the chemotherapy-starting date to the date of CR documentation were not also different between two groups (median 31.5 days on Group 1 vs 27.0 days on Group 2) (P = 0.29). The median number of transfusion requirement during the induction therapy was not different in the red blood cells (10 units, each) and platelets (16.5 units in Group 1 vs 18.0 units in Group 2; P 〉 0.05). Thirty patients received the recombinant human granulocyte colony-stimulating factor (G-CSF) three days after termination of chemotherapy. The duration of G-CSF administration was not different between two groups (P = 0.86). However, the frequency of septicemia and septic shock after induction chemotherapy was statistically significantly higher in Group 1 (54.5% and 9.5%, respectively) compared to that in Group 2 (36.3% and 0.5%, respectively) (P 〈 0.01). We also observed a higher incidence of clinically-documented invasive fungal infection in Group 1 (45.5%) compared to Group 2 (15.0%), although the difference was not statistically significant (P = 0.095). The incidence of other regimen-related toxicities including renal dysfunction, hepatic dysfunction and heart failure was not different between two groups. Overall survival and disease-free survival also were not different between the groups. In conclusion, the attenuated dose of idarubicin can be recommended for the remission induction chemotherapy for the elderly de novo AML patients with normal karyotype since it is associated with lower incidence of sepsis and septic shock with comparable CR rate.

Description: In ANLL, during intensive induction,invasive fungal infections (IFI) related to prolonged neutropenia,mucosal damage,steroids, geographical and center variations is the main factor wich can influence disease outcome The optimal prophylactic regimen has not yet to be identified. The AmB lipid formulations let to treat IFI in refractory or intolerant patients. The efficacy of these drugs appears to be related both to improved tissue penetration along with sustained bioactivity of drug levels in lung, brain, kidneys, liver and spleen.(Anaissie et al.2004).On this basis,, in a cohort of adult (〉18y) ANLL patients,during induction,we applied a pilot study for IFI prophylaxis in the aim to test the efficacy and safety of a single large dose of L-AmB.The primary endpoint was to evaluate the incidence of documented or suspected fungal infection during and up to four weeks after prophylaxis discontinuation.PATIENTS: From September 2004 to May 2005 18 consecutive adult ANLL (4 APL) patients −12 M,6 F, median age 56 y (range 39–75)- entered in this study. Intensive induction chemotherapy included standard /high dose cytosine-arabinoside + antracyclines +etoposide or fludarabine and retinoic acid + antracyclines in the 4 APL. METHODS: The criteria of inclusion were:1)neutropenia (PMN

Description: Children with the severe deficiency phenotype of leukocyte adhesion deficiency (LAD-1) suffer recurrent, life-threatening bacterial infections due to defective adherence and migration of their leukocytes. LAD-1 is caused by heterogeneous molecular defects in the leukocyte integrin CD18 molecule. Dogs with the canine form of leukocyte adhesion deficiency (CLAD), like children with severe deficiency LAD-1, experience severe bacterial infections, and typically die within the first few months of life from infection. CLAD represents a disease-specific, large animal model for evaluating new therapeutic approaches for the human disease LAD. In these studies, we tested a retroviral-vector mediated gene therapy approach in CLAD. Autologous CLAD CD34+ bone marrow hematopoietic stem cells were pre-stimulated overnight with growth factors cIL-6, cSCF, hFlt3-L, and hTPO, then incubated with retroviral vector PG13/MSCV-cCD18 over 48 hours on recombinant fibronectin. Transduction of the CLAD CD34+ cells was measured by flow cytometry for CD18+ cells and ranged from 11% to 21%. The transduced cells were re-infused (0.26 − 1.49 x 106 CD18+ cells / kg) into the dogs following the administration of two different non-myeloablative conditioning regimens: 5 CLAD dogs received autologous, gene-corrected CD34+ cells following 200 cGy total body irradiation (TBI) and 2 CLAD dogs received autologous, gene-corrected CD34+ cells following 10 mg/kg busulfan. Peripheral blood samples were analyzed by flow cytometry for CD18 expression following the re-infusion of the transduced CD34+ cells. The frequency of CD18+ gene-corrected leukocytes in the peripheral blood ranged from 0.04% to a high of 4.44% at 6 – 11 months post-gene transfer. Two of the five dogs in the first group and one of the two dogs in the second group that received CD18+ gene-corrected cells are alive and well on no prophylactic treatment at 9 – 14 months of age. Of note, the CLAD dog receiving busulfan conditioning has the highest level of CD18+ gene-corrected cells (4.44% at 6 months post-infusion), with the levels increasing at monthly intervals since the second month following re-infusion. These results contrast markedly with those seen in untreated CLAD dogs that die or are euthanized within the first few months of life due to intractable infection. These studies indicate that a clinically applicable non-myeloablative regimen of either 200 cGy TBI or 10 mg/kg busulfan facilitates the engraftment of sufficient autologous, CD18-gene corrected cells to correct the lethal disease phenotype in CLAD. No evidence of monoclonality has been detected by LAM-PCR in any of the dogs with therapeutic levels of gene-corrected cells. In future studies we will optimize the transduction protocol in order to increase the number of CD34+ gene-corrected cells for infusion, as well as closely monitor the gene-corrected animals for any evidence of insertional mutagenesis or other complications related to the therapy. Together, these findings support the use of either of two clinically applicable, non-myeloablative conditioning regimens prior to the infusion of autologous, CD18 gene-corrected cells in gene therapy clinical trials for LAD.

Description: The result of cytogenetics is one of the most important prognostic factors on the prognosis of AML. HDAC, auto PBPCT and allogeneic BMT after 1 or 2 times of post remission therapy based on 4 prognostic groups(APL: Acute promyelocytic leukemia, GPG: Good prognosis group, IPG: Intermediate prognosis group, PPG: Poor prognosis group by MRC definition) were underwent based on cytogenetics data. We studied CR, relapse, toxic death, DFS and OS. Inclusion criteria were age

Description: Leukemias are composed of a hierarchy of cells only a fraction of which have stem cell like properties, and are capable of self-renewal. MLL fusion proteins produced by translocations involving the Mixed Lineage Leukemia (MLL) gene on chromosome 11q23 confer stem cell-like properties on committed hematopoietic progenitors. This provides an opportunity to determine if global cellular reprogramming is necessary for leukemia stem cell (LSC) generation from committed progenitors or if induction of a more limited self-renewal signature in committed progenitors is sufficient. We transduced murine IL-7R− Lin− Sca-1− c-Kit+ CD34+ FcγRII/IIIhi granulocyte macrophage progenitors (GMPs) with retroviruses encoding the MLL-AF9 fusion protein, which led to the development of acute myelogenous leukemia. From the leukemias we isolated a population of IL-7R− Lin− Sca-1− c-Kit+ CD34int. FcγRII/IIIint. LSCs which can transplant the disease when fewer than 20 cells are injected into secondary recipients. We used hierarchical clustering, K-means clustering and principal component analysis to compare gene expression profiles of the LSC population to the normal lin− sca-1+ c-kit+ HSC-enriched population, IL-7R− Lin− Sca-1− c-Kit+ CD34+ FcγRII/IIIlo common myeloid progenitors (CMPs), IL-7R− Lin− Sca-1− c-Kit+ CD34− FcγRII/III− megakaryocyte erythroid progenitors (MEPs) and GMPs and found that the global gene expression profile most resembles the normal GMP from which they arose. However, a leukemia self-renewal signature was identified that shows significant overlap with a group of genes normally highly expressed in HSCs whose expression decreases during the transition to normal committed progenitors. Supervised analysis and gene set enrichment analysis (GSEA) demonstrated approximately 300 genes in the leukemia self-renewal signature. This is only a subset of the approximately 1500 genes that are highly expressed in the normal HSC-enriched population that show decreased expression in CMPs, MEPs, and GMPs. Next, we determined if this 300-gene leukemia stem cell signature is directly regulated by MLL-AF9 or if there is a hierarchy of gene expression. Assessment of gene expression changes 48 hours after MLL-AF9 expression in isolated GMPs demonstrated increased expression of 23/300 genes in the leukemia self-renewal signature. Of interest, there is a high degree of similarity between the 23 MLL immediate response genes and human MLL-rearranged AMLs including HOXA5, HOXA7, HOXA9, HOXA10, MEIS1 and genes not previously known to have a role in MLL-mediated leukemogenesis such as myocyte enhancer factor 2C (MEF2C). Detailed loss-of-function studies using shRNA and dominant negative mutants show inhibition of MEF2C reduces LSC colony formation and serial replating in semi-solid culture to less than 20% of control. Furthermore shRNA mediated inhibition of MEF2C has a significant impact on proliferation of human MLL-AF9 dependent leukemia cell lines, but not cell lines from other subtypes of AML. These data demonstrate LSCs can be generated from committed progenitors without widespread reprogramming of gene expression, and a leukemia self-renewal signature is activated in the process. We have used this program to identify MEF2C as playing a role in MLL-AF9 induced AML. Identification of this program provides an opportunity to further assess its importance in normal tissue homeostasis and neoplastic self-renewal/proliferation, and defines the progression from normal hematopoietic progenitor to leukemia stem cell.

Description: Targeted therapy is increasingly incorporated into the chemotherapy regimens of adult ALL. While the prognosis of BCR/ABL + (Philadelphia) ALL is well recognized; the significance of CD 20 postivity is unknown. We report the characteristics of 76 patients treated at Emory University between January 1999 and March 2005, divided into 2 groups based on CD 20 expression, as determined by flow cytometry. The CD 20+ patients presented with a higher WBC count and LDH, and lower platelet counts (table). In the CD 20+ group, 65% had pre B cell ALL and 8% had high grade B cell leukemia/lymphoma, whereas the CD 20- group consisted of 30% pre B cell ALL, 24% T cell ALL, and 10% had high grade leukemia/lymphoma. Of the 35 Ph+ ALL patients, 27 were CD20+ (20%) and 18 CD20- (51%). Patients were treated on clinical trials L20, ALL2, and more recently with the Hyper-CVAD regimen (n=32). Only 5 of the CD 20+ patients received rituximab in addition to chemotherapy. With a median follow-up of 320 days, 46% of patients in each group are alive, 35% and 38% relapsed in the CD 20+ and CD 20- group, respectively. In summary, we found that CD 20+ ALL patients present with higher WBC count and LDH, and a lower platelet count; although, due to our small sample size, these differences were not significant. Further follow up is needed to provide mature results on survival and remission. CD 20+ CD 20- p= Total numbers 26 50 Median Age (yrs) 45.2 (18–74) 44 (19–76) NS Median WBC (103/μl) 29.1 10 0.09 Median Platelet (103/μl) 35 65 0.11 Median LDH (IU) 894 500 0.05 Normal Cytogenetics 13 (50%) 21 (42%) NS Ph+ ALL 7 (26%) 18 (35%) NS CR within 60 days 20 (77%) 27 (54%) NS Primary Refractory 2 (8%) 6 (12%) 0.7 Relapse 9 (35%) 19 (38%) NS Alive at last follow up 12(46%) 23 (46%) NS Expired by d270 8 (36%) 11 (25%) NS

Description: Higher birthweight, maternal history of miscarriage and low birth order have been associated with increased risk of childhood leukaemias and some solid tumours. No study has investigated these factors together and differences in disease etiology between girls and boys have been generally overlooked. In a retrospective case-control study, 732 childhood (≤15 yr) cancer cases from the population-based Northern Region Young Persons’ Malignant Disease Registry (NRYPMDR) whose hospital birth records could be accessed and 3723 controls matched for date and hospital of birth were compared. We examined maternal reproductive history and birthweight for gender-specific associations using conditional logistic regression. In univariate analysis, maternal history of miscarriage showed an association with all cancers (OR = 1.29; 95% CI = 1.05 to 1.62, P = 0.02). In individual cancer groups, this association was significant for acute lymphoblastic leukaemia (ALL) (n=225, OR = 1.56; 95% CI = 1.07 to 2.27, P = 0.02), and marginally significant in osteosarcoma and neuroblastic tumours (neuroblastoma and ganglioneuroblastoma). There was no significant association with birth order. Being first born was a weak risk factor for ALL in boys only (OR=1.3, 95% CI = 0.8 to1.8). In boys but not in girls, the risk of ALL increased with birthweight (OR = 1.06 per 100 gr increase; 95% CI = 1.01 to 1.11, P = 0.01). When birthweights were normalized using UK standards for gestational age and gender, the associations were not more marked. A multivariate model for ALL confirmed the independence of associations with miscarriage history and birthweight. Gestational age was not a risk marker and did not explain the associations with birth weight and miscarriage history. Consideration of gender unravelled significant associations of maternal reproductive history and size at birth with childhood cancer markedly different between girls and boys. Most notably, associations with birth weight and miscarriage and the weak association with being first-born in childhood ALL were all stronger in males. The findings for birthweights normalized for gestational age suggested that size at birth rather than in utero growth trajectory is of etiologic importance in childhood ALL.

Description: The optimal treatment of patients with childhood acute lymphoblastic leukaemia (ALL) depends on establishing accurate diagnosis. Our investigations seek to strategically develop the application of microarray gene expression profiling to identify ALL patients with clinically homogenous presentations but which may respond differently to established treatment regimens. We have determined the gene expression profiles of ALL bone marrow (BM) samples taken from patients at diagnosis. Data analysis has focussed on the use of a novel and innovative statistical technology, Gene-RaVE. This series of patent protected algorithms builds a multinomial regression model using Bayesian variable selection. Gene-RaVE leads to the generation of a parsimonious and simple diagnostic gene expression signature, but which provides increased predictive ability over current analysis approaches. We describe our analysis of both Affymetrix (HU133A) and cDNA (10.5K) microarray gene expression profiles generated from diagnostic BM from 〉100 ALL patients covering the broad ALL subtypes including T and B lineage as well as T cell lymphoma leukaemia. Comparison of gene expression data failed to identify clearly distinguishing profiles between patients identified as ‘standard risk’ from ‘medium risk’ according to BFM95 clinical criteria. Gene expression profiles from a cohort of ALL patients, identified as ‘standard risk’ at diagnosis, were compared on the basis of their overall clinical outcome: relapse within 2 yrs vs non-relapse. Using a range of analyses including t-test, Gene-RaVE, discriminant analysis approaches and principle component analysis, we have discovered that small subsets of genes (

Description: An increased incidence of obesity and cardiovascular morbidity and mortality was recently observed in adult survivors of childhood malignancies younger than 45 years. The purpose of this study was to investigate the presence of early indicators of the dysmetabolic syndrome in a population of young survivors of acute lymphoblastic leukemia (ALL) in childhood. Patients and methods: Our study included 58 patients (31 males), aged 5–24 years (mean 12.9 years) with ALL, who had finished their treatment by the same protocol at least 2 years before the study (mean 5.1 years, range 2–10 years). Ten patients had received cranial irradiation (18 Gy). No patient had a history of thyroid disease or diabetes mellitus or received hormonal substitution. We obtained a detailed history of their diet habits and level of physical activity and measured their body mass index (BMI) and blood pressure (BP). Complete blood counts and blood chemistry tests were also obtained. Finally, bone density was measured in the lumbar spine and femur with DEXA. Results: Diet enriched in lipids and low carbohydrates was reported by 43/58 (74%), while absence of any athletic activity by 38/58 (65%) of the patients. Frank obesity with a BMI 〉30 or its equivalent for age and gender was observed in 12/58 (21%) of the patients, while 25/58 (43%) were overweight, with a BMI 〉2595th percentile) was observed in 13/58 patients (22%), while in 25/58 (43%) it was 〉 the 75th percentile. Dyslipidemia characterized by one, two, or three indices (serum cholesterol, triglycerides or LDL concentrations 〉95th percentile) was respectively detected in 20/58 (35%), 13/58 (22%), and 7/58 (12%) of the patients. Reduced serum HDL concentrations (

Description: Introduction: Abnormal eosinophils or eosinophilia in the bone marrow (BM) or peripheral blood (PB) are often found in acute myeloblastic leukemia (AML), especially in core binding factor (CBF) leukemias, including AML with t(8;21)(q22;q22) and inv(16)(p13;q22)/t(16;16)(q13;q22). However, it is still unclear why eosinophilia is frequently found in CBF leukemia. Recently, a Fip1-like1 (FIP1L1)- Platelet derived growth factor receptor (PDGFR) α fusion gene was demonstrated in idiopathic hypereosinophilic syndrome (HES) and chronic eosinophilic leukemia. This observation suggests that the activation of PDGFR may be linked to eosinophil differentiation of malignant cell progenitors. On the other hand, the signaling of PDGFR also has a very important role in malignant cell growth, survival, and differentiation. A two-hit model for the pathogenesis of AML, which seems to be caused by inactivating mutations in transcription factors and genetic lesions in tyrosine kinase resulting in constitutive activation, has been proposed. Moreover, it was shown that additional constitutive activation of RTKs causes leukemia in AML1/ETO- or CBFβ/MYH11-transgenic mice. In view of these findings, we investigated whether genetic abnormality of PDGFRα/β genes was associated with leukemogenesis or eosinophilia of CBF leukemia. Method: Twenty-two samples of BM or peripheral blood PB from cases of AML with karyotypic abnormality of t(8;21) or inv(16) were obtained from Japanese adult de novo patients (〉16 years old). The expression of the FIP1L1-PDGFRα fusion gene and PDGFRα/β genes was studied by RT-PCR. The FIP1L1-PDGFRα fusion gene was detected by nested PCR. For analysis of the JM and TK2 domains of PDGFRα/β, exons 11–13 and 17–21 of PDGFRα and exons 11–14 and 16–20 of PDGFRβ were amplified, respectively. Confirmed cDNA products were subjected to direct sequencing. Result: Among 22 cases of CBF leukemia with eosinophilia, 11 cases were inv(16) and 11 were t(8;21). Regarding the cases with inv(16), 9 were M4Eo and 2 were M2 subtype in FAB classification. The median percentage of eosinophils in their BM was 10.7% (range 5.6–31.8%). All cases with t(8;21) were diagnosed as M2 subtype and the median percentage of eosinophils in their BM was 1.8% (range 0.2–6.9). In this study, no FIP1L1-PDGFRα fusion gene was found. The PDGFRα gene was expressed in 19 of 22 (86.4%) cases and expression of the PDGFRβ gene was detected by RT-PCR in all tested cases. Association between the expression of the PDGFRα/β genes and the eosinophil blood count was not seen. The sequencing of JM and TK2 domains of cDNA detected three types of single nucleotide alterations in PDGFRα/β genes; one was in codon 603 in the JM domain of PDGFRα in 9 cases, changing GCG to GCA (G2203A), both of which encode the same amino acid, alanine (Ala). The second was in the TK2 domain of PDGFRα, in which an abnormality was detected in the third nucleotide of codon 824 in 7 cases, changing GTC to GTT (C2866T), both of which encode valine (Val). The third was in codon 867 in the TK2 domain of PDGFRβ in 1 case, changing TTA to TTG (A2957G), both of which encode the same amino acid, leucine (Leu). However, all of them were silent changes. Moreover, all three variants were present in cDNA extracted from BM samples of patients in remission and were previously described as single nucleotide polymorphisms (SNPs). Conclusion: PDGFRα/β genes do not appear to play a significant pathogenetic role in eosinophilia or leukemogenesis of CBF leukemia.

Description: Sezary Syndrome (SS) is a rare form of Cutaneous T-Cell Lymphoma (CTCL) characterised by a distinct metastatic pattern mainly involving skin and blood. Chemokine and chemokine receptors have been implicated in the spreading process of many cancers including various forms of non-Hodgkin T-cell lymphomas (NHL). In this study we report that chemokine receptor CXCR4 is over-expressed by both circulating and skin-homing neoplastic T-lymphocytes of SS patients and is functionally active as demonstrated by the migration of freshly isolated Sezary (SzS) cells along the chemical gradient of its natural ligand SDF-1. To shed light on the regulation of CXCR4/SDF1 interaction, we also investigated the enzymatic activity of CD26/dipeptidylpeptidase IV (DPPIV) since SDF-1 is efficiently inactivated by CD26, in physiological condition.. This is of particular relevance because one of the hallmark of the circulating SzS cells is the loss of CD26 from the cell surface. We first demonstrated that the CD26 negative phenotype is similarly maintained also in the skin-homing neoplastic T lymphocytes; we then observed that the addition of exogenus soluble CD26 reduces the migratory response of SS cells to SDF-1 whereas the inhibition of the CD26 peptidase activity in Hut78, a CD26-positive CTCL cell line, enhances the SDF-1-induced migration of these cells. We finally showed that SS individuals exhibit a reduced activity of the soluble CD26 as revealed by the measurements performed on the patients derived plasma. Our findings suggest that the SDF-1-CXCR4 axis could play an important role in skin homing of SS through the regulatory activity of CD26.

Description: Background: Single-agent rituximab was shown to prolong overall response rate (ORR) in marginal zone lymphomas. However, despite the confirmed activity of this immuno-biologic agent a significant number of adverse events were reported in this commonly considered indolent lymphoma entity. Methods: To reduce the impact of rituximab toxicity we investigated the activity of low-dose rituximab infusions in 3 consecutive elderly patients (over 70 years) with nodal marginal zone lymphomas. All patients (Ann Arbor stage IV with bone marrow (BM) involvement, age range from 71 to 84 years, normal LDH and WHO performance status = 0) were treated with weekly low-dose rituximab (75 mg/m2) administred for 6 consecutive weeks. Results: All patients complleted the treatment program. No complete response were observed. All patients confirmed a partial response (PR) with reduction of symptoms and disease related BM involvement. The median response duration was 14 months. At a median follow-up of 18 months, 2 patients relapsed and one patient (chemiotherapy naive) is still responding. The 3 patients did not experienced any adverse events and depsite the older age of patients, no expected infusion-related side effects were recorded. Conclusion: This investigative report demostrated the efficacy and the well tolerability of low-dose rituximab (75 mg/m2 administreted weekly for 6 consecutive weeks) in elderly patients with well defined diagnosis of nodal marginal zone lymphoma. These observations can lead to the hypothesis that the dose and the schedule of rituximab should be redefined in this lymphoma entity.

Description: Marginal Zone Lymphoma (MZL) is a low grade malignant B cell lymphoproliferative disorder that comprises 2% of all lymphomas. Often an enlarged spleen and villous lymphocytes are present. Many patients however may appear to have chronic lymphocytic leukemia or other lymphoproliferative disorders. Immunophenotyping can differentiate these low grade lymphoproliferative disorders. MZL frequently is CD 20 + but CD5, CD 10, and CD23−. Treatment regimens include observation, chemotherapy, splenectomy, and immunotherapy with or without chemotherapy. 20 patients over the past six have been identified with MZL based on pathologic and immunophenotypic characteristics. 6 patients have had no treatment and have had stable disease from 9 months to over 4 years. 2 patients underwent splenectomy from 3 months to 2 years from diagnosis and have stable disease. 1 patient was diagnosed at splenectomy and relapsed 5 1/2 years later and remitted with rituxin. 1 patient received weekly rituxin for 4 weeks and has been in remission for 40 months.1 patient received weekly rituxin for 4 weeks, relapsed 9 months later, and remitted with rituxin and then maintenance rituxin. 1 patient responded to weekly rituxin for 4 weeks and is receiving maintenance rituxin. 1 patient responded to weekly rituxin for 4 weeks and then had residual disease irradiated. 1 patient remains in remission after 50 months after a cervical node was irradiated. 1 patient with blastic MZL remitted with CHOP-rituxin. 2 patients progressed after rituxin and required chemotherapy. A 92 yo patient has had a partial response to rituxin. An 82 yo patient died from complications from MZL. 15/20 patients have so far avoided chemotherapy. Rituxin may be an appropriate treatment for MZL, but clinical trials will be necessary to confirm this approach.

Description: We previously reported that TEL-FGFR3 in a patient with peripheral T-cell lymphoma and AML conferred IL-3 independency to Ba/F3 cells and activates MAPKs, p38, PI3K, Stat3 and Stat5 through its constitutive tyrosine kinase (TK) activity in TEL-FGFR3 transfected Ba/F3 cells (TF-V5). Both FGFR3 TK specific inhibitor SU5402 and PI3K inhibitor LY294002 inhibited cell growth of TF-V5 in a dose dependent manner (IC50=5μM, 10μM respectively). SU5402(25μM) resulted in G1 arrest (90%) and induced 80% of apoptosis to TF-V5 after 24hr. LY294002(50μM) decreased expression of c-Myc, Cyclin D3 and CDK4 in TF-V5 and induced G1 arrest (90%) and apoptosis about 20% to TF-V5 after 24hr. SU5402 completely suppressed expression of Bcl-xL and Bcl-2 after 24hr. LY294002 partially suppressed these expressions. As LY294002 did not affect the phosphorylation of STAT5 and STAT3, we hypothesized that these distinct apoptosis inducing abilities between SU5402 and LY294002 might be caused by the difference of Bcl-xL and Bcl-2 expression levels. Recent report suggests that FGFR3 activates Stat5 through recruitment of Pyk2 (proline-rich tyrosine kinase 2)-Src to its juxta-membrane (JM) domain. Therefore, we examined whether PP1/PP2 which are known as Src kinase inhibitors inhibit TF-V5 cell growth. PP1 and PP2 inhibited TEL-FGFR3 induced cell proliferation in a dose dependent manner (IC50=15μM, 15μM respectively). In contrast, growth of mock with IL-3 was not inhibited at a high concentration of PP1or PP2(30μM). PP1/PP2 did not affect auto-phosphorylation of TEL-FGFR3. Interestingly, PP1/PP2 inhibit Tyr phosphorylation of Pyk2 except Tyr402 and markedly suppressed activation of Stat3, Stat5 without affecting MAPKs and PLC gamma activation. PP1/PP2 suppressed expression of Bcl-xL and Bcl-2 partially but not of Bax and induced apoptosis 20% and 30% respectively. PP1/PP2 partially blocks cell cycle at G1 phase. When TF-V5 was co-treated with PP1(30μM)+LY294002(50μM) or PP2(30μM)+LY294002(50μM), apoptosis was induced in 60–70% of TF-V5 cells which remained at G1 phase after 24hr. Although PP1+LY294002 or PP2+LY294002 did not suppress Bcl-xL and Bcl-2 completely like SU5402, PP1/PP2 induced N-terminal cleavage form of Bax. Conclusion: PP1/PP2 induces apoptosis in TF-V5 by activating Bax and by Pyk2-Src kinase inactivation that leads to the change of Bax/Bcl-2 or Bax/Bcl-XL ratio. Combination of PP1/PP2 and LY294002 induce high rate of apoptosis under G1 arrest in TF-V5 and may represent a novel approach against TEL-FGFR3 associated hematopoietic malignancies.

Description: Cyclophosphamide is used in high doses as a part of the conditioning regimen prior to stem cell transplantation. It is usually given for two or four consecutive days, primarily to facilitate engraftment of donor cells. Cyclophosphamide is a prodrug that is activated in liver by a 4-hydroxylation reaction catalyzed by cytochrome P450 (CYP) enzymes. Several studies have shown that cyclophosphamide induces its own metabolism, which affects its pharmacokinetic parameters after repeated dose (Chang TK, Yu L et al. Cancer Res1997; 57:1946–54 and Schuler U, Ehninger G et al. Cancer Chemother Pharmacol1987; 20:248–52). In the present study, we aimed to investigate the effect of repeated doses of cyclophosphamide on the CYPs in rat. mRNA, protein, and enzyme activity levels were investigated. Animals received (200 mg/kg, i.v.) at time 0, 20, 48 h. Additionally another group of animals was treated at 0, 6, 20, 26, 48 and 54 h. At each time point three animals were killed 30 min after the administration (Xie H, Afsharian P et al. Xenobiotica2005; 35:239–51). mRNAs of CYP2B1 and 2B2 were significantly induced up to 458- and 8.3-fold at 6h, 983- and 102-fold at 26 h, and 342- and 33-fold at 54 h. CYP2B protein levels were increased and their peaks was observed at 20 and 48 h. Microsomal activity of CYP2B was determined at three different concentration of cyclophosphamide (1, 0.5, 0.1 mM) by measuring the formation rate of 4-hydroxy-cyclophosphamide (4-OH-CPA). The microsomal activity increased as reflected in an increase in cyclophosphamide 4-hydroxylation at all concentrations used using microsomes from rats treated at 6, 20 and 48 h. A significant increase of 4-hydroxylation of cyclophosphamide (0.1 mM) by 2.9-, 4.4- and 4.2- fold, respectively compared to the control rats. However, a decrease in the hydroxylation rate was observed using microsomes from rats treated at 26 and 54 h and in all concentrations used that might due to hepatotoxicity effect of cyclophosphamide when administered in short time period of repeated dose. A significant (p

Description: Castleman’s Disease (CD) is a distinct pathologic entity with clinical heterogeneity. with hyperimmune or immune dysregulation, There is no known standard treatment. A variety of therapies have been tried including Surgery, Radiation, steroids, single agent chemotherapy, multi agent chemotherapy, Rituximab and monoclonal antibody to IL-6. Plasmaphersis which was successful in this patient has not been tried before in CD. Case report 31 year old female with no significant past medical illness admitted with 10 days history of fever, abdominal pains. CT scan of chest and abdomen showed small mediastinal and left axillary lymphadenopathy. Physical examination revealed a palpable 2 cm rubbery mobile non tender left axillary node and mild tenderness over right upper quadrant, otherwise unremarkable Laboratory examination on admission showed BUN 17 mg/dL, Creatinine 1.4 mg/dL, Potassium 5.5 meq/L, Calcium 8.4 mg/dL, Total Protein 6.6 g/dL, Albumin 2.9 g/dL, Bilirubin 1 mg/dL, Alkaline phosphatase 427 U/L, AST 27 U/L, ALT 26 U/L, GGT 288 U/L. WBCs 12,400, with 70% Neutrophils, 7%Bands, 10%Lymphocytes, 11% Monocytes and 2%. Eosinophils, HGB 12 g/dL, Platelets 630,000. Peripheral smear was normal. ESR 65 mm/hr. ANA-negative. Serum Complement C4 22 mg/dL and C3 116 mg/dL. Monospot test- negative. Toxo titer was negative. PT 15.5” and PTT 38”. Serum Protein Electrophoresis and seum immunofixation were normal. Urine Bence Jones Protein was negative. Serum Cryoglobulin was negative. Urinalysis was normal. Hepatitis profile was negative. Hepatobiliary scan was normal. Hospital Course- She continued to have fever despite negative cultures. Renal functions worsened over the first few days of admission, BUN and Creatinine increased to 27 mg and 2.3 mg respectively. Left axillary lymph node biopsy showed Hyaline vascular Castleman’s disease like changes. Biopsy of right kidney was done showed Chronic Thrombotic microangiopathy with predominant glomeruler involvement. Bone marrow biopsy and Immunohistochemistry of the aspiration were normal. HHV- 8 antibody was negative. HIV screening was also negative. She was started on Prednisone 1mg/kg/day without clinical or laboratory improvement. BUN increased to 101mg/dL and Creatinine to 3.8mg/dL with generalized anasarca and reduced urine out put. Hgb dropped to 6.7gms/dl, Platelets 27,000 and WBCs 18,700 with neutrophilia. Peripheral smear showed no schistocytes at any time. Since her condition was worsening despite high dose of steroids, patient was started on daily Plasmaphersis with noticeable daily clinical and laboratory improvement. After 26 sessions of Plasmapheresis all the hematological and renal abnormalities improved with clearance of anasarca and normal urine out put. At the time of discharge - CBC showed WBCs 13,000, Hgb 11.1 g/dL, Platelets 123,000. BUN 25mg/dL and Creatinine 0.5 mg/dL. The repeat CT scan revealed no lymphadenopathy. Two year later she remains in complete remission with normal renal function and normal blood counts. Conclusion Plasmaphersis can help in controlling the Hyper immune or immune dysregulation, consequences of Castleman’s disease and may be an option in treating severe systemic manifestations of CD.

Description: More than 56,000 new cases of non-Hodgkin’s lymphoma (NHL) will be diagnosed this year in the United States. Despite advances in treatment modalities such as radiation, biologic agents, and cytotoxic chemotherapeutic regimens, only 25–30% of these patients will be cured of their disease. Standard salvage regimens such as DHAP, ESHAP, ICE and EPOCH have proven efficacy at the cost of increasing toxicity and hospitalization costs. The reported response rates for these are on the order of 50–75% as first-line salvage regimens. However, as our aging patient population develops worsening performance status and co-morbidities, it seems appropriate to develop effective lymphoma treatments, which have fewer toxicities and lower costs for administration. One approach is to combine non-myelosuppressive therapies. One non-myelosuppressive agent, which has efficacy in lymphoma, is gallium nitrate. Investigation of gallium nitrate for cancer treatment dates back to the 1970’s. While it is currently approved for the treatment of bisphosphonate resistant hypercalcemia of malignancy, it has also been shown to inhibit ribonucleotide reductase and bind transferrin and potentially complex with the transferrin receptor, which is highly expressed in intermediate and aggressive histology lymphomas. It appears that the binding of the transferrin receptor on the lymphocyte as well as its inhibition of ribonucleotide reductase, eventually impairs iron metabolism, which is a necessary component of the intracellular cytochrome systems/mitochondrial function and ultimately oxidative phosphorylation. The current study is a phase II clinical trial investigating the combination of gallium nitrate, rituximab and dexamethasone (GaRD) for relapsed or refractory DLBCL, MCL or transformed follicular lymphomas. The gallium nitrate is given at 200mg/m2 CIV days 1–7, rituximab 375mg/m2 IVPB day 1 and dexamethasone 40 mg po days 1–4. Eligible patients must have proven relapsed or refractory disease and have a SWOG PS

Description: Lymphomatoid papulosis (LyP) is a CD30+ cutaneous lymphoma according to the WHO classifications and regarded as a condition of uncertain malignant potential. The incidence of LyP in children is relatively low compared to that in adults. It is correlated with malignant lymphomas in 5–20% of adult LyP-patients. In children, an increased risk for the development of a malignant lymphoma is observed. The clinical course is often chronic. Most common treatment for LyP is topical steroids, antibiotics, phototherapy and low-dose methotrexate. Prognosis is excellent with a survival of 100% even for patients with malignant lymphomas. Case-report: An 8-years old boy suffered from reddish nodules (maximum 7 cm in diameter) on his right forearm and his left leg. Immunohistochemical analysis identified a CD30+large-T-cell-type-NHL of the skin. Topic steroids were effective but six months later he showed axillar lymph-node swelling. Immunohistochemically it was classified as anaplastic large cell T-type-lymphoma (ALCL) without signs of systemic involvement. Combination chemotherapy according to the European ALCL-trial (high-risk-group) was given for six months. All nodules (cutaneous and lympoid/axilla) resolved within a few weeks. Two months after cessation of chemotherapy a new skin-nodule on the left forearm appeared. The immunohistochemical diagnosis was now LyP. No specific therapy was given. In the following two months he developed two further solid and painful skin-lesions. In this situation we decided to start a therapeutic approach with subcutaneous mistleote (MT), which was injected close to one of these lesions. The skin-nodules decreased after the first dose and MT injections into all nodules were continued. Within the following two weeks the skin-lesions resolved completely and subcutaneous MT therapy was continued. Two months later, he developed two new nodules responding within a few days to increased dose of MT. While continuation therapy with MT the boy was without clinical signs both of the LvP and ALCL for nearly two years. Subsequently MT therapy was terminated. Three weeks after cessation the cutaneus LyP generally reactivated all over the body with typical nodules (maximum 1 cm in diameter). Subcutaneus MT therapy was restarted and the cutnaneus LyP regressed within 2 weeks completely without additional therapy.

Description: Objective: While busulfan is a commonly used chemotherapeutic agent in the treatment of many hematological diseases, its effectiveness against neuroblastoma is still in question. This study aims to assess the degree of apoptosis and cell death in neuroblastoma cell lines and primary neuroblastoma tumors when exposed to varying doses of busulfan. Materials and Methods: Cultures from established cell lines SKN-SH, SKN-DOX-R, IMR-5, and NGP (n=4), as well as cultures from primary tumors (n=2) were seeded at 106 cells/ml in RPMI640 supplemented with 10% fetal bovine serum (FBS) and transferred to 24-well plates, where cells were exposed to 1ml of busulfan at 0, 0.001, 0.005, 0.01, 0.05, and 0.1mg/ml per well. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 for 72 hours. Wells were sacrificed after 0, 6, 24, 48 and 72 hours and tested with Annexin V and PI; 10,000 events were measured by flow cytometry. The percentage of apoptotic and dead cells was plotted in a graph and a t-test was performed against the untreated control. Results: After 24 hours, there was a significant decrease in cell viability of each dose when compared to the control untreated cells (p

Description: Methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MTR) are key enzymes in folate metabolism, which is essential for DNA methylation and synthesis. It is known that polymorphisms in its genes have been associated with some forms of cancer including lymphoma. Previous studies have shown MTHFR 677TT was associated with decreased risk of non-Hodgkin’s lymphoma(NHL). However, recent two reports have shown MTHFR 677TT was associated with increased risk. To evaluate the association between the MTHFR C677T and MTR A2756G polymorphisms and risk of non-Hodgkin’s lymphoma, large-scale population-based case-control study was conducted in Chonnam University Hwasun Hospital between March 1997 and June 2005. Three hundreds sixty-five patients with histologically comfirmed lymphoma and 1,162 controls were evaluated. Genotyping was done using PCR-RFLP. The cases consisted of 203 diffuse large B-cell lymphomas(DLBCL), 77 T-cell lymphomas, 62 other B-cell lymphomas, and 23 unclassifiable lymphomas. The MTHFR 677CT and 677TT genotypes were inversely associated with NHL and DLBCL, respectively. Using subjects with the MTHFR 677CC as reference group, the odds ratio of MTHFR 677CT and 677TT were (0.70, 95% CI 0.54–0.90) and (0.46, 95% CI 0.32–0.68) for NHL. The association was more evident for DLBCL (OR 0.56, 95% CI 0.40–0.78 for 677CT; OR 0.40, 95% CI 0.24–0.65 for 677TT). Dose-response effect was evident for the MTHFR T-allele (p 〈 0.01). There was no significant association of MTR A2756G with NHL. These results suggest that the MTHFR polymorphism may play an important role in the pathogenesis of NHL, particularly DLBCL.

Description: The population of new ANHL patients is heterogeneous in terms of their biological parameters and the IPI scoring system is widely used to make the disease prognosis. Recently heterogeneity of ANHL patients in terms of their QoL has been shown. There are four groups of patients with the following grades of QoL impairment: none, mild, significant, and critical impairment. We aimed to study the distribution of new ANHL patients according to the grades of QoL impairment and to identify the relationship between the QoL impairment groups and the IPI groups. 73 new aggressive NHL lymphoma patients (Stage IIB-IV, mean age 55.2 SD=17.6, males/females – 39/34) were involved in this study. QoL assessment was conducted before treatment using SF-36. The method of integral profiles was used to calculate the integral QoL index (IQLI). Patients were stratified using IQLI. Gamma correlation between ranges of QoL impairment groups and IPI groups was used. The following distribution of ANHL patients was demonstrated on the basis of different grades of QoL impairment: 9% - no impairment (IQLI - 0.6); 12% - mild impairment (0.3); 24% - significant impairment (0.13); 55% - critical impairment (0.02). The distribution of patients in these groups who were classified as high-intermediate or high risk according to the IPI was 28%, 67%, 83%, and 90%, respectively. High correlation between ranges of QoL impairment groups and IPI groups was observed: Gamma correlation – 0.6 (p

Description: Background. Recent reports have shown that Rituximab added to conventional chemotherapy may significantly improve the prognosis of CD20-positive Diffuse Large B-cell Lymphoma (DLB-CL). However, patients with unfavorable clinical presentation still have a poor outcome. Other studies have documented an increased anti-lymphoma activity upon addition of Rituximab to intensified treatments with autologous peripheral blood stem cell (PBSC) transplantation. Based on these premises, a prospective multicenter study has been performed on the use of a Rituximab-supplemented high-dose sequential (R-HDS) chemotherapy schedule with PBPC autografting in patients with unfavorable DLB-CL, defined as score 2 and 3 (intermediate-high or high) according to the age-adjusted International Prognostic Index (aaIPI). Methods. The R-HDS regimen included: (i) an initial debulkying with 3 APO courses; (ii) a high-dose (hd) phase consisting in the sequential administration, at 15–20 day intervals, of hd- cyclophosphamide (7gr/sqm, with two Rituximab doses at 375 mg/sqm), hd-Ara-C (2gr/sqm b.i.d. for 6 days with Rituximab), and hd etoposide + Cisplatin; (iii) a final myeloablative phase (hd-Mitoxantrone + L-Pam) with PBSC autografting and 2 more doses of Rituximab. Involved-field radiotherapy was scheduled on areas of previous bulky disease or residual lesions. Six Centers affiliated to the GITIL group (Gruppo Italiano Terapie Innovative nei Linfomi) participated in the multicenter study. Patient enrollment started in November 1999 and was closed in September 2004. Results. Overall, 112 previously untreated patients aged ≤ 60 years, with CD20-positive DLB-CL and aaIPI score 2 (74 pts) or 3 (38 pts), entered the study protocol and are evaluable. There were 5 early toxic deaths (3 sepsis in the hd-phase, 1 pneumonia and 1 leucoencephalopathy from JC-virus infection after autografting) and one late toxic death due to pneumonia which occurred at 10 mos. after R-HDS. The TRM was 5.3%. Ninety patients (80 %) achieved Complete Remission (CR). At a median follow-up of 24 mos, 90 patients (80%) are alive and 83 (74%) are in continuous CR (CCR), leading to a 5.3-yr event-free survival (EFS) projection of 71%. There was a trend towards a better outcome in aaIPI 2 vs. 3, although the difference was not statistically significant. Conclusions. The CR, OS and EFS rates observed after R-HDS compare favorably with the poor outcome anticipated in aaIPI 2–3 patients managed with conventional chemotherapy. The results of this phase II study prompted an ongoing phase III GITIL multicenter study to compare R-CHOP vs. R-HDS in younger patients with high-risk DLB-CL.

Description: The standard cytoreduction for bone marrow transplantation (BMT) of pts with HGBpathies has been the combination of Busulfan (BU) and Cyclophosphamide However, for pts with advanced disease, high rates of graft rejection and toxicity were reported. For such high risk pts, a BU and Fludarabine (FLU) cytoreductive combination was used at low dose in the context of non-myeloablative BMT and was associated with a poor outcome with high rates of graft rejection. In view of the low toxicity associated with the BU-FLU combination, we used both agents at higher, potentially myeloablative doses for the cytoreduction of six pts with high risk HGBpathies. Between 12/00 and 04/05, 4 pts with thalassemia (Thal) and 2 pts with sickle cell disease (SCD), including 2 males and 4 females aged 4.6–15.3 years were transplanted using this regimen. All 4 pts with Thal had advanced Lucarelli Class 2 disease, while the 2 pts with SCD had stroke, recurrent vaso-occlusive crises (VOC), sickle lung disease and alloimmunization (n=1) and recurrent VOC, acute chest syndrome and osteomyelitis (n=1). Cytoreduction included intravenous BU (0.8–1 mg/Kg/dose x 14), FLU (30 mg/m2/day x 5) and Rabbit ATG (2.5 mg/Kg x 2). GvHD prophylaxis consisted of Tacrolimus (n=3) or cyclosporine (n=3) and methotrexate (n=5) or Steroids (n=1). Pts received an unmodified BMT from their HLA-identical sibling with total nucleated cell doses of 0.7–5.7 x 108 cells/Kg. The regimen was well tolerated with minimal toxicity. With a median follow-up of 21.5 mo (range 3–55 mo), all 6 pts are disease- and transfusion- free. There was no graft rejection and no GvHD. Chimerism status for pts with Thal was 98–100% donor, 5 mo to 2 years post BMT, while for the 2 pts with SCD, it was mixed with 50 and 80% donor cells at 3 mo and 2 years post BMT. The pt with 50% donor cells received a low graft cell dose (0.7 x 108 nucleated cells/Kg). One additional pt with SCD and a history of 2 strokes and moya-moya disease received BU FLU + melphalan (70 mg/m2 x 2). This pt had minimal post BMT toxicity, and is engrafted with 100% donor cells at 1 year post BMT. In summary, the combination of high dose BU and FLU for pts with high risk HGBpathies was well tolerated and induced full engraftment for pts with Thal, but mixed chimerism for pts with SCD. Nevertheless, all pts so treated survive with normal hematologic function. The addition of melphalan to BU FLU was also well tolerated and could be beneficial in attaining complete myeloablation and full chimerism in pts with SCD.

Description: Matched related donor (MRD) bone marrow transplantation is the treatment of choice for pediatric patients with severe aplastic anemia; however, only 25% of patients will have an HLA-identical sibling. Alternative donor transplants may be an option for these patients, but such therapies have been associated with greater incidences of graft failures and graft-versus-host disease (GVHD). We retrospectively analyzed 32 pediatric patients who have undergone either bone marrow or peripheral blood stem cell transplant for severe aplastic anemia at our institution from April 1997 to April 2005. These patients had a total of 34 transplants. One patient had a MRD transplant followed by a matched unrelated donor (MUD) transplant eight years later, while another patient had a HLA-mismatched unrelated donor (MMUD) transplant followed by a transplant from a haplo-identical parent. Of the remaining 30 patients, 12 received MRD transplants, whereas 18 patients received alternative donor transplants - 11 MUD, 3 haplo-identical donors, and 4 MMUD. The median age at transplant was 9 years (range 1.5 to 18.4 yrs). All patients who received alternative donor transplants had previously failed therapy, including antithymocyte globulin (ATG) and cyclosporine. For MRD transplants, the conditioning regimen most often utilized cyclophosphamide 50 mg/kg x 4 days and ATG 30 mg/kg x 3 days. For alternative donor transplants, the conditioning regimen most often utilized cyclophosphamide 50 mg/kg x 4 days, Campath 3–10 mg x 4 days (dependent upon patient’s weight) or ATG 30 mg/kg x 3 days, and TBI (single fraction 200 cGy for MUD; two fractions 200 cGy for MMUD). Alternative donor recipients who received ATG in their preparative regimen were transplanted between December 1997 and March 2001 (n=9), whereas patients who received Campath were treated between November 2001 and April 2005 (n=11). GVHD prophylaxis was either FK506 or cyclosporine +/− mini-methotrexate. The overall survival for MRD patients was 91.7% versus 80% for alternative donor patients at a median follow-up of 47 months (range 3 to 100 months). Of the 32 patients, there were 5 deaths: pulmonary failure with extensive, chronic GVHD (n=1); poor graft function with infection (n=1); and infection (n=3). For patients receiving alternative donor transplants, the overall survival for the Campath group was 81.8% vs. 77.8% in the ATG group. None of the Campath patients developed extensive, chronic GVHD compared to 3/9 ATG patients. In conclusion, alternative donor transplantation using Campath or ATG in the preparatory regimens can establish donor engraftment and offers a curative therapy for pediatric severe aplastic anemia patients with survival similar to that of patients receiving matched sibling transplants. Although follow-up is shorter, Campath may be associated with a reduced incidence of extensive, chronic GVHD and further investigation is warranted.

Description: Although the success of imatinib mesylate therapy represents an exciting advance in targeted cancer therapy, it has still to be determined whether responses to this p210 inhibitor in chronic myeloid leukemia (CML) patients will be durable. In fact most of clinical studies agree on the evidence of a persistent molecular disease in the majority of treated patients and altough the absolute level of bcr-abl transcript may vary over the treatment, yet a molecular complete response is of rare observation. In addition, discontinuation of imatinib exerts always in rapid loss of response. In accordance to this the persistence of malignant progenitors in patients in complete cytogenetic response (CCR) after short term imatinib treatment, has been recently demonstrated. In particular, Bathia et al. showed in 12/15 patients studied after a median time of 10 months of imatinib treatment a median of 11% of residual CML CD34+ progenitors in the bone marrow (by FISH Dual Fusion bcr/abl analysis)while only 3/15 patients had no detectable residual CD34+ cells. Less is known about residual Ph+/CD34+ cells surviving after a prolonged therapy with this targeting drug. Thus, we evaluated the amount of bone marrow residual CD34+ cells in 17 CML patients in stable CCR after a long lasting treatment with imatinib. At the time of evaluation, the patients were on conventional dose (400mg) Imatinib for a median time of 48 months (range 36–58 months) having achieved a CCR status (conventionally defined as the complete absence of t(9;22) on caryotypic analysis) within 3 to 6 months of treatment. However all of them still showed molecular disease as detected by nested RT-PCR. Bone marrow CD34+ cell-enriched populations were selected from mononuclear cells using immunomagnetic column separation and were evaluated after cytospin by FISH using a bcr-abl Dual Color Extra Signal Probe(LSI bcr-abl ES, Vysis), that is able to detect bcr-abl fusion in interphase nuclei with a false positive signal rate close to 0. A minimum of 100 CD34+ nuclei per each sample were evaluated. Interestingly, in 8/17 patients no Ph+/CD34+ cells were detected, while in the remaining 9/17 patients a median of 2% (range 0.5–11%) of bcr-abl positive progenitors were still observed. In this small selected serie of patients prolonged treatment with imatinib appears to be correlated with a lower, yet detectable, amount of residual bone marrow Ph+/CD34+ cells when compared to previously published data. This result could be partly explained with the different specificity and sensitivity of the probe used (bcr/abl ES

Description: The quantitative assay for free light chains [FLC] has been reported to be sensitive and specific for detecting and monitoring free light chain diseases such as multiple myeloma. To evaluate the sensitivity of FLC for monitoring patients in complete remission for early detection of relapse, the measurement of more than 250 serum free light chains were performed with the commercial available Freelite TM kit [Binding Site] in 26 patients who achieved complete remission with negative immunofixation after dose reduced allogeneic stem cell transplantation. The patient groups were divided in those who remained immunofixation negative [n=12, group 1] during follow-up of at least 1 year and those who had been immunofixation negative but became positive during follow-up [n=9, group 2] and those who had achieved near complete remission with positive immunofixation but then became immunofixation negative during follow-up [n=5, group 3]. In group 1 the measuring of 105 FLC concentration and kappa/lambda ratio was performed in 12 patients. In 10 patients [83 %] free light concentration of kappa or lambda remained within the normal range during follow-up of more than 1 year. In 2 patients [17 %] kappa or lambda FLC concentration was above the normal range, but remained stable without any signs of increasing amount. Group 2 consisted of 9 patients who had been immunofixation negative but became positive during follow-up. In all patients an increase of the corresponding free light chain could be observed in serum. In 4 patients a very close monitoring of immunofixation and free light assay was performed and an at least 25 % increase of the free light concentration in serum was observed at a median of 97 days before immunfixation became positive. In group 3 five patients who had been immunofixation positive became negative during follow-up. In all of the patients the free light concentration was within the range at time of negative immunofixation. The corresponding free light concentration dropped down and reached normal level at a median of 38 days before the patients had achieved negativity of immunofixation. These results suggest that serum free light chain assay allows monitoring of patients with complete remission and might detect early relapse before immunofixation becomes positive. Thus, an early increase of free light chain assay in immunofixation negative patients after allogeneic transplantation might be an useful guide for adoptive immunotherapy strategies to prevent clinical relapse.

Description: Study Objective: To estimate the probability of finding an HLA matched cord blood unit, taking into account ethnicity, and the required size of national inventory of cord blood units that would provide a 6/6 or 5/6 HLA-A, -B, -DRB1 match for 80% of US patients. Methods: The study was limited to patients and cord blood units in the NYBC inventory through October 1, 2004 that had HLA-A, -B and -DRB1 typing completed at low resolution for class I and high resolution for DRB1. Of 16,222 search requests, 10,007 patients had high resolution DRB1 typing and of 26,675 cord blood units in our bank, 20,773 are DRB1 typed, thus far, at high resolution. One-third of the inventory would provide ≥ 2.0 x 107 TNC for a 70 kg adult. The patients had relatively low frequencies of the common HLA haplotypes, suggesting a reduction because suitable matches were found among marrow donor registries. Patient ethnicity was similar to that of the US population while donors were more likely to include members of ethnic minorities (20% African-American, 21% Hispanic, 8% Asian), as expected (we collect cord blood in some maternity hospitals that serve a high proportion of non-Caucasoid expectant mothers). A simulated search was performed to find matches for all patients in the current inventory and repeated to find potential grafts with mismatches in the GvHD direction only (no rejection mismatch). Grafts with only GvHD mismatches have had outcomes similar to a 6/6 match (Blood2003; 102: 478a). Results: All except 94 patients (0.9%) had one or more cord blood units that provided a 4/6 match or better, 61% had a 5/6 match and 9% a 6/6 match. The probability in finding well-matched units almost doubled when we included both 6/6 matches and units mismatched in the GvHD direction only. The chance of finding a 5/6 or 6/6 match varied with patient ethnicity and was lowest for African-Americans (44% and 1.7%, respectively), despite the relatively high proportion of African-American donors to the inventory. As expected, patients were more likely to find a suitable match within their own ethnic group. When the inventory search was restricted in size (randomly selected units) to 5,000, 10,000, 15,000 and the full inventory of 20,773 units, the chance of finding a 5/6 or 6/6 match increased (36%, 48%, 55% and 61%, respectively) demonstrating a progressively diminishing improvement in match success. Conclusions: These empirical comparisons indicate that most patients can find a suitably matched cord blood unit, defined as either a 5/6 or 6/6 HLA match, within our relatively small inventory of cord blood units. The probability of finding a match being highest in the patient’s own ethnic group underscores the need to enrich cord blood inventories with cord blood units donated by ethnic minorities. A projection based on these analyses suggest that 55–60,000 units would provide a 5/6 or 6/6 match for 80% of patients assuming that the ethnic distribution of donors and patients is maintained. However, cord blood units must provide a cell dose ≥ 2.0 x 107 TNC/Kg for single unit transplants. Thus, some 155–170,000 cord blood units may be required to also provide 70 Kg adults with ≥ 80% chance of finding a suitable match with an adequate cell dose. Inventory requirements might decrease if, for example, transplants with well matched units or two unit transplants lessen the cell dose limits, as preliminary data now suggests.

Description: We analyzed 150 consecutive patients (pts) transplanted from unrelated donors (URD) at single institution- Silesian Medical Academy in Katowice- with use of the same standard operating procedure from February 1997 until December 2004 for CML (74 pts), AML (28), ALL (27), SAA (9), MDS (7), MM (1), NHL (1), PNH (1), OMF (1), bi-phenotypic AL (1). 92 pts were transplanted from matched donors, including 59 with complete 10 alleles (HLA-A,B,C,DRB1,DQB1) high resolution (HR) DNA-typing; and 33 with first class match established on base of low-resolution (LR) (13), serological (7) or un-complete typing without HLA-C (13) and second class HR typing. 58 pts were transplanted from mismatched donors (first class typing was HR in 43, LR in 14 and serological in 1 pt): 22 with single allelic mismatch (2 HLA-A, 7-B, 6-C, 7-DQB1), 33 with single HLA-C antigen mismatch and 3 with double mismatches (2 B+C, 1 C+C). Survival advantage at 4 years, although without statistical significance (p=0.16), was observed in the group of pts transplanted from 10/10 alleles matched donors (36+/− 11%) over those with mismatched donors (24+/− 11%). Oppositely, pts transplanted from matched donors who were not completely typed in HR did not achieve better survival (23+/− 17%). Poorest survival (13+/− 12%, p=0.007) was observed in patients transplanted from mismatched homozygous donors (n=10). The risk of acute GVHD grade 3–4 was increased (p=0.007) in pts with mismatched donors (31+/− 6%) when compared to matched completely typed in HR (10+/− 4%). Also the rate of graft failure tended to be lower in pts with matched than mismatched donors (5.1% versus 10.2%, p=0.25). In contrast, relapse rate was lower in mismatched (23+/− 10%) than in HR matched pts (34+/− 12%, p=0.55) what may reflect better GVL effect in mismatched transplant recipients. Unexpectedly, the rate of chronic GVHD was similar in pts with 10/10 alleles matched and mismatched donors (40+/− 10% versus 42+/− 9%, p=0.75). These results indicate that complete high resolution HLA class I typing is necessary for adequate selection of unrelated donors. Class I HLA-B and -C mismatches influence both survival and serious a-GVHD incidence. 10/10 alleles matched mismatched p survival at 4 years 36+/− 11% 24+/− 11% 0.16 aGVHD grade 3–4 10+/− 4% 31+/− 6% 0.007 graft failure rate 5.1% 10.2% 0.25 relapse rate 34+/− 12% 23+/− 10% 0.55 cGVHD 40+/− 10% 42+/− 9% 0.75 10/10 alleles matched mismatched homozygous donors p survival at 4 years 36+/− 11% 13+/− 12% 0.007

Description: Mobilization with chemotherapy (CT) plus hematopoietic growth factors (HGFs) is superior to mobilization with HGFs only. For stem cell mobilization after chemotherapy HGFs are typically given once or twice a day for approximately 14 days. The aim of this study was to evaluate whether mobilization of peripheral blood stem cells (PBSC) with DT-PACE plus pegfilgrastim was equivalent to mobilization with DT-PACE + filgrastim. Patients analyzed were enrolled in two consecutive studies 2001–12 (N=97) and 2003–41 (N=72) and included only patients who had received ≥ 2 cycles of prior therapy, but no prior transplant. Both protocols employed a single cycle of induction with DT-PACE (Lee et al. JCO2003;21:2732) followed by stem cell collection with either filgrastim 5μg/kg bid until completion of stem cell collection (2001–12) or pegfilgrastim 6mg on days 6 and 13 (2003–41). Patients then proceeded with tandem transplants, one consolidation cycle with DT-PACE and two years of maintenance with thalidomide (100mg daily) and dexamethasone (4 mg/day, days 1–4 q 3 weeks). Group characteristics were compared using the Kruskall-Wallis test. Time to hematopoietic recovery after transplantation was analyzed with Kaplan-Meier plots, and comparison was performed using the logrank statistic. The two studies had comparable characteristics, except that more patients ≥ 65 years were enrolled in 2003–41 (35% vs 13%; p=.001). The median number of collection days was 2 in both studies (p=.8). The median number of CD34 cells/kg (x106) collected per day was 9.7 (2001–12) vs 12.7 (2003–41) (p=.2). The total number of CD34 cell/kg (x106) collected was 20.4 (2001–12) versus 25.4 (2003–41) (p=.2). The median number of CD34 cells/kg (x106) infused after the first transplant was 4.1 (2001–12) and 4.5 (2003–41) (p=.5). The time to recover ANC 〉 500/μl and platelets 〉 20,000/μl without transfusion was shorter for 2003–41 patients (13 versus 15 days; p〈 .0001) (Figure 1). Mobilization of PBSC with pegfilgrastim is easier for patients because they only receive 2 injections and is as effective as filgrastim when combined with DT-PACE. Moreover, mobilization with pegfilgrastim may result in a more rapid hematologic recovery after transplantation. Cumulative incidence analysis Recovery of ANC to 500 and Platelets 〉 20K 2003-41 vs. 2001-12, Transplant 1 Cumulative incidence analysis Recovery of ANC to 500 and Platelets 〉 20K 2003-41 vs. 2001-12, Transplant 1